Clearing drug interferences in myeloma treatment using mass spectrometry.

OBJECTIVE: To explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients. DESIGN & METHODS: 86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs. RESULTS: Mass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix. CONCLUSION: This proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.

Comparison of MALDI-TOF mass spectrometry analysis of peripheral blood and bone marrow-based flow cytometry for tracking measurable residual disease in patients with multiple myeloma.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) may soon replace routine electrophoretic methods for monitoring monoclonal proteins in patients with multiple myeloma. To further evaluate the clinical utility of this assay, we compared the performance of MALDI-TOF-MS head-to-head with an established bone marrow-based measurable residual disease assay by flow cytometry (Flow-BM-MRD), using Memorial Sloan Kettering Cancer Center's 10-color, single-tube method. Our results suggest that MALDI-TOF-MS adds value to bone marrow-based MRD testing and may be most useful for early detection of relapse in peripheral blood compared to current electrophoretic methods.

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.

A transplant "immunome" screening platform defines a targetable epitope fingerprint of multiple myeloma.

Multiple myeloma (MM) is a hematological cancer for which immune-based treatments are currently in development. Many of these rely on the identification of highly disease-specific, strongly and stably expressed antigens. Here, we profiled the myeloma B-cell immunome both to explore its predictive role in the context of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and to identify novel immunotherapeutic targets. We used random peptide phage display, reverse immunization, and next-generation sequencing-assisted antibody phage display to establish a highly myeloma-specific epitope fingerprint targeted by B-cell responses of 18 patients in clinical remission. We found that allogeneic HSCT more efficiently allowed production of myeloma-specific antibodies compared with autologous HSCT and that a highly reactive epitope recognition signature correlated with superior response to treatment. Next, we performed myeloma cell surface screenings of phage-displayed patient transplant immunomes. Although some of the screenings yielded clear-cut surface binders, the majority of screenings did not, suggesting that many of the targeted antigens may in fact not be accessible to the B-cell immune system in untreated myeloma cells. This fit well with the identification of heat-shock proteins as a class of antigens that showed overall the broadest reactivity with myeloma patient sera after allogeneic HSCT and that may be significantly translocated to the cell surface upon treatment as a result of immunogenic cell death. Our data reveal a disease-specific epitope signature of MM that is predictive for response to treatment. Mining of transplant immunomes for strong myeloma surface binders may open up avenues for myeloma immunotherapy.

Pulmonary Extranodal Marginal Zone Lymphoma with Macroglobulinemia and Mixed Cryoglobulinemia Developed in a Patient with Chronic Hepatitis C.

We report a 65-year-old woman with a chronic hepatitis C virus infection who developed pulmonary extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissues complicated with macroglobulinemia and mixed cryoglobulinemia. She was treated with immunochemotherapy which resulted in the reduction of both the tumors and the serum immunoglobulin (Ig) M level. This case exemplifies an extensive stimulation upon immune system with derangement in the production of immunoglobulines associated with EMZL, and suggests that it is necessary to consider the possibility of B-cell lymphoma when IgM paraprotein is detected.

Inability to Measure M-Protein With Capillary Zone Electrophoresis (CAPPILLARYS 2) in Tracings With NonDiscernable Peaks.

BACKGROUND: We performed a retrospective study to illustrate the challenges with quantifying monoclonal (M)-protein in the cases of serum protein capillary zone electrophoresis (SPCZE) where no discernable peak is apparent. MATERIALS AND METHODS: We retrospectively reviewed 160 serum immunofixation electrophoresis (SIFE) that were performed at Memorial Hermann Hospital-Texas Medical Center between October 2013 and November 2013 and we identified the positive SIFE results. The corresponding SPCZE of the positive SIFE were retrieved and evaluated for the ability to quantify M-proteins in them. We define the ability to quantify M-protein as the ability for the operator of the SPCZE to identify a discernable peak and to be able to manually gate the area under the peak. RESULTS: Twenty-two cases of SIFE detected a monoclonal immunoglobulin. Of the corresponding 22 SPCZE, we could not quantify the M-protein in 6 (27.3%) of the cases. CONCLUSION: We have shown several cases where we were not able to quantify the M-protein with SPCZE. This poses a challenge in the diagnosis and management of these patients.

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.

Human amyloidogenic light chains directly impair cardiomyocyte function through an increase in cellular oxidant stress.

Primary amyloidosis is a systemic disorder characterized by the clonal production and tissue deposition of immunoglobulin light chain (LC) proteins. Congestive heart failure remains the greatest cause of death in primary amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Amyloid cardiomyopathy is largely unresponsive to current heart failure therapies, and is associated with a median survival of less than 6 months and a 5-year survival of less than 10%. The mechanisms underlying this disorder, however, remain unknown. In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy (cardiac-LC), specifically alter cellular redox state in isolated cardiomyocytes, marked by an increase in intracellular reactive oxygen species and upregulation of the redox-sensitive protein, heme oxygenase-1. In contrast, vehicle or control LC proteins isolated from patients without cardiac involvement did not alter cardiomyocyte redox status. Oxidant stress imposed by cardiac-LC proteins further resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intracellular calcium handling. Cardiomyocyte dysfunction induced by cardiac-LC proteins was independent of neurohormonal stimulants, vascular factors, or extracellular fibril deposition, and was prevented through treatment with a superoxide dismutase/catalase mimetic. This study suggests that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.

[A case with multiple myeloma in which serum forms gel precipitation upon exposure to air].

We present the case of a 69-years-old man who was admitted to hospital with multiple myeloma. IgG-kappa type monoclonal protein was detected in the serum. When we separated the serum obtained from blood sample of the patient and the lid of the collecting tube was opened, the patient's serum became gelled immediately. When the lid of the collecting tube remained closed, the patient's serum did not become gelled even at 4 degrees C. Moreover, the gelled serum of the patient did not resolve at 56 degrees C. Taken together, these results indicated that gel formation of the patient's serum may not be due to cryoglobulin. It was found that the pH of the patient's serum elevated to pH 8.0 quickly after exposed to air. It was also found that the patient's serum, but not the sera of other IgG-kappa multiple myeloma patients, became gelled as soon as PBS of pH 8.0 was added. These results highly suggest that the patient's serum becomes gelled at pH 8.0. However, the isoelectric focusing of isolated precipitation in the patient showed fractions around the pH 8.5-8.7 zone, which was different from the pH at which the precipitation began to form. We think that this may be the first report of a multiple myeloma patient whose serum becomes gelled after exposed to air.

Lack of SC/pIgR-mediated epithelial transport of a human polymeric IgA devoid of J chain: in vitro and in vivo studies.

Three human polymeric IgA (pIgA) myeloma proteins of tetrameric size were compared for their J-chain content, their in vitro secretory component (SC)-binding ability, and their capacity to be transcytosed by polymeric immunoglobulin receptor (pIgR)-expressing epithelial cells in vitro and rat hepatocytes in vivo. One of the three pIgA preparations, pIgA-L, was shown to lack J chain and was unable to combine with purified free human and rat SC, whereas pIgA-G and pIgA-C contained J chain and combined readily with SC. Furthermore, pIgA-L was not transferred into rat bile after intravenous injection, and was hardly transported apically by polarized Madin-Darbey canine kidney cell monolayers expressing the human pIgR, whereas pIgA-G and pIgA-C were efficiently transported in both test systems. Together with our recent demonstration that antibodies to human J chain block the SC/pIgR-mediated epithelial transport of pIgA, these data unanimously confirm the proposed key role of J chain in the epithelial transport of polymeric immunoglobulins into exocrine secretions.

Separation of human plasma/serum proteins by capillary isoelectric focusing in the absence of denaturing agents.

Human plasma/serum proteins were separated by capillary isoelectric focusing in the absence of denaturing agents. Proteins focused in a fused silica capillary were mobilized by replacing the catholyte sodium hydroxide to acetic acid. The performance of the separation of human plasma proteins has been examined by changing the duration of the step of isoelectric focusing, carrier ampholyte concentration, and plasma protein concentration. The separation patterns of plasma proteins were compared with those obtained by micro two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) run in the absence of denaturants in order to locate the major plasma proteins on the capillary electropherograms. Using the established electrophoretic conditions and the results of peak identification, proteins in the sera of IgG myeloma patients were analyzed by capillary isoelectric focusing. The advantages of capillary isoelectric focusing in plasma protein analysis compared with 2-D PAGE are discussed.

Structure and function of IgE myeloma protein VL from an atopic patient.

In a woman suffering from IgE myeloma, hay fever and polyvalent respiratory and skin allergy the IgE monoclonal protein VL was isolated and investigated with respect to structural and functional properties. The amino acid sequence of 22 isolated peptides--especially of the biologically significant C2-C3 part--corresponded with that originally described by Bennich et al. (Immunol Rev 1978;41:3-23; Prog Immunol 1974;13:49-58). However, in mass spectrometry the sugar residues on ASN 99 (219) and 252 (371) were deficient in sialic acids. The native IgE VL protein precipitated with high intensity all mannose-specific lectins as concanavalin A (Con A) and was able to release histamine after triggering by these lectins. The same lectins also elicited more histamine release and more positive skin reactions in atopic than in healthy persons. In sera from atopic patients the binding of IgE on Con A Sepharose 4B column was stronger than in normal persons. It is suggested that changes in the IgE glycosylation state may contribute to IgE-mediated pictures of clinical allergy by the nonimmunological pathway.

Homogenous IgA monomers, dimers, trimers and tetramers from the same IgA myeloma serum.

Starting from two IgA1 myeloma sera, the isolation of monoclonal monomeric, dimeric, trimeric and tetrameric IgA in a high state of purity and size homogeneity for each serum is described. The method combined repetitive gel filtrations on Ultrogel AcA22 with affinity chromatography on Jacalin-Sepharose. These various forms of pure polymeric IgA obtained from the same monoclonal IgA should allow a precise comparison of their respective structure and reactivity with different IgA-binding proteins, such as IgA Fc-receptors, the polymeric Ig receptor, and lectins.

Hybrid myeloma cells which secrete heterodimeric IgG: a model to study the N-linked glycan.

Fundamental questions remain unanswered regarding the effect of the acceptor polypeptide structure on the fine structure of the N-linked glycan of glycoproteins and conversely, the effect of the glycan structure of IgG on the function and structure of the protein. The construction of myeloma hybrids capable of secreting multiple IgG which differ with regard to the fine structure of their N-linked oligosaccharides would be a valuable model for studying these questions. P3X63Ag8 analogous glycan of the IgG2b secreted by Sp2/HLBu. Fusion hybrids of these cells secrete parental IgG1, and to a lesser degree IgG2b, as well as a heterodimeric IgG containing both the gamma 1 and gamma 2b chains. The oligosaccharide of each chain is identical in structure to the appropriate parental IgG. Such cells allow for the analysis of acceptor properties that influence glycan fine structure, as well as the role of glycan structure on the stability of the IgG.

Carbohydrate analysis of immunoglobulin G myeloma proteins by lectin and high performance liquid chromatography: role of glycosyltransferases in the structures.

The carbohydrate structures and the enzymatic basis for glycosylation of IgG by bone marrow plasma cells were determined in 7 patients with monoclonal gammopathy of undetermined significance and 22 patients with IgG MM. Lectin-binding analysis showed that in all cases of monoclonal gammopathy of undetermined significance and normal controls the IgG heavy chains bound to Ricinus communis agglutinin more strongly than to concanavalin A. In contrast, the IgG in 11 of the 17 advanced cases of MM (stages II and III) studied reacted to concanavalin A more strongly. Structural analysis showed that the reduced R. communis agglutinin binding capacity of these MM IgGs was due to hypogalactosylation of IgG. The galactosyltransferase and N-acetylglucosaminyltransferase III activities of the bone marrow myeloma cells from 5 MM cases were found to have a low enzyme activity ratio of galactosyltransferase to N-acetylglucosaminyltransferase III which reflects the hypogalactosylation. This indicates that the difference in the carbohydrate moieties observed in myeloma proteins is due to variations in the activities of the two glycosyltransferases.

Biclonal gammopathy of kappa light chain type in a case of multiple myeloma.

This immunoglobulin abnormality was found in the serum of a 33 years old man presenting with anemia, weight loss, bone pain and a single bone lesion. The cellulose acetate electrophoresis of his serum showed 2 homogeneous bands migrating in the gamma and alpha-2 regions. Agar gel immunoelectrophoresis using anti-whole normal serum revealed 2 abnormal precipitin arcs of gamma-2 and alpha-2 respective mobility. These 2 M-components precipitated with anti-human kappa light chain antiserum but failed to react with lambda-light chain specific and H-chains specific anti-sera. Furthermore, the 2 monoclonal kappa chains were still demonstrated in the supernatant of the se-rum treated with 50% saturated ammonium sulphate. The 2 kappa chains were isolated from the supernatant using goat anti-kappa antibodies bound to Sepharose-4B. Their SDS-polyacrylamide gel electrophoresis pattern showed a single protein band of 25 Kd molecular weight. This suggest the presence of 2 monomeric kappa chains of different clonal origin. Subsequently, the difference in their electrophoretic mobility was probably due to different V-kappa sequences. However, it is also possible that the unusual alpha-2 mobility is a consequence of a moderate glycosylation without increase in molecular weight.

Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses.

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.