Comprehensive Assessment of M-Proteins Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.

BACKGROUND: Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests. METHODS: We developed a MALDI mass spectrometry-based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays. RESULTS: MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to <1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were <20% across all samples having M-protein concentrations >0.045 g/dL, with the ability to detect M-proteins <0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite(™) assay. CONCLUSIONS: MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored.

Screening Method for M-Proteins in Serum Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.

BACKGROUND: Current recommendations for screening for monoclonal gammopathies include serum protein electrophoresis (PEL), imunofixation electrophoresis (IFE), and free light chain (FLC) ratios to identify or rule out an M-protein. The aim of this study was to examine the feasibility of an assay based on immunoenrichment and MALDI-TOF-MS (MASS-SCREEN) to qualitatively screen for M-proteins. METHODS: Serum from 556 patients previously screened for M-proteins by PEL and IFE were immunopurified using a κ/λ-specific nanobody bead mixture. Following purification, light chains (LC) were released from their heavy chains by reduction. MALDI-TOF analysis was performed and the mass-to-charge LC distributions were visually examined for the presence of an M-protein by both unblinded and blinded analysts. RESULTS: In unblinded analysis, MASS-SCREEN detected 100% of the PEL-positive samples with an analytical sensitivity and specificity of 96% and 81% using IFE positivity as the standard. In a blinded analysis using 6 different laboratory personnel, consensus was reached in 92% of the samples. Overall analytical sensitivity and specificity were reduced to 92% and 80%, respectively. FLC ratios were found to be abnormal in 28% of MASS-SCREEN-negative samples, suggesting FLC measurements need to be considered in screening. CONCLUSIONS: MASS-SCREEN could replace PEL in a panel that would include FLC measurements. Further studies and method development should be performed to validate the clinical sensitivity and specificity and to determine if this panel will suffice as a general screen for monoclonal proteins.

Laboratory testing requirements for diagnosis and follow-up of multiple myeloma and related plasma cell dyscrasias.

Monoclonal immunoglobulins are markers of plasma cell proliferative diseases and have been described as the first (and perhaps best) serological tumor marker. The unique structure of each monoclonal protein makes them highly specific for each plasma cell clone. The difficulties of using monoclonal proteins for diagnosing and monitoring multiple myeloma, however, stem from the diverse disease presentations and broad range of serum protein concentrations and molecular weights. Because of these challenges, no single test can confidently diagnose or monitor all patients. Panels of tests have been recommended for sensitivity and efficiency. In this review we discuss the various disease presentations and the use of various tests such as protein electrophoresis and immunofixation electrophoresis as well as immunoglobulin quantitation, free light chain quantitation, and heavy-light chain quantitation by immuno-nephelometry. The choice of tests for inclusion in diagnostic and monitoring panels may need to be tailored to each patient, and examples are provided. The panel currently recommended for diagnostic screening is serum protein electrophoresis, immunofixation electrophoresis, and free light chain quantitation.

Monitoring free light chains in serum using mass spectrometry.

BACKGROUND: Serum immunoglobulin free light chains (FLC) are secreted into circulation by plasma cells as a by-product of immunoglobulin production. In a healthy individual the population of FLC is polyclonal as no single cell is secreting more FLC than the total immunoglobulin secreting cell population. In a person with a plasma cell dyscrasia, such as multiple myeloma (MM) or light chain amyloidosis (AL), a clonal population of plasma cells secretes a monoclonal light chain at a concentration above the normal polyclonal background. METHODS: We recently showed that monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) can be used to identify and quantify a monoclonal light chain (LC) in serum and urine above the polyclonal background. This was accomplished by reducing immunoglobulin disulfide bonds releasing the LC to be analyzed by microLC-ESI-Q-TOF mass spectrometry. Here we demonstrate that the methodology can also be applied to the detection and quantification of FLC by analyzing a non-reduced sample. RESULTS: Proof of concept experiments were performed using purified FLC spiked into normal serum to assess linearity and precision. In addition, a cohort of 27 patients with AL was analyzed and miRAMM was able to detect a monoclonal FLC in 23 of the 27 patients that had abnormal FLC values by immunonephelometry. CONCLUSIONS: The high resolution and high mass measurement accuracy provided by the mass spectrometry based methodology eliminates the need for κ/λ ratios as the method can quantitatively monitor the abundance of the κ and λ polyclonal background at the same time it measures the monoclonal FLC.

Monoclonal antibody therapeutics as potential interferences on protein electrophoresis and immunofixation.

BACKGROUND: The use of therapeutic recombinant monoclonal antibodies (mAbs) has triggered concerns of mis-diagnosis of a plasma cell dyscrasia in treated patients. The purpose of this study is to determine if infliximab (INF), adalimumab (ADA), eculizumab (ECU), vedolizumab (VEDO), and rituximab (RITU) are detected as monoclonal proteins by serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). METHODS: Pooled normal sera were spiked with various concentrations (ranging from trough to peak) of INF, ADA, ECU, VEDO and RITU. The peak concentration for VEDO and RITU was also added to samples with known monoclonal gammopathies. All samples were analyzed by SPEP (Helena Laboratories) and IFE (Sebia); sera containing peak concentrations of mAbs were reflexed to electrospray-time-of-flight mass spectrometry (AbSciex Triple TOF 5600) for the intact light chain monoclonal immunoglobulin rapid accurate mass measurement (miRAMM). RESULTS: For all mAbs tested, no quantifiable M-spikes were observed by SPEP at any concentration analyzed. Small γ fraction abnormalities were noted on SPEP for VEDO at 300 µg/mL and RITU at 400 µg/mL, with identification of small IgG κ proteins on IFE. Using miRAMM for peak samples, therapeutic mAbs light chain accurate masses were identified above the polyclonal background and were distinct from endogenous monoclonal gammopathies. CONCLUSIONS: MAbs should not be easily confounded with plasma cell dyscrasias in patients undergoing therapy except when a SPEP and IFE are performed within a couple of days from infusion (peak). In ambiguous cases the use of the miRAMM technology could precisely identify the therapeutic mAb distinct from any endogenous monoclonal protein.

Monitoring IgA multiple myeloma: immunoglobulin heavy/light chain assays.

BACKGROUND: The use of electrophoresis to monitor monoclonal immunoglobulins migrating in the ß fraction may be difficult because of their comigration with transferrin and complement proteins. METHODS: Immunoassays specific for IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ heavy/light chain (HLC) were validated for use in the clinical laboratory. We assessed sample stability, inter- and intraassay variability, linearity, accuracy, and reference intervals for all 6 assays. We tested accuracy by verifying that the sum of the concentrations for the HLC-pairs accounted for the total immunoglobulins in each of 129 healthy sera, and that the HLC-pair ratios (rHLCs) were outside the reference interval in 97% of 518 diagnostic multiple myeloma (MM) samples. RESULTS: We assessed diagnostic samples and posttreatment sera in 32 IgG and 30 IgA patients for HLC concentrations, rHLC, and total immunoglobulins and compared these nephelometry results with serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). In sample sets from patients with IgG MM, the sensitivity of SPEP was almost the same as for rHLC, and no additional advantage was conferred by running HLC assays. In pre- and posttreatment samples from patients with IgA MM, the SPEP, rHLC, and IFE identified clonality in 28%, 56%, and 61%, respectively. In addition, when M-spikes were quantifiable, the concentration of the involved HLC was linearly related to that of the SPEP M-spike, with a slope near 1. CONCLUSIONS: The use of IgA HLC assays for monitoring ß-migrating IgA monoclonal proteins can substitute for the combination of SPEP, IFE, and total IgA quantification.

Monitoring M-proteins in patients with multiple myeloma using heavy-chain variable region clonotypic peptides and LC-MS/MS.

Multiple myeloma is a disease characterized by a clonal expansion of plasma cells that secrete a monoclonal immunoglobulin also referred to as an M-protein. In the clinical laboratory, protein electrophoresis (PEL), immunofixation electrophoresis (IFE), and free light chain nephelometry (FLC) are used to detect, monitor, and quantify an M-protein. Here, we present an alternative method based on monitoring a clonotypic (i.e., clone-specific) peptide from the M-protein heavy chain variable region using LC-MS/MS. Tryptic digests were performed on IgG purified serum from 10 patients with a known IgG M-protein. Digests were analyzed by shotgun LC-MS/MS, and the results were searched against a protein database with the patient specific, heavy chain variable region gene sequence added to the database. In all 10 cases, the protein database search matched multiple clonotypic peptides from each patient's heavy chain variable region. The clonotypic peptides were then used to quantitate the amount of M-protein in patient serum samples using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The response for the clonotypic peptide observed by SRM correlated with the M-protein observed by PEL. In addition, the clonotypic peptide was clearly observed by SRM in samples that were negative by IFE and FLC. Monitoring clonotypic peptides using SRM has the capacity to redefine clinical residual disease because of its superior sensitivity and specificity compared with current analytical methods.

Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy.

A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.

Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography--tandem mass spectrometry.

BACKGROUND: Measurement of IgG subclasses is a useful tool for investigation of humoral immune deficiency in the presence of total IgG within reference intervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We describe an LC-MS/MS method that can multiplex all 4 subclasses along with total IgG by use of either IgG subclass-specific peptide stable isotope-labeled internal standards or a surrogate digest standard for quantification and does not rely on antigen/antibody reactions. METHODS: We combined serum with labeled internal peptide standards and intact purified horse IgG. Samples were denatured, reduced, alkylated, and digested. We analyzed the digested serum by LC-MS/MS for IgG subclasses 1-4 and total IgG. RESULTS: We assayed 112 patient sera by LC-MS/MS and immunonephelometry. The mean of the slopes and R(2) values for IgG1, IgG2, IgG3, IgG4, and IgG were 1.18 and 0.93, respectively. Interassay imprecision for the LC-MS/MS method was <15% for total IgG and subclasses and was slightly improved by use of a calibrator peptide from an exogenous horse IgG. Summed total IgG correlated with the measured total IgG within 10%. Reference intervals and analytical measuring range were all similar to our previous validation data for the immunonephelometry assays. CONCLUSIONS: Total IgG and IgG subclasses 1, 2, 3, and 4 can be quantified by LC-MS/MS with performance comparable to nephelometry.

Elevated serum monoclonal and polyclonal free light chains and interferon inducible protein-10 predicts inferior prognosis in untreated diffuse large B-cell lymphoma.

The detection of serum free light (FLC) is useful in the diagnosis of several hematological diseases. The role and biological relevance of monoclonal or polyclonal FLC elevations in predicting long-term outcome in diffuse large B-cell lymphoma (DLBCL) is unknown. We determined the relationship of the type of FLC elevations to outcome, tumor genotype, and pattern of serum cytokine elevations in 276 patients with untreated DLBCL. Elevated FLC was an adverse prognostic factor through 6 years of follow-up (monoclonal, Event free survival (EFS) HR = 3.56, 95% CI: 1.88-6.76, P <0.0001; polyclonal, EFS HR = 2.56, 95% CI: 1.50-4.38, P = 0.0006). About 73% of DLBCL tumors with monoclonal FLC elevations were activated B-cell type (ABC) versus 33% from patients with normal FLC. Only ABC-DLBCL lines secreted kappa FLC in vitro and this secretion could be inhibited by the NF-κB inhibitor bortezomib. Patients with monoclonal FLC had significantly (all P <0.001) increased serum levels of IL-12, sIL-2Rα, IL-1R, and IP-10. Patients with polyclonal elevations of FLC had higher levels of IL-6 (P = 0.033), IL-8 (P =0.025), sIL2Rα (P = 0.011), and IL-1R1 (P = 0.041). The combination of elevated FLC and a CXC superfamily chemokine IP-10 predicted a particularly inferior outcome characterized by late relapse. These increased abnormal FLC and cytokines are potentially useful biomarkers for prognosis and selecting agents for untreated DLBCL.

Elevated monoclonal and polyclonal serum immunoglobulin free light chain as prognostic factors in B- and T-cell non-Hodgkin lymphoma.

The serum immunoglobulin free light chain (FLC) assay quantitates free kappa (κ) and lambda (λ) light chains. FLC elevations in patients with diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and chronic lymphocytic leukemia (CLL) are associated with an inferior survival. These increases in FLC can be monoclonal (as in myeloma) or polyclonal. The goal was to estimate the frequency of these elevations within distinct types of B-cell and T-cell non-Hodgkin lymphoma (NHL) and whether the FLC measurements are associated with event-free survival (EFS). We studied serum for FLC abnormalities using normal laboratory reference ranges to define an elevated κ or λ FLC. Elevations were further classified as polyclonal or monoclonal. Four hundred ninety-two patients were studied: 453 B-cell and 34 T-cell NHL patients. Twenty-nine % (142/453) of patients had an elevated FLC of which 10% were monoclonal elevations. Within B-cell NHL, FLC abnormalities were most common in lymphoplasmacytic (79%), mantle cell (68%), and lymphomas of mucosa associated lymphoid tissue (31%); they were least common in follicular (15%). The hazard ratio (HR) for EFS in all patients was 1.41 (95% CI; 1.11-1.81); in all B-cell NHL the HR was 1.44 (95% CI 1.11-1.96); in all T-cell NHL the HR was 1.17 (95% CI 0.55-2.49). FLC abnormalities predicted an inferior OS (HR = 2.75, 95% CI: 1.93-3.90, P < 0.0001). The serum FLC assay is useful for prognosis in both B-cell and T-cell types of NHL. In B-cell NHL further discrimination between a monoclonal and polyclonal elevation may be helpful and should be analyzed in prospective clinical trials.

Monoclonal and polyclonal serum free light chains and clinical outcome in chronic lymphocytic leukemia.

Free light chains (FLCs) are the most commonly detected paraproteins in chronic lymphocytic leukemia (CLL). We examined the types of FLC abnormalities and prognostic utility of the FLC assay compared with standard prognostic biomarkers in a prospective cohort of 339 patients with newly diagnosed CLL. Three types of FLC abnormalities were identified: monoclonal elevated FLC (elevated κ and/or λ with abnormal FLC ratio), polyclonal elevated FLC (elevated κ and/or λ with normal FLC ratio), and ratio-only FLC abnormality (normal range κ and λ with abnormal FLC ratio). One hundred sixty-five patients (49%) had a FLC abnormality with approximately equal distribution among monoclonal elevation, polyclonal elevation, and ratio-only abnormality. All FLC abnormalities were associated with poor time to first treatment: monoclonal FLC (hazard ratio [HR], 4.99; 95% confidence interval [CI], 2.94-8.48), polyclonal FLC (HR, 2.40; 95% CI, 1.24-4.64), ratio-only FLC (HR, 2.57; 95% CI, 1.40-4.69). Monoclonal FLC and polyclonal FLC were associated with poor overall survival compared with patients with normal FLC. Results remained significant after adjusting for Rai stage. The FLC assay is a simple, widely available clinical test with similar prognostic utility as routinely used prognostic biomarkers for CLL. Among persons with FLC abnormalities, the type of abnormality affects prognostic significance.

Increased prevalence of light chain monoclonal gammopathy of undetermined significance (LC-MGUS) in first-degree relatives of individuals with multiple myeloma.

Previously, we reported increased risk of heavy-chain (HC) monoclonal gammopathy of undetermined significance (MGUS) among first-degree (1°) relatives of multiple myeloma (MM) or HC-MGUS probands. This study investigated whether there was comparable risk for light-chain (LC) MGUS among 911 relatives of the same HC-MGUS/MM probands versus a reference population of 21 463. Seventeen 1° relatives had LC-MGUS (adjusted prevalence = 1·7%, 95% CI = 0·9­2·6%). There was increased risk of LC-MGUS in relatives of MM probands (RR = 3·4, 95% CI = 2·0­5·5). We saw no increased risk in relatives of HC-MGUS probands. We conclude that the prevalence of LC-MGUS is significantly higher among 1° relatives of MM probands compared to the reference population.

Obesity is associated with an increased risk of monoclonal gammopathy of undetermined significance among black and white women.

Obesity and black race have been associated with excess risk of multiple myeloma. The association of obesity with monoclonal gammopathy of undetermined significance (MGUS) is unknown. Further, it is not known whether the increased risk of multiple myeloma and MGUS in blacks is related to socioeconomic status, genetic susceptibility, or both. We screened 1000 black and 996 white women (range, 40-79 years) of similar socioeconomic status for MGUS; the aim of the study was to assess MGUS risk in relation to obesity and race. A total of 39 (3.9%) blacks and 21 (2.1%) whites had MGUS. On multivariate analysis, obesity (odds ratio [OR] = 1.8; P = .04), black race (OR = 1.8; P = .04), and increasing age (> 55 vs < 43 years; OR = 2.5; P = .03) were independently associated with an excess risk of MGUS. Our findings support the hypothesis that obesity is etiologically linked to myelomagenesis. The 2-fold excess of MGUS among blacks compared with whites of similar socioeconomic status supports a role for susceptibility genes in MGUS.

Impact of optimal follow-up of monoclonal gammopathy of undetermined significance on early diagnosis and prevention of myeloma-related complications.

Monoclonal gammopathy of undetermined significance (MGUS) is associated with a long-term risk of progression to multiple myeloma (MM) or related malignancy. To prevent serious myeloma-related complications, lifelong annual follow-up has been recommended, but its value is unknown. We reviewed all patients from southeastern Minnesota seen at Mayo Clinic between 1973 and 2004 with MGUS who subsequently progressed to MM. Of 116 patients, 69% had optimal follow-up of MGUS. Among these, abnormalities on serial follow-up laboratory testing led to the diagnosis of MM in 16%, whereas MM was diagnosed only after serious MM-related complications in 45%. In the remaining, workup of less serious symptoms (25%), incidental finding during workup of unrelated medical conditions (11%), and unknown (3%) were the mechanisms leading to MM diagnosis. High-risk MGUS patients (≥ 1.5 g/dL and/or non-IgG MGUS) were more likely to be optimally followed (81% vs 64%), and be diagnosed with MM secondary to serial follow-up testing (21% vs 7%). This retrospective study suggests that routine annual follow-up of MGUS may not be required in low-risk MGUS. Future studies are needed to replicate and expand our findings and to determine the optimal frequency of monitoring in higher-risk MGUS patients.

Serum immunoglobulin free light-chain measurement in primary amyloidosis: prognostic value and correlations with clinical features.

Immunoglobulin free light chains (FLCs) are the precursors of amyloid fibrils in primary amyloidosis (AL). We studied the relationship between FLC levels and clinical features in 730 patients with newly diagnosed AL. The plasma cell clone was λ in 72% patients, and κ in 28% patients. κ-AL had more GI tract and liver involvement, where as renal involvement was more with λ-AL. While the overall survival (OS) was similar for κ and λ-AL, the median OS for those without an identifiable serum heavy chain was significantly shorter (12.6 vs 29.9 months; P = .02). The OS was shorter among those with a higher dFLC (involved FLC-uninvolved FLC; κ > 29.4 mg/dL or λ > 18.2 mg/dL using median for cutoff); 10.9 vs 37.1 months; P < .001. In multivariate analysis, dFLC was independent of other prognostic factors. The type of light chain impacts the spectrum of organ involvement and the FLC burden correlates with survival in AL.

Prevalence and risk of progression of light-chain monoclonal gammopathy of undetermined significance: a retrospective population-based cohort study.

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) is defined by expression of heavy-chain immunoglobulin (IgH) and is the precursor lesion for 80% of cases of multiple myeloma. The remaining 20% are characterised by absence of IgH expression; we aimed to assess prevalence of a corresponding precursor entity, light-chain MGUS. METHODS: We used a population-based cohort, previously assembled to estimate MGUS prevalence, of 21,463 residents of Olmsted County, MN, USA, aged 50 years and older. We did a serum free light-chain assay on all samples with sufficient serum remaining, and immunofixation electrophoresis was done for all samples with an abnormal free light-chain ratio or abnormal protein electrophoresis results from the original study. Light-chain MGUS was defined as an abnormal free light-chain ratio with no IgH expression, plus increased concentration of the involved light chain. We calculated age-specific and sex-specific prevalence and rates of progression to lymphoproliferative disorders for light-chain and conventional MGUS and assessed incidence of renal disorders in patients with light-chain MGUS. FINDINGS: 610 (3.3%) of 18,357 people tested had an abnormal free light-chain ratio, of whom 213 had IgH expression that was diagnostic of conventional MGUS. 146 of the remaining 397 individuals had an increase of at least one free light chain and met criteria for light-chain MGUS. Prevalence of light-chain MGUS was 0.8% (95% CI 0.7-0.9), contributing to an overall MGUS prevalence of 4.2% (3.9-4.5). Risk of progression to multiple myeloma in patients with light-chain MGUS was 0.3% (0.1-0.8) per 100 person-years. 30 (23%) of 129 patients with light-chain MGUS were diagnosed with renal disease. INTERPRETATION: We define a clinical entity representing the light-chain equivalent of conventional MGUS and posing a risk of progression to light-chain multiple myeloma and related disorders. FUNDING: US National Cancer Institute.