Intact Monoclonal Immunoglobulin Research Articles

B-181 Multi-centre Analytical Performance Evaluation of the EXENTâ Solution for Identification and Quantitation of Monoclonal Immunoglobulins

Abstract Background Current routine methodologies for monoclonal immunoglobulin measurements may not be sufficiently sensitive to reflect the depth of response seen in patients since the introduction of novel therapies; mass spectrometry may offer a valuable, sensitive alternative approach. Here we describe the preliminary analytical performance characteristics of the EXENT® solution (in development by The Binding Site, part of Thermo Fisher scientific) that combines specific immunoprecipitation steps and mass spectrometry for the identification and quantification of IgG, IgA and IgM intact monoclonal immunoglobulins. Each intact monoclonal immunoglobulin clone can be tracked using its unique m/z value. Methods The Lower Limit of Measuring Interval (LLMI) was established for each immunoglobulin type following EP17-A2:2012. Linearity studies were performed according to CLSI EP06-A2:2020 using high and low pools of IgG, IgA, and IgM monoclonal samples and with additional linearity testing below 1 g/L for each specificity to more effectively demonstrate low-end linearity. Within run, between run, between analyzer, between lot and total precision for M protein concentrations and for molecular mass (m/z) of the monoclonal peaks were assessed according to CLSI EP5-A3-2014. Interference was tested following ED3:2018 using 20 potential interferents against 7 samples including high and low IgG, IgA and IgM monoclonal samples. Results The initial indication of the EXENT solution performance, in development, are set out below. Results suggest an analytical sensitivity of the assays around 15 mg/L at the LLMI for all specificities. Linearity over a range of 0.014–88.9 g/L for IgG, 0.011–68.4 g/L for IgA, and 0.11–74.2 g/L for IgM. Coefficients of variation (CVs) for M protein concentrations in precision studies were <15% for all specificities and samples. Mass/charge values were within ±1.1 to ±1.5 m/z in total precision studies, and within ±2.7 to ±3.9 m/z in between lot precision studies, respectively, for M proteins with an m/z value ranging from 11 360.6 to 11 698.5 m/z. No significant interference effects were observed when testing the 20 interferents including intralipid (20 g/L), triglyceride (15 g/L), bilirubin (400 mg/L), rheumatoid factor (200 IU/mL) and haemoglobin (10 g/L). Conclusion The new EXENT solution demonstrates the potential for a wide measuring interval and the ability to detect M proteins with very low concentration. Also, it could provide stable and reproducible performance for the detection and typing of monoclonal immunoglobulins.

Clinicopathological manifestations of coexistent monoclonal immunoglobulin deposition disease and immunotactoid glomerulopathy.

Combination of monoclonal immunoglobulin deposition disease (MIDD) and immunotactoid glomerulopathy (ITG) is a rare form of monoclonal immunoglobulin (MIg)-associated renal disease. We retrospectively reviewed the native kidney biopsy specimens at Peking University People’s Hospital from 2011 to 2020. Five patients were diagnosed as MIDD + ITG. Their clinical and pathological characteristics were studied. The typical clinical features were nephritic syndrome and renal dysfunction with prominent anemia, but hematuria was mild. Unlike single MIDD and single ITG, on light microscopy, segmentally distributed mesangial nodular sclerosis on the basis of mesangial matrix hyperplasia was the major lesion. Others including membranoproliferative glomerulonephritis (MPGN)-like lesion, glomerular basement membrane thickness, and mild to moderate mesangial and endothelial proliferations might presented at the same time and in the same glomeruli. On immunofluorescence, MIg, usually monoclonal light chains, deposited along glomerular basement membranes and tubular basement membranes, while the intact MIg or monoclonal heavy chain deposited in the mesangial regions. Corresponding to the depositions on immunofluorescence, punctate “powdery” deposits along glomerular basement membranes and tubular basement membranes under electronic microscopy indicated the presence of MIDD. Microtubular substructures (diameters of 20–50 nm) exhibiting hollow cores arranged in parallel arrays in mesangial regions indicated the presence of ITG. Patients treated with bortezomib-based regimen seemed to have better outcomes. In conclusion, MIDD + ITG is a rare combination form of MIg-associated renal disease. Accurate diagnosis requires the comprehensive pathological investigations.

Normalization of the Serum Free Light Chain Ratio in Patients with Intact Immunoglobulin Multiple Myeloma Who Achieve Less Than Complete Response

BackgroundNormalization of the serum free light chain ratio (rFLC) following achievement of complete response (CR) denotes stringent CR in multiple myeloma (MM) and is associated with favorable prognosis. In contrast, the clinical significance of this phenomenon in patients not achieving CR has not been energetically studied. Only a few studies have reported better progression free survival (PFS) and overall survival (OS) in patients who achieved normalization of the rFLC at their best response than in those who did not. As monoclonal intact immunoglobulin (Ig) and monoclonal FLC are independent biomarkers for the neoplastic plasma cell burden in intact immunoglobulin MM (IIMM), it is not clear whether patients who failed to achieve CR but normalized the rFLC also had improved outcomes. Here, we retrospectively analyzed the prognostic significance of normalization of the rFLC in IIMM patients who did not achieve CR.Patients and methodsConsecutive patients with IIMM diagnosed and treated at the Kameda Medical Center from Jun 2007 to April 2017 were identified. IMWG response criteria were used to assess response. We excluded patients with negative immunofixation for monoclonal M-protein in the serum or urine at diagnosis and those who achieved CR. The FLC assay (FREELITE, The Binding Site, Birmingham, UK) was performed at least monthly as a part of routine clinical management. The following ranges were used for normal FLC values: κ FLC, 0.33-1.94 mg/dL; λ FLC, 0.57-2.63 mg/dL; and rFLC, 0.26-1.65.ResultsThere were 239 patients diagnosed with symptomatic MM, including 191 patients with IIMM and 48 patients with light chain MM (LCMM). Among 191 patients with IIMM, 78 achieved CR by chemotherapy; the remaining 113 patients were included in this analysis. No patient with LCMM achieving less than CR (n = 15) obtained normalization of rFLC during the whole treatment. The median observation period was 21.9 months (m) [range, 0.2-118.9 m] and the median age was 77.0 yr (range, 43.8-90.7 yr). Sixty-four of the 113 patients (56.6%) obtained a normal rFLC during the course of treatment. Patients who achieved a normal rFLC were significantly younger (median age, 75.3 vs 79.2 yr, P = 0.014), more frequently received autologous stem cell transplantation (15.6% vs 0%, P = 0.005), and had better response [very good partial response (VGPR) rate: 57.8% vs 22.4%, P < 0.001] than those who did not. No significant difference was seen between patients with and without normalization of the rFLC with regard to sex, heavy- or light-chain type, ISS category, revised ISS category, albumin, β2 microglobulin, or creatinine levels, hemoglobin concentration, lactate dehydrogenase abnormality, induction regimen, or administration of maintenance therapy. Patients with a normal rFLC had significantly longer PFS and OS than those without on univariate analysis (median PFS, 24.2 vs 6.6 m, P = 0.001; median OS, 59.6 vs 20.2 m, P < 0.001). Normalization of the rFLC retained its prognostic significance for both PFS and OS on multivariate analysis (both P < 0.001). We further examined the impact of achievement of a normal rFLC on PFS and OS in patients with a high or low baseline rFLC. Using receiver operating characteristic curve analysis, the optimal cutoff was 69.8; patients were divided into two groups based on the cutoff. As expected, patients with a higher baseline rFLC who achieved a normal rFLC had longer PFS and OS (median PFS, 29.7 vs 4.3 m, P < 0.001; median OS, 93.6 vs 15.8 m, P < 0.001), but those with a lower baseline rFLC did not (median PFS, 21.4 vs 7.1 m, P = 0.484; median OS, 53.9 vs 34.9 m, P = 0.120). In addition, patients with a lower baseline rFLC were more likely to achieve normal rFLC than those with a higher baseline rFLC, although the difference was not statistically significant (P = 0.074).ConclusionThis study demonstrated that achieving normalization of the rFLC during the course of treatment is robustly predictive for both PFS and OS in patients with IIMM who do not achieve CR. As monoclonal FLC is secreted independently of monoclonal intact Ig from neoplastic plasma cells, normalization of the rFLC also confers improved prognosis of patients with IIMM who had high levels of rFLC. These data indicate that serial measurement of FLC at all levels of response may be a valuable tool for accurate prognostication in IIMM even when achieving only VGPR or worse response to therapy. [Display omitted] DisclosuresKitadate:Fujimoto: Research Funding; Novartis: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama kagaku: Research Funding; Eisai: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Pfizer: Research Funding.

A Method to Identify Patients with Multiple Myeloma Who Are at Risk of Developing Concomitant Amyloidosis

INTRODUCTION : Multiple myeloma (MM) is a plasma cell dyscrasia that is characterized by the presence of both a clonal plasma cell population in the bone marrow and intact monoclonal immunoglobulin and/or free light chain (LC) proteins in the serum. It has been shown that MM may be an ominous precursor to light chain-associated (AL) amyloidosis, a devastating protein misfolding disorder characterized by the systemic deposition of extracellular amyloid fibrils composed of LC components. In the US, there are ~30,000 new cases of MM every year, and it is estimated that 10 - 15% of these patients will progress to develop amyloidosis.Light chain amyloid may deposit in any organ or tissue, but the heart, liver, spleen, kidneys and nerves are most commonly involved. The accumulation of amyloid in organs, especially the heart and kidneys, results in architectural damage leading to progressive organ dysfunction and death. It has been documented that concomitant MM with amyloidosis results in a considerably poorer prognosis than MM alone.Currently, there is no way of identifying those MM patients who will progress to develop LC-associated amyloidosis, but the ability to identify at-risk patients would be of clinical value because anti-amyloid immunotherapeutic agents are on the horizon. If such patients are identified prior to the onset of amyloid-associated clinical symptoms and organ damage, physicians could implement closer monitoring and begin a maintenance dose of amyloid-clearing antibody reagents.Early detection of amyloid is critical for enhancing patient survival; thus, we have investigated an approach to discern which MM patients have a greater likelihood of developing amyloidosis, based on the amyloid-related properties of the monoclonal light chain. We have previously shown that LC derived from MM and AL patients, when radiolabeled, can be recruited by synthetic amyloid-like fibrils. We observed that MM LC were recruited less effectively than AL LC, suggesting that there is disease specific reactivity of LC with amyloid fibrils (Martin et al . 2017 PLoS One, 12(3): e0174152.). We have since adapted this approach to a more facile and practical microplate assay measuring the ability of LC to compete with monomeric protein for fibril elongation. We have been able to discern patient populations with this technique.MATERIALS & METHODS : For these studies, 96-well microplates were coated with 0.83 μM synthetic amyloid-like fibrils (rVλ6Wil) and dried at 37°C overnight. After washing and a “blocking” step, a 100 μL solution of urinary LC protein-either AL amyloidosis or MM patient-derived-at increasing serial concentrations (0.05 - 5 µg/mL) mixed with biotinylated rVλ6Wil protein monomer (5 nM) was added to the wells. After 1 h incubation, the amount of fibril-recruited biontinyl-rVλ6Wil monomer was measured. Concentration gradients for MM and AL LC were measured and compared using a one-tailed Student's t-test.RESULTS : The data indicate that, in this competition assay, AL-associated urinary LC inhibited rVλ6Wil monomer recruitment more effectively than MM-derived LC. Our concentration-dependent gradient analysis discerned proteins from the two disease populations with statistical significance (p < 0.007). Notably, LC from a patient initially diagnosed with MM, who later developed amyloidosis, behaved as an AL LC in this assay, indicating the ability of this technique to predict the amyloidogenic propensity of LC.DISCUSSION : We have studied the capacity of intact, patient-derived LC to inhibit synthetic fibril elongation as a means of predicting the risk of amyloidosis. The assay was able to discriminate between LC proteins derived from MM and AL patients. This approach, in conjunction with other metrics, may be useful in discerning those patients with MM, or perhaps LC-associated monoclonal gammopathy of undetermined significance (LCMGUS), who are at risk of developing amyloidosis based on LC activity in our recruitment assay. We are currently adapting this technique for use with patient serum. [Display omitted] DisclosuresMartin:University of Tennessee Research Foundation: Patents & Royalties. Wall:University of Tennessee Research Foundation: Patents & Royalties.

Distinguishing therapeutic monoclonal antibodies from patient monoclonal proteins by electrospray time-of-flight mass spectrometry

Monoclonal gammopathies are a group of disorders characterised by proliferation of clonal plasma cells. Monoclonal intact immunoglobulin (Ig), free light chain and heavy chain proteins are routinely detected using electrophoresis and are important for diagnosis, monitoring and patient management. With the introduction of therapeutic monoclonal antibodies (tmAb), which are humanised monoclonal Ig proteins, for treatment of multiple myeloma and other disorders, protein electrophoresis is subject to interference from these therapeutics. They present as additional bands in the electrophoresis, complicating the interpretation of this investigation. The tmAbs cannot be distinguished from endogenous monoclonal proteins by current methods, which may lead to mis-diagnosis, and therefore incorrect patient management. We have developed an electrospray time-of-flight mass spectrometry (TOF MS) method which is able to detect tmAbs in patient serum and differentiate these from endogenous patient clones using accurate mass assignment of Ig light chains. The unique light chain mass of the tmAb can be identified and distinguished from patient monoclonal light chains. TOF MS also offers the advantage of being more sensitive than protein electrophoresis, enabling a lower limit of detection to be achieved.

Diagnosis of haematological diseases with monoclonal gammopathy

AbstractDespite the complexity of lymphoproliferative disorders, the presence of monoclonal immunoglobulins has been providing diagnostic guidance to the haematologist for more than a century. Clinicians may be alerted for the possible presence of serum monoclonal immunoglobulins produced in excess by increased erythrocyte sedimentation rate. Thereafter the diagnosis of monoclonal gammopathy could be proven by serum protein electrophoresis followed by immunofixation to properly identify the M‐protein. In a quarter of the cases, no intact monoclonal immunoglobulin could be detected in the sera, however, free light chains produced in excess may be identified using specialized immunoassays, so called free light chain analysis.Idiopathic monoclonal gammopathy of unknown significance (MGUS) may be present in 1% of the population above age 50 and up to 10% for individuals over 80 years of age. Due to the high frequency of MGUS, between 50‐65% of all monoclonal proteins detected fall into this category and most asymptomatic patients remain undetected for many years. MGUS patients have a 1% per year chance for transformation into malignant monoclonal gammopathies. Presently, it is accepted that most multiple myeloma and amyloidosis patients have their disease developed by transformation from preexisting MGUS. Additionally, some nonmalignant hematological disease also frequently exhibit monoclonal immunoglobulin synthesis, most notably Gaucher’s disease.From the field of neoplastic hematological diseases, smouldering and symptomatic multiple myeloma as well as Waldenström’s macroglobulinaemia almost always exhibit significant amounts of serum M‐protein and/or monoclonal free light chain excess. In case of solitary plasmocytoma, primary AL amyloidosis, cryoglobulinaemia and plasma cell leukemia, the correlation with gammopathy is less strict. Monoclonal gammopathy is less common in different types of non‐Hodgkin lymphoma. Within lymphomas lymphoplasmocytic lymphoma and marginal zone lymphoma more commonly secrete monoclonal immunoglobulins, while other B‐cell lymphomas have 14% cumulative chance for specific gammopathy. In the case of B‐cell chronic lymphocytic leukemia (the most common leukemia), intact monoclonal gammopathy has a 2% appearance, while abnormal free light chains may be detected in a third of the cases. Sporadically monoclonal gammopathy may be detected in chronic myelomonocytic leukemia, refractory anaemia, large granular cell leukemia and angioimmunoblastic lymphoma. Notably, in many of these diseases, the amount of secreted monoclonal immunoglobulin and/or free light chains correlate with the tumor burden and could be used for disease monitoring, especially in indolent hematological neoplasias.

Monoclonal immunoglobulin mediates complement activation in monoclonal gammopathy associated-C3 glomerulonephritis

BackgroundC3 glomerulonephritis (C3GN) is a rare disease caused by inherited or acquired complement alternative pathway (CAP) dysregulation, which could also be secondary to monoclonal gammopathy of undetermined significance (MGUS). Herein, we described a patient presenting with C3GN and monoclonal gammopathy, and the pathogenic association between the two diseases was further explored in vitro.Case presentationA 76-year-old Chinese man presented with low serum C3 level, haematuria and nephrotic syndrome, and experienced rapid worsening of renal function over a period of 10 months. His serum and urine immunofixation electrophoresis both revealed a monoclonal IgGλ. A bone marrow puncture showed plasma cell dyscrasias with the highest plasma cell count of 5.25%. Kidney biopsy showed the presence of C3 glomerulonephritis, with exclusive deposits of C3 visible on immunofluorescence, a membranoproliferative pattern on light microscopy and electron dense deposits in sub-epithelial, intramembranous, sub-endothelial and mesangial regions by electron microscopy. The patient was positive for C3 nephritic factor (C3NeF) activity and anti-CFH autoantibodies, and all became negative during disease remission. The anti-CFH autoantibodies purified from the patient’s plasma exchange fluids were proven to be a monoclonal IgGλ, and could inhibit CFH binding to C3b and accelerate the formation of C3 convertase indirectly by interfering with the formation-impeding activity of CFH. No deficiency of candidate genes, especially variants in CFH, was detected in our patient. Based on the pathological and laboratory findings, the diagnosis of monoclonal gammopathy of renal significance (MGRS)-associated C3GN was finally made.ConclusionsThis is the first demonstration that intact monoclonal immunoglobulin (IgGλ) could act as an anti-CFH antibody and lead to MGRS-associated C3GN by activating the CAP.

Heavy and Light Chain Monitoring in High Risk Smoldering Multiple Myeloma Patients Included in the GEM-CESAR Trial: Comparison with Conventional and Minimal Residual Disease IMWG Response Assessment

Heavy and Light Chain Monitoring in High Risk Smoldering Multiple Myeloma Patients Included in the GEM-CESAR Trial: Comparison with Conventional and Minimal Residual Disease IMWG Response Assessment

A Longitudinal Evaluation of Euroflow and Combined Quantitative Immunoprecipitation (QIP) and Free Light Chain (FLC) Mass Spectometry (MS) in Functional High Risk Multiple Myeloma

A Longitudinal Evaluation of Euroflow and Combined Quantitative Immunoprecipitation (QIP) and Free Light Chain (FLC) Mass Spectometry (MS) in Functional High Risk Multiple Myeloma

Research Advance in Light Chain Escape of Multiple Myeloma -Review

More than 80% of patients with MM is present as intact monoclonal immunoglobulin (Ig). Usually, the patients with intact immunoglobulin MM (IIMM) show parallel fluctuations of their intact Ig and FLCs or BJP. In the era of novel agents, including thalidomide, lenalidomide and bortezomib, the natural disease development and classic relapse patterns have been changed, the relapse was characterized by an increase in sFLC or BJP without a corresponding increase in paraprotein level, a phenomenon termed "light chain escape", indicates a worse outcome in patients with MM. This review focuses on the mechanism, clinical significance and early diagnosis of light chain escape.

Free Light Chain Escape in Multiple Myeloma : an Exceptional Phenomenon

Background : Free Light Chain (FLC) escape has been described for the first time in 1971 by Hobbs, who reported biochemical relapses of multiple myeloma (MM) with only Bence Jones proteinuria, in patients followed for an intact monoclonal immunoglobulin (Ig) MM. Indeed, FLC escape is defined as an increase of FLC without corresponding increase of the intact monoclonal Ig. In the era of novel-agent based therapy and autologous stem cell transplantation (ASCT), the patterns of disease progression may change, including a potential increased rate of FLC escape. Brioli and al (Blood 2014;123(22):3414-9) reported that 10% of the cases at relapse presented with FLC escape, and that FLC evaluation at relapse could represent an interesting marker of impact from intraclonal heterogeneity on myeloma outcome. We analysed the relapse patterns of patients treated according to the IFM 2009 clinical trial.Methods: Patients treated according to the IFM 2009 clinical trial (Lenalidomide bortezomib dexamethasone RVD + / - ASCT, Attal and al, ASH 2015) were analyzed. In patients presenting with an intact monoclonal Ig at diagnosis, serum and urine electrophoresis + FLC were compared in a central lab at the time of diagnosis and at the time of progression, in order to identify FLC escape.Results: 700 patients with symptomatic de novo MM were enrolled in the IFM DFCI 2009 clinical trial. Among them, 318 had a progressive disease, assessed with a centralized biochemistry analysis and 267 patients of them with an intact monoclonal Ig were included in this study. A vast majority (250 patients, 94%) showed an increase of the initial serum monoclonal component up to 5 g/L or up to 25% from the NADIR (IMWG criteria for progressive disease). 8 patients progressed with new bone lesions or plasmocytoma (3%) without any biological markers of progressive disease... Finally, 9 patients (3%) were identified as FLC escape as they did not exhibit an increased intact monoclonal Ig but they had an increased serum FLC and/or an increased Bence Jones proteinuria. Three of them had a serum and urinary measurable disease on diagnosis and they relapsed with both an increase of FLC and an increase of Bence Jones proteinuria. The six other patients presented at diagnosis only with an urinary measurable disease: 3 of them relapsed with an increase of FLC, without any Bence Jones proteinuria, the three others relapsed both on FLC and urines. Isotype of monoclonal component was IgG (6 patients), IgA (2) or IgD (1); light chain was Kappa for 6 patients and Lambda for the three others. Four patients were treated with RVD alone, and 5 patients with RVD + ASCT. The relapse occurred in a median of 2 years after diagnosis (5 months - 3.5 years).Conclusion: Based on a very large study of patients treated into a phase 3 clinical trial with centralized assessment of response and relapse, we are showing that FLC escape in a very rare phenomenom, observed in 3% of the cases. The low frequency of FLC escape does not lead to a systematic monitoring of intact Ig MM by FLC. DisclosuresAttal:amgen: Consultancy, Research Funding; celgene: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Avet-Loiseau:sanofi: Consultancy; janssen: Consultancy; amgen: Consultancy; celgene: Consultancy. Moreau:Takeda: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria.

Minimal tumour burden in haematological diseases: a step forward with quantitative assessment of Bence-Jones in nephelometry?

Minimal tumour burden in haematological diseases: a step forward with quantitative assessment of Bence-Jones in nephelometry?

Aggressive FLC Escape in a Patient with IgD Myeloma.

Background. Some patients who are stable or in remission from a myeloma secreting intact monoclonal immunoglobulin (+/− associated free light-chains (FLCs)) relapse with production of FLC. This FLC escape is one of the illustrations of the intraclonal heterogeneity of multiple myeloma. Results. We report FLC escape in a patient with IgD myeloma characterized by a severe outcome. We discuss parameters that negatively impacted prognosis in this patient, including bone lesions, biochemical parameters, and genomic abnormalities. Conclusion. This case illustrates the selective pressure exerted by therapeutic drugs and the variable sensitivity of subclones to these drugs; it also highlights the importance of FLC monitoring in treated MM patients.

Clinical course of light-chain smouldering multiple myeloma (idiopathic Bence Jones proteinuria): a retrospective cohort study

Bence Jones proteinuria is a disorder that is defined by the excretion of monoclonal light-chain protein. About 15-20% of patients with multiple myeloma secrete monoclonal light chains only, without expression of the normal immunoglobulin heavy chain, which constitutes light-chain multiple myeloma. The definition, prevalence, and progression of these premalignant phases of light-chain multiple myeloma have not been fully characterised. We aimed to identify a subset of patients with idiopathic Bence Jones proteinuria who had a high risk of progression to light-chain multiple myeloma analogous to that seen in patients with smouldering multiple myeloma. In this retrospective cohort study, we studied all patients seen at the Mayo Clinic (Rochester, MN, USA) within 30 days of diagnosis of idiopathic Bence Jones proteinuria between Jan 1, 1960, and June 30, 2004. Inclusion criteria were monoclonal light chain in the urine (≥0·2 g/24 h), absence of intact monoclonal immunoglobulin (M protein) in the serum, and no evidence of multiple myeloma, light-chain amyloidosis, or other related plasma-cell proliferative disorders. The primary endpoint was progression to symptomatic multiple myeloma or light-chain amyloidosis. We examined the cumulative probability of progression and the association of potential risk factors on progression rates to identify patients with a high risk of progression to multiple myeloma or light-chain amyloidosis. We identified 101 patients with idiopathic Bence Jones proteinuria. During 901 total person-years of follow-up, 27 (27%) patients developed multiple myeloma and seven (7%) developed light-chain amyloidosis. The major risk factors for progression were amount of urinary excretion of M protein per 24 h, proportion of bone marrow plasma cells, presence of a markedly abnormal free-light-chain ratio (<0·01 or >100), and reduction of all three uninvolved immunoglobulins. Based on the risk of progression, monoclonal light-chain excretion of 0·5 g/24 h or greater or at least 10% bone marrow plasma cells, or both, in the absence of end-organ damage was used to define light-chain smouldering multiple myeloma. The cumulative probability of progression to active multiple myeloma or light-chain amyloidosis in patients with light-chain smouldering multiple myeloma was 27·8% (95% CI 14·2-39·2) at 5 years, 44·6% (27·9-57·4) at 10 years, and 56·5% (36·3-70·2) at 15 years. Light-chain smouldering multiple myeloma as defined in this study is associated with a high risk of progression to symptomatic light-chain multiple myeloma, and this subset of patients needs careful observation and could benefit from clinical trials of early intervention. Jabbs Foundation (Birmingham, UK), US National Cancer Institute, and Henry J Predolin Foundation (Madison, WI, USA).

Comparison of the free and total light chain assays in serum and urine samples with immunofixation electrophoresis for detecting monoclonal proteins in patients with monoclonal gammopathy

ABSTRACT Monoclonal protein (M-protein) is produced by a malignant clone of plasma cells. Detected in serum and/or urine, this typically indicates multiple myeloma (MM) or other monoclonal gammopathy (MG). In a majority of MM cases, with the production of intact monoclonal immunoglobulin (Ig), malignant plasmocytes and/or B lymphocytes often produce excessive amounts of free light chains (FLCs). Excessive synthesis of FLCs lowers the ability of renal proximal tubules to re-absorb FLCs, which results in abnormally high levels of FLCs in the urine (Bence Jones protein, BJP). In laboratory practice, there are tests available for the quantitative measurement of only FLCs κ and λ or for total light chains (TLCs). These tests measure both free forms and bound in the (Ig) molecules forms as light chains that are evident in the serum and in urine. The purpose of this study was to evaluate the FLCs and TLCs approaches in screening serum and urine samples of patients with MM, doing so in comparison to the results of immunofixation (IFE) assessment. A second purpose was to assess the suitability of the collected material for obtaining the most reliable results. The results of serum FLCs (sFLCs) assays suggest that this approach is of the highest reliability and diagnostic usefulness in the detection of MG with excess production of FLCs, in comparison to other available tests. In our work, when κ band light chains were detected in serum IFE (sIFE), 91% patients had their FLCs concentrations beyond the reference range, whereas 89% patients had increased λ FLCs when λ band light chains were detected in sIFE. We also found abnormal sFLC κ/λ ratios in 86.4% and 88.9% of all subject patients who had κ or λ band light chains detected in their sIFE, respectively.

Evaluation Of Immunoglobulin Variations (Clonal Changes) In Symptomatic Multiple Myeloma (MM) Patients' Course

Changes in the production of monoclonal intact immunoglobulin (M-Ig) or of serum free light chains (sFLC) during symptomatic MM patients' relapse eventually reflect molecular myeloma cells changes and / or reveal clonal cell competition.We aimed to study changes in Ig production (CIg) in symptomatic MM patients and to evaluate related frequency and corresponding specific disease characteristics. Patients and Methods232 symptomatic MM patients with available follow-up sFLC measurements, were retrospectively studied. Light chain escape (LCE) was defined as sFLC increase with stable or falling M-Ig concentrations, M-Ig escape (MCE) as decreasing sFLC with increasing M-Ig, de-differentiation (DD) as clinical relapse with normal or decreased M-Ig and sFLC and Clonal Domination (CD) as normalization of formerly increased IgG, IgA or FLC in relapsing patients presenting increase of another component. Survival was calculated from diagnosis or from CIg date to last follow-up or death, survival curves were plotted by Kaplan-Meyer Method and assessed by the log-rank test. ResultsThere were 94 women and 128 men, median aged 66 years; 29%, 42%, 29% and 22%, 30%, 48% were in Durie-Salmon and ISS stages I, II, III and 1, 2, 3 respectively. MM type was IgG MM in 59%, IgA in 21%, light chain only in 17%, IgD in 2% and non-secretory in1%. Median survival of the whole cohort was 46,4 months. CIg was observed in 39 patients (17%), consisting in LCE in 15 patients (6%), MCE in 7 (3%), DD in 5 (2%) and CD in 10 (4%); two additional patients (1 IgD and 1 LC) transiently produced another monoclonal component that was IgG in both cases, while in stringent complete remission. In CD patients, the dominated clone was IgG in 9 out of 10 patients, while the dominating one was LC in 8 and IgA in 2. LCE and MCE were more frequent in IgG patients. The median number of treatment lines received prior CIg was 5 for LCE, 4 for MCE, 2 for DD and 1 for CD. LCE and MCE patients had all received novel agents and/or ASCT. The median time from CIg to last follow-up or death was 2,6 months (2,2-3) for LCE, 3,3 months (2,2-4,4) for MCE, 6,3 months (1,1-11,6) for DD and 31,1 months (23,6-38,6) for CD. Patients presenting LCE, MCE and DD had a considerably shorter survival after CIg compared to patients presenting CD (p=0,0002). However because CIg was usually a late event in the course of the disease the overall survival of CIg patients was 60,6 months.In conclusion, LCE, MCE and DD are late events in the course of MM, mainly observed in patients whose previous treatments included with novel drugs. They reflect a very aggressive disease behavior with shortened survival thereafter, probably due to the emergence of a new resistant clone. CD was mainly observed in patients secreting low IgG levels and FLCs, and possibly reflect IgG clone remission in biclonal patients, given that thereafter, the disease behaves as a usual multiple myeloma, secreting however the other clone. Disclosures:No relevant conflicts of interest to declare.

An Intact Immunoglobulin IgAλ Multiple Myeloma Patient Exhibiting Light Chain Escape

AbstractAbstract 5008A 48 year old man was referred to the Institute of Hematology and Transfusion Medicine, Warsaw, Poland in April 2008 with anemia (Hemoglobin; 10. 4 g/dl) and mild renal impairment (eGFR; 75. 4 mL/min/1. 73m2). An initial diagnostic monoclonal protein screen (serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE) and serum free light chain (FLC) analysis) revealed an IgAλ monoclonal protein (0. 8g/dL) with monoclonal serum FLC and an abnormal serum FLC κ/λ ratio (0. 0001; RI, 0. 26–1. 65). A bone marrow biopsy at that time confirmed 60% involvement of monoclonal λ - restricted plasma cells; a bone survey did not detect any osteolysis. The patient was diagnosed with multiple myeloma (MM) (ISS stage I, Durie and Salmon stage IA) and was initially treated with 6 cycles of vincristin, doxorubicin and dexamethasone (VAD). The patient responded well to the induction treatment and subsequently underwent a successful autologous stem cell transplantation (ASCT).The patient was monitored for 3 years subsequent to the ASCT with both serum and urine electrophoresis, serum FLC analysis (Freelite) and heavy chain/light chain (HLC) immunoassays (Hevylite). Sixteen months following the ASCT the dFLC (involved λ FLC– uninvolved κ FLC) concentration began to increase, the FLC κ/λ ratio became abnormal with a trace of λ Bence Jones protein (BJP) detected by urine IFE. However, both SPE and IFE were normal and the HLC ratio (IgAλ/IgAκ) was within the normal range. During the next 9 months the dFLC continued to increase and a λ BJP could now be clearly detected on the urine IFE. 27 months following the ASCT the patient sustained a pathological fracture of the tibiae and was referred to our centre 4 months later. At this point, the dFLC concentration was highly elevated (3168 mg/L) with a λ BJP detectable by both serum and urine IFE. However, there was no detectable monoclonal intact immunoglobulin by serum IFE or HLC analysis, indicating disease relapse by a separate FLC clone; referred to as light chain escape (LCE). A bone marrow biopsy revealed 15% involvement of λ restricted plasma cells; this time a bone survey identified osteolysis. The patient was diagnosed with progression of multiple myeloma and received 6 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD regimen). He responded well to treatment and 3 years following the ASCT achieved a CR as indicated by a normalized κ/λ FLC ratio, negative immunofixation with 1–3% bone marrow plasma cells. The patient is now well and able to continue with normal life.In this case study the increase in the dFLC levels was the first indication of disease progression and highlights the importance of monitoring intact immunoglobulin MM patients with serum FLC immunoassays for early detection of LCE. Disclosures:Endean:The Binding Site Group Ltd: Employment. Harding:Binding Site: Employment.

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...

Changes in serum‐free light chain rather than intact monoclonal immunoglobulin levels predicts outcome following therapy in primary amyloidosis

Current response criteria for light-chain amyloidosis (AL) relegate FLC response to a subsidiary status relative to serum M-protein response. Given that light chains form the substrate for amyloid fibril formation, we hypothesized that changes in FLC might better predict outcome compared to changes in intact immunoglobulin levels. Two patient cohorts were studied, 347 patients who underwent an autologous stem-cell transplant (SCT) and 96 patients treated with melphalan/dexamethasone. We identified the lowest value following therapy for intact serum M-protein and the difference between involved and uninvolved FLC (FLC-diff). We first examined the relative contribution of M-protein and FLC-diff on the overall survival (OS), and found that FLC reduction, rather than M-protein reduction, significantly impacted OS. The median OS was not reached among those with a 50% decrease in FLC-diff compared to 20 months for the remainder. On regression analysis, a 90% reduction in FLC-diff following SCT best predicted being alive at 3 or 5 years. The median OS among those with a 90% decrease was not reached compared to 37.4 months for the rest P < 0.001. The current study supports the notion that FLC response is a more useful measure of hematological response than M-protein response. It also highlights the importance of achieving at least a 90% reduction in the FLC-diff to improve the outcome of patients with light-chain AL.

Investigations of Chemical Modifications of Amino-Terminated Organic Films on Silicon Substrates and Controlled Protein Immobilization

Fourier transform infrared spectroscopy by grazing-angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV-visible spectroscopy, and fluorescence microscopy were employed to investigate chemical modifications of amino-terminated organic thin films on silicon substrates, protein immobilization, and the biological activity and hydrolytic stability of immobilized proteins. Amino-terminated organic films were prepared on silicon wafers by self-assembling 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene. Surface amino groups were derivatized into three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. UV-visible absorption measurements and fluorescence microscopy revealed that more than 40% of surface amino groups were chemically modified. Protein immobilization was carried out on modified APTES films containing these linkers via coupling with primary amines (-NH(2)) in intact monoclonal rabbit immunoglobulin G (IgG), the aldehyde (-CHO) of an oxidized carbohydrate residue in IgG, or the sulfhydryl (-SH) of fragmented half-IgG, respectively. FTIR spectra contain vibrational signatures of these functional groups present in modified APTES films and immobilized IgGs. Changes in the APTES film thickness after chemical modifications and protein immobilization were also observed by ellipsometric measurements. The biological activity and long-term hydrolytic stability of immobilized IgGs on modified APTES films were estimated by fluorescence measurements of an adsorbed antigen, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (FITC-Ab). Our results indicate that the FITC-Ab binding capacity of half-IgG immobilized via maleimide groups is greater than that of the oxidized IgG and the intact IgG immobilized via hydrazide and NHS ester groups, respectively. In addition, IgGs immobilized using all coupling chemistries were hydrolytically stable in phosphate-buffered saline (PBS).