Application of Capillary Electrophoresis in Monoclonal Gammopathies and the Cutoff Value of Monoclonal Protein in Differential Diagnosis of Multiple Myeloma and Other Monoclonal Gammopathies.

OBJECTIVE: Monoclonal protein (MP) exists in various diseases, and capillary electrophoresis (CE) has been widely used to detect MP. However, there is not much research on the application value of MP in the differential diagnosis of monoclonal gammopathies. This study aimed to explore MP's cutoff value for the differential diagnosis of multiple myeloma (MM) and other monoclonal gammopathies (MGs). METHODS: A retrospective analysis of 8167 cases was conducted. Serum MP was detected by CE, and the patients' clinical information was collected from the clinical database of our hospital. RESULTS: 985 cases had MP with high peaks, and 91.1% were diagnosed with malignant diseases. The MP showed small peaks in 471 cases, and only 24.4% were diagnosed with malignant diseases. Among the MPs, the IgG-κ type was the most common type, followed by the IgG-λ, IgA-κ, IgA-λ, free λ light chain, IgM-κ, free κ light chain, double clone, and IgM-λ types. Differences in the MP of the IgG, IgA, IgM, and FLC types between the MM group and MGUS group were statistically different (P<0.01). The MP of the IgG, IgA, and FLC types showed clear specificity and sensitivity in discriminating MM from other monoclonal gammopathies in ROC curve analysis. Serum IgM had statistical significance in the differential diagnosis between WM and other MGs (P<0.01). However, there was no statistical significance in the differential diagnosis between MM and other MGs (P=0.140). The cutoff values of the MP of the IgG, IgA, and FLC types were >18.67g/L, >13.86g/L, and >10.15g/L, respectively, for the differential diagnosis of MM and other MGs. The cutoff value of the MP of IgM for the WM diagnosis was >37.75 g/L. CONCLUSION: CE has good clinical application value in the diagnosis of monoclonal gammopathies, and MP can be used in the differential diagnosis of MM and other monoclonal gammopathies.

Reporting Apparent Biclonal Immunoglobulin-A Monoclonal Proteins with Identical Light Chains.

It is generally not well appreciated that Immunoglobulin A (IgA) can present as apparent biclonal monoclonal protein (M-protein) comprising monomers and polymers, despite the fact that Kyle pointed this out in 1999. In this clinical communication, we report on 3 patients with Ig-A multiple myeloma (MM) who displayed this phenomenon. Of these 3 patients, 2 had identical kappa light chains suggesting biclonality, while the other had 2 lambda light chains. Because the Ig-A M-proteins are clustered in the beta-area, accurate densitometric quantification is made difficult. Hence, we suggest assaying Ig-A levels and free light chains in concert with the M-protein levels to monitor these patients on therapy. In conclusion, when encountering biclonal Ig-A M-proteins with identical light chains, the laboratorian should add the 2 values and present one composite number to the clinician.

Distinguishing Drug from Disease by Use of the Hydrashift 2/4 Daratumumab Assay.

BACKGROUND: Daratumumab, a monoclonal antibody used to treat relapsed or refractory multiple myeloma, can interfere with protein electrophoresis and immunofixation assays. False-positive immunofixation results due to daratumumab can cause uncertainty regarding the status of a patient's disease and lead to potential misclassification of their response to therapy. The Hydrashift 2/4 Daratumumab assay (Sebia) was recently cleared by the Food and Drug Administration for resolving daratumumab interference on immunofixation. Here, we evaluate the performance of the Hydrashift assay in multiple myeloma patients receiving treatment with daratumumab-based regimens. METHODS: Waste serum samples from multiple myeloma patients (n = 40) receiving daratumumab were analyzed by standard immunofixation and the Hydrashift assay. Results from these tests were compared and were evaluated along with pretreatment serum protein electrophoresis and immunofixation results, if available. RESULTS: The Hydrashift assay shifted the migration of daratumumab in patient samples. In 27 cases, the patient's M protein was distinguishable from daratumumab by standard immunofixation. In these cases, the Hydrashift assay confirmed that the IgGκ band was daratumumab and helped identify the presence of treatment-related oligoclonal bands. There were 11 instances in which the patient's IgGκ M protein comigrated with daratumumab. In all 11 cases, the Hydrashift assay confirmed the presence of residual M protein. Finally, in 2 patients whose pretreatment immunofixation results were not available, the Hydrashift assay confirmed that the IgGκ band visible on immunofixation was due to daratumumab alone. CONCLUSIONS: The Hydrashift 2/4 Daratumumab assay is a useful tool to clarify the source of an IgGκ band on immunofixation and allow a patient's M protein to be viewed without interference.

Development of a Targeted Mass-Spectrometry Serum Assay To Quantify M-Protein in the Presence of Therapeutic Monoclonal Antibodies.

M-protein diagnostics can be compromised for patients receiving therapeutic monoclonal antibodies as treatment in multiple myeloma. Conventional techniques are often not able to distinguish between M-proteins and therapeutic monoclonal antibodies administered to the patient. This may prevent correct response assessment and can lead to overtreatment. We have developed a serum-based targeted mass-spectrometry assay to detect M-proteins, even in the presence of three therapeutic monoclonal antibodies (daratumumab, ipilimumab, and nivolumab). This assay can target proteotypic M-protein peptides as well as unique peptides derived from therapeutic monoclonal antibodies. We address the sensitivity in M-protein diagnostics and show that our mass-spectrometry assay is more than two orders of magnitude more sensitive than conventional M-protein diagnostics. The use of stable isotope-labeled peptides allows absolute quantification of the M-protein and increases the potential of assay standardization across multiple laboratories. Finally, we discuss the position of mass-spectrometry assays in monitoring minimal residual disease in multiple myeloma, which is currently dominated by molecular techniques based on plasma cell assessment that requires invasive bone marrow aspirations or biopsies.

Heavy+light chain monitoring correlates with clinical outcome in multiple myeloma patients.

Novel anti-myeloma agents have improved patient response rates, which are historically based on reductions of the M-protein. These methods can be inaccurate for quantifying M-proteins at low concentrations. We compared the consistency and clinical impact of response assignment by electrophoretic and heavy+light chain (HLC) immunoassays post-consolidation in 463 newly diagnosed patients. The two methods gave similar assignments in patients with partial (PR; 79% agreement) or complete response (⩾CR; 92%). However, in patients achieving very good PR (VGPR) there was poor concordance between methods (45%). Median progression-free survival (PFS) for standard VGPR patients was 34.5 months; HLC responses stratified these patients further into PR, VGPR and ⩾CR, with median PFS of 21.3, 28.9 months and not reached, respectively; P<0.001. At this time, abnormal HLC ratios had better concordance with multiparametric flow cytometry (sensitivity 10-4) (37 and 34% positive, respectively), compared to immunofixation (62% positive). In addition, HLC-pair suppression was identified in 38% of patients and associated with shorter PFS (30.6 months vs not reached; P<0.001). We conclude that HLC monitoring could augment electrophoretic assessments in patients achieving VGPR. The prognostic significance of HLC responses might partly depend on the patients' ability to recover their immune system, as determined by normalisation of HLC measurements.

Light chain multiple myeloma: an evaluation of its biochemical investigations.

Multiple myeloma is a type of plasma cell dyscrasia, characterised by presence of paraprotein or monoclonal (M)-protein in serum or urine. The M-protein may consist of an intact immunoglobulin, the heavy chain only or the light chain only. The latter, designated as light chain multiple myeloma (LCMM) makes up almost 20% of myelomas. Clinical manifestation is often heralded by hypercalcaemia, renal impairment, normocytic normochromic anaemia and bone lesions, reflecting end-organ damage, collectively known as the acronym CRAB. In particular, free light chain nephrotoxicity accounts for the high prevalence of renal impairment seen in LCMM. This case illustrates a typical presentation of LCMM with focal discussion on its initial and diagnostic, as well as prognostic biochemical investigations.

Immunoglobulin A nephropathy in a patient with IgG kappa light-chain myeloma.

Paraproteins can cause a variable set of pathologic changes in the kidney. The introduction of novel anti-plasma cell agents capable of reversing renal failure have revolutionized the management of paraprotein-mediated kidney injury. Activation of the transcription factor nuclear factor kB (NF-kB) has been shown to be involved in the development of human glomerulonephritis (GN). Inhibitors of NF-kB may provide potential agents for treatment of immune complex GN. In this paper, we report a patient with IgA nephropathy and IgG kappa myeloma, who responded dramatically to chemotherapy targeted toward myeloma. Our findings support the idea that drugs modulating NF-kB may add another dimension to the management of IgA nephropathy.

Pure Red Cell Aplasia Associated with Monoclonal Gammopathy of Undetermined Significance and Literature Review.

BACKGROUND: Pure red cell aplasia (PRCA) is an uncommon disease which involves an almost complete absence of the erythroid lineage in bone marrow (BM) and causes severe anemia. Cases due to monoclonal gammopathy occurring in plasma cell disorder have been infrequently reported. Here we report a case of PRCA associated plasma cell disorder, especially monoclonal gammopathy of undetermined significance (MGUS). METHODS: A 55-year-old male visited the ER due to general weakness. At his initial visit he exhibited severe anemia. Mild intravascular hemolysis was suspected. For anemia evaluation, BM examination was performed. In BM aspiration, almost no erythroid precursor cells were observed. Also, plasma cells were relatively elevated, at 7.2%. Serum electrophoresis and immunofixation revealed paraproteinemia of 5.1 g/L (IgG and lambda). No hypercalcemia, renal insufficiency or lytic bone lesions were found. This unusual case showed MGUS accompanied by PCRA. We were also able to assume the erythroid cell-specific restriction due to paraprotein, because we ruled out possible causes of PRCA. RESULTS: We discovered several reported cases associated with plasma cell dyscrasia. However, most of these cases involved plasma cell myeloma, characterized by high immunoglobulin burden. Our case demonstrates that PRCA is also observed in cases with MGUS, where immunoglobulin burden is low. CONCLUSIONS: It is not yet accurately known, what parts of erythroid precursors are targeted by M-protein nor what the mechanism is. Therefore, additional research into this matter is necessary.

Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

Levels of uninvolved immunoglobulins predict clinical status and progression-free survival for multiple myeloma patients.

Multiple myeloma (MM) is characterized by the enhanced production of the same monoclonal immunoglobulin (M-Ig or M protein). Techniques such as serum protein electrophoresis and nephelometry are routinely used to quantify levels of this protein in the serum of MM patients. However, these methods are not without their shortcomings and problems accurately quantifying M proteins remain. Precise quantification of the types and levels of M-Ig present is critical to monitoring patient response to therapy. In this study, we investigated the ability of the HevyLite (HLC) immunoassay to correlate with clinical status based on levels of involved and uninvolved antibodies. In our cohort of MM patients, we observed that significantly higher ratios and greater differences of involved HLC levels compared to uninvolved HLC levels correlated with a worse clinical status. Similarly, higher absolute levels of involved HLC antibodies and lower levels of uninvolved HLC antibodies also correlated with a worse clinical status and a shorter progression-free survival. These findings suggest that the HLC assay is a useful and a promising tool for determining the clinical status and survival time for patients with multiple myeloma.

Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients.

AIM: To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. METHODS: Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. RESULTS: HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P<0.001 and P=0.002, respectively) and an abnormal serum FLC ratio (for both P<0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P=0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P<0.001 and P=0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P=0.021). In a multivariate analysis, an abnormal HLC ratio and ß2-microglobulin level >3.5mg/L were independent risk factors for survival. CONCLUSION: The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome.

Detecting monoclonal immunoglobulins in human serum using mass spectrometry.

Established guidelines from the International Myeloma Working Group recommend diagnostic screening for patients suspected of plasma cell proliferative disease using protein electrophoresis (PEL), free light chain measurements and immunofixation electrophoresis (IFE) of serum and urine in certain cases. Plasma cell proliferative disorders are generally classified as monoclonal gammopathies given most are associated with the excess secretion of a monoclonal immunoglobulin or M-protein. In clinical practice, the M-protein is detected in a patients' serum by the appearance of a distinct protein band migrating within regions typically occupied by immunoglobulins. Given each M-protein is comprised by a sequence of amino acids pre-defined by somatic recombination unique to each clonal plasma cell, the molecular mass of the M-protein can act as a surrogate marker. We established a mass spectrometry based method to assign molecular mass to the immunoglobulin light chain of the M-protein and used this to detect the presence of M-proteins. Our method first enriches serum for immunoglobulins, followed by reduction to separate light chains from heavy chains, followed by microflow LC-ESI-Q-TOF MS. The multiply charged light chain ions are converted to their molecular mass and reconstructed peak area calculations are used for quantification. Using this method, we term "monoclonal immunoglobulin Rapid Accurate Molecular Mass" or miRAMM, the presence of M-proteins can be reliably detected with superior sensitivity compared to current gel-based PEL and IFE techniques.

A descriptive study of plasma cell dyscrasias in Egyptian population.

BACKGROUND: Plasma cell dyscrasias (PCDs) refer to a spectrum of disorders characterized by the monoclonal proliferation of lymphoplasmacytic cells in the bone marrow and, sometimes, tissue deposition of monoclonal immunoglobulins or their components. These disorders include multiple myeloma (MM) and Waldenström's macroglobulinemia, as well as rare conditions such as light-chain deposition disease (LCDD) and heavy-chain diseases (HCDs). The worldwide annual incidence of MM is estimated at 86,000, which is approximately 0.8% of all new cancer cases. PURPOSE: Our retrospective study aims to highlight the immunologic and epidemiological features of PCDs mainly MM in Egyptian patients and compare our results with those of other populations. METHODS: Two hundred seventeen Egyptian patients with PCD were enrolled in the study. Serum, urine protein electrophoresis and immunofixation were used to demonstrate M protein. RESULTS: One hundred thirty-eight patients (63.6%) had IgG monoclonal band, 38 patients (17.5%) had IgA, 12 patients (5.5%) had Waldenström's macroglobulinemia (IgM monoclonal band) and 29 patients (13.4%) were light chain myeloma. One hundred fifty-one (70%) were Kappa chain positive and 66 patients (30%) were lumbda positive. Conventional cytogenetics was available for 40 patients; of them12 patients (30%) showed 13q-. Mean OS was 37.5months (1-84months). Survival analysis was statistically insignificant according to age, sex and ISS or type of treatment (P value>0.05). CONCLUSION: Long term follow up is required to further define the role of different therapeutic lines of treatment including ASCT in the various stages of PCD based on OS data.

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes.

BACKGROUND: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. DESIGN AND METHODS: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters. RESULTS: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. CONCLUSIONS: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

Targeted idiotype-fusion DNA vaccines for human multiple myeloma: preclinical testing.

OBJECTIVES: A homodimeric fusion DNA vaccine targeting idiotype (Id) to antigen-presenting cells (APC) induced robust tumor protection in a mouse model of multiple myeloma (MM). Similar Id vaccine molecules were generated for four patients with MM with three main objectives: (i) do the vaccine molecules induce bona fide anti-Id immune responses in mice? (ii) does targeting of the vaccine molecules to APC enhance immune responses? (iii) can anti-Id antibodies, generated as by-product in vaccinated mice, be used to establish sensitive assays for complete remission (CR) prior to patient vaccination? METHODS: Chimeric vaccine molecules targeting patient Id to mouse major histocompatibility complex (MHC) class II molecules were genetically constructed for four patients with MM. RESULTS: DNA vaccination of mice with chimeric vaccines targeting patient Id to mouse MHC class II molecules elicited antibodies specific for the patient's myeloma protein. Targeting MHC class II greatly enhanced anti-Id responses. Mouse anti-Id antibodies were used to establish myeloma protein-specific enzyme-linked immunosorbent assays (ELISAs) that were between 75 and 1500 times more sensitive than conventional serum protein electrophoresis and immunofixation. CONCLUSIONS: These results pave the way for testing targeted DNA Id vaccines in patients in CR. Id- and patient-specific ELISA could be established affording evaluation of CR depth beyond current serological methods.

Identification and characterization of myeloma-associated antigens in Trichinella spiralis.

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4(+), CD8(+) T lymphocyte, and CD19(+) B lymphocyte were significantly increased (P<0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P<0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P>0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.

Recognition of galactose-deficient O-glycans in the hinge region of IgA1 by N-acetylgalactosamine-specific snail lectins: a comparative binding study.

Aberrancies in IgA1 glycosylation have been linked to the pathogenesis of IgA nephropathy (IgAN), a kidney disease characterized by deposits of IgA1-containing immune complexes in the glomerular mesangium. IgA1 from IgAN patients is characterized by the presence of galactose (Gal)-deficient O-glycans in the hinge region that can act as epitopes for anti-glycan IgG or IgA1 antibodies. The resulting circulating immune complexes are trapped in the glomerular mesangium of the kidney where they trigger localized inflammatory responses by activating mesangial cells. Certain lectins recognize the terminal N-acetylgalactosamine (GalNAc)-containing O-glycans on Gal-deficient IgA1 and can be potentially used as diagnostic tools. To improve our understanding of GalNAc recognition by these lectins, we have conducted binding studies to assess the interaction of Helix aspersa agglutinin (HAA) and Helix pomatia agglutinin (HPA) with Gal-deficient IgA1. Surface plasmon resonance spectroscopy revealed that both HAA and HPA bind to a Gal-deficient synthetic hinge region glycopeptide (HR-GalNAc) as well as various aberrantly glycosylated IgA1 myeloma proteins. Despite having six binding sites, both HAA and HPA bind IgA1 in a functionally bivalent manner, with the apparent affinity for IgA1 related to the number of exposed GalNAc groups in the IgA1 hinge. Finally, HAA and HPA were shown to discriminate very effectively between the IgA1 secreted by cell lines derived from peripheral blood cells of patients with IgAN and that from cells of healthy controls. These studies provide insight into lectin recognition of the Gal-deficient IgA1 hinge region and lay the groundwork for the development of reliable diagnostic tools for IgAN.

Laboratory characterizations on 2007 cases of monoclonal gammopathies in East China.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor beta1 (TGFbeta1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFbeta1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonal immunoglobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain kappa 6.1%, lambda 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (kappa/lambda ) 94.4%, quantitation of Igs 83%, light chain quantitation 80.9%, and urine light chain ratio (kappa/lambda) 58.0%. Plasma TGFbeta1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C>T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitation and urine light chain determination. Although TGFbeta1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFbeta1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study.