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Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4), encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here, we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years, we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma, autoimmune diseases, and blood traits. Seven variants map to the IGH locus, three to the Fcγ receptor (FCGR) locus, and two to the human leukocyte antigen (HLA) region, affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f), G2m(n) and G3m(b*) allotypes, and IgG1, IgG2 and IgG3, respectively. Additionally, we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3, ZPBP2, GSDMB, ORMDL3). Interestingly, the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics., (© 2024. The Author(s).)

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, V H regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of C H 1-4 domains while peptides from the 40 kDa H chain only mapped to C H 1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of C L and V L genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Camels belong to a group of animals, where the structure of placenta does not allow intrauterine transfer of maternal immunoglobulins to the fetus and maternal immunity is exclusively transferred by colostrum to the newborn calf. There are few studies on the passive transfer of maternal immunity in the dromedary camel. This study determined total immunoglobulin G concentration, heavy chain antibody (HCAbs) levels, and neutrophils to lymphocytes ratio (NLR) in female camels and their newborn calves. For this, samples were collected from nine she-camels (blood and colostrum) and their calves (blood). IgG concentration and HCAb level were determined in mother serum and colostrum as well as in calf serum using ELISA. The NLR was calculated after the estimation of relative fractions of neutrophils and lymphocytes in collected blood samples using a blood cell analyzer. Both IgG and HCAbs were higher concentrated in camel colostrum than in mother serum. At parturition and before the first colostrum intake, calf serum did not contain any measurable concentration of IgG and only low levels of HCAbs. After colostrum consumption, a rise in IgG and HCAb levels was observed in calf serum. For total IgG, a minimum was reached on day 30 postnatum. While a significant increase in IgG concentration was seen on day 60 postnatum, no significant rise was measured in HCAbs at that age. Only post-colostrum IgG levels in calf serum correlated positively with IgG levels in mother colostrum. Directly after birth, newborn calves showed significantly higher NLR than their mothers. This indicates a pro-inflammatory nature of the calf immune response. The decrease of the NLR on day 60 postnatum may argue for the maturation of the calf immune response at this age.

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients., Competing Interests: Conflict of interest disclosure None of the authors declared a conflict of interest., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Although no therapies are approved for light chain (AL) amyloidosis, cyclophosphamide, bortezomib, and dexamethasone (CyBorD) is considered standard of care. Based on outcomes of daratumumab in multiple myeloma (MM), the phase 3 ANDROMEDA study (NCT03201965) is evaluating daratumumab-CyBorD vs CyBorD in newly diagnosed AL amyloidosis. We report results of the 28-patient safety run-in. Patients received subcutaneous daratumumab (DARA SC) weekly in cycles 1 to 2, every 2 weeks in cycles 3 to 6, and every 4 weeks thereafter for up to 2 years. CyBorD was given weekly for 6 cycles. Patients had a median of 2 involved organs (kidney, 68%; cardiac, 61%). Patients received a median of 16 (range, 1-23) treatment cycles. Treatment-emergent adverse events were consistent with DARA SC in MM and CyBorD. Infusion-related reactions occurred in 1 patient (grade 1). No grade 5 treatment-emergent adverse events occurred; 5 patients died, including 3 after transplant. Overall hematologic response rate was 96%, with a complete hematologic response in 15 (54%) patients; at least partial response occurred in 20, 22, and 17 patients at 1, 3, and 6 months, respectively. Renal response occurred in 6 of 16, 7 of 15, and 10 of 15 patients, and cardiac response occurred in 6 of 16, 6 of 13, and 8 of 13 patients at 3, 6, and 12 months, respectively. Hepatic response occurred in 2 of 3 patients at 12 months. Daratumumab-CyBorD was well tolerated, with no new safety concerns versus the intravenous formulation, and demonstrated robust hematologic and organ responses. This trial was registered at www.clinicaltrials.gov as #NCT03201965., (© 2020 by The American Society of Hematology.)

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest related to this article., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Systemic lupus erythematosus (SLE) is characterized by the overproduction of high-affinity autoreactive antibodies. Here, we show that more than 65.8% of 222 recombinant antibodies derived from 8 SLE patients can be secreted as heavy chain-only antibodies (HCAbs) when expressed in HEK-293T cells. The secretion of HCAbs follows the conventional endoplasmic reticulum-Golgi apparatus pathway, despite triggering a weaker unfolded protein response (UPR). Many of the purified SLE HCAbs remain autoreactive and have an even higher affinity for dsDNA, Sm, nucleosome, and cardiolipin than HCAbs from healthy individuals. Extended analyses of the CDR3 region and the heavy chain variable (VH) region of HCAb F3 show that the VH region is responsible for IgH secretion, while the CDR3 region determines its reactivity. Such a high frequency of HCAb secretion cannot fully concur with our current understanding of antibody assembly and secretion. The presence of a large proportion of autoreactive HCAbs in SLE reveals a novel mechanism for the generation of autoreactive antibodies in lupus., (Copyright © 2020 Xu, Yang, Zhuo, Yu, Liao, Li, Yue, Su and Zhang.)

Introduction: Immune thrombocytopenia (ITP) is an acquired hemorrhagic disease due to antiplatelet antibodies, that will become a chronic disease in 70% of adults. Most of chronic ITP patients display clonal restriction of antiplatelet antibodies. To date, there is no biomarker able to predict the evolution of the disease. The objective of the study is to determine whether Hevylite® and/or Freelite® assays are prognostic factors for progression to chronic ITP., Methods: This is a retrospective, monocentric, prognostic study of a biomarker, performed using frozen samples stored in a serum library. Freelite® and a Hevylite® assays were performed on the samples collected at diagnosis for adult patients with newly diagnosed ITP at the University Hospital of Poitiers between 2014/01/01 and 2017/05/01. To predict the evolution into a chronic disease, a ROC curve analysis was performed on four variables: IgGκ, IgGκ/IgGλ ratio, IgGκ - IgGλ, and κ/λ ratio., Results: Thirty-two patients were included and analyzed. No patient had an abnormal κ/λ ratio. Three patients had an abnormal IgGκ/IgGλ ratio. The following variables IgGκ, IgGκ/IgGλ, IgGκ - IgGλ, and κ/λ ratio were not able to predict progression to chronic ITP in our study., Conclusion: This study did not reveal any prognostic value of the Freelite® and Hevylite® tests on the evolution of ITP into a chronic disease., (Copyright © 2019 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier Masson SAS. All rights reserved.)

Introduction: Heavy and light chain amyloidosis is an extremely rare condition. There are few reports referring to the clinical impact of cardiac involvement in heavy and light chain amyloidosis, and the significance of myocardial impairment has not yet been completely explained., Patient Concerns: A 66-year-old Japanese man was admitted to our hospital presenting with nephrotic syndrome and congestive heart failure., Diagnosis: Kidney and endoscopic gastric mucosal biopsy demonstrated congophilic hyalinization in most of the glomeruli and surrounding vessel walls, which were highly positive for immunoglobulin A and lambda. Finally, the patient was diagnosed as an atypical multiple myeloma with systemic heavy and light chain amyloidosis., Interventions: The patient was referred to hematology for further treatment and was moved to another hospital for the administration of chemotherapy using melphalan and dexamethasone., Outcomes: The patient was still alive after 15-month follow-up from the initial diagnosis., Conclusion: Initial screening and follow-up for cardiac involvement are important for heavy and light chain amyloidosis. Further investigation for the prognosis of heavy and light chain amyloidosis is required to improve the strategies of diagnosis and treatment options for patients with this disease.

Background: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation., Aims: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL., Results: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months., Conclusion: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients. A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3-13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing-Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ). For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x-8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x-0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape. Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE., (Copyright © 2019 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Antibodies and Fc-fusion antibody-like proteins have become successful biologics developed for cancer treatment, passive immunity against infection, addiction, and autoimmune diseases. In general these biopharmaceuticals can be used for blocking protein:protein interactions, crosslinking host receptors to induce signaling, recruiting effector cells to targets, and fixing complement. With the vast capability of antibodies to affect infectious and genetic diseases much effort has been placed on improving and tailoring antibodies for specific functions. While antibody:antigen engagement is critical for an efficacious antibody biologic, equally as important are the hinge and constant domains of the heavy chain. It is the hinge and constant domains of the antibody that engage host receptors or complement protein to mediate a myriad of effector functions and regulate antibody circulation. Molecular and structural studies have provided insight into how the hinge and constant domains from antibodies across different species, isotypes, subclasses, and alleles are recognized by host cell receptors and complement protein C1q. The molecular details of these interactions have led to manipulation of the sequences and glycosylation of hinge and constant domains to enhance or reduce antibody effector functions and circulating half-life. This review will describe the concepts being applied to optimize the hinge and crystallizable fragment of antibodies, and it will detail how these interactions can be tuned up or down to mediate a biological function that confers a desired disease outcome.

Exosomes are vehicles in the body fluid that participate in many biological processes, especially immune responses. In this study, we employed comparative proteome analysis to investigate the roles of serum exosomes during viral infection in neonates using porcine epidemic diarrhea virus (PEDV), a devastating enteric virus in newborn piglets, as a model virus. Serum exosomes were first isolated from newborn piglets infected with PEDV or mock-infected newborn piglets, followed by label-free LC-MS/MS-based comparative quantitative proteomic analysis. Among the 441 detected proteins, 10 complement proteins were found in the serum exosomes, and significantly decreased expression levels of the C3, C6, and CFB complements were measured in PEDV-infected serum exosomes compared to those in mock-infected serum exosomes. After confirmation by Western blot, we then investigated the function of these exosomes in PEDV infection and discovered that exosomes from mock-infected newborn piglets restricted PEDV infection. However, this inhibition disappeared after the exosomes were heat-inactivated, suggesting that complements are key antiviral molecules. Our findings improve the understanding of antiviral responses mediated by exosomes in neonatal piglets and facilitate the discovery of novel antiviral drugs.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N -glycosylation on the CH2 domain, and the remodeling of the N -linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N -glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N -glycosylation. Although IgGs usually have a single N -glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N -glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N -glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N -linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N -glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N -glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N -glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911., (© 2019 Chandler et al.)

Background: Immunoglobulin heavy-and-light-chain amyloidosis (AHL amyloidosis) is a newly established disease entity where both the immunoglobulin heavy-chain and light-chain compose amyloid fibrils. The immunoglobulins responsible for the amyloid fibrils are generally identified by immunostaining and/or laser microdissection-liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS). However, both techniques do not biochemically differentiate immunoglobulins that formed amyloid fibrils from non-responsible immunoglobulins., Case Presentation: We herein report a case of 67-year-old female patient with renal amyloidosis due to lymphoplasmacytic lymphoma secreting monoclonal immunoglobulin M (IgM)-kappa. Renal immunostaining monotypically positive for IgM-kappa and LMD-LC-MS/MS identification of mu heavy-chain and kappa light-chain were consistent with the diagnosis of AHL amyloidosis. In order to confirm that both the immunoglobulin heavy-chain and light-chain were forming amyloid fibrils, we performed LC-MS/MS of renal amyloid fibrils isolated by the traditional amyloid purification method. The additional LC-MS/MS identified kappa light-chain only without any heavy-chain component. These results were suggestive that amyloid fibrils were composed by kappa light-chain only and that the mu heavy-chain identified by immunostaining and LMD-LC-MS/MS was derived from the non-specific co-deposition of monoclonal IgM-kappa., Conclusion: The case was AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition. While immunostaining and LMD-LC-MS/MS are irreplaceable techniques to classify amyloidosis, confident exclusion of the present condition should be required to diagnose AHL amyloidosis.

The aim of the study was to investigate the relationship between tacrolimus (TAC) immunosuppressive treatment and serum concentrations of immunoglobulin heavy/light chain pairs (sHLC) and free light chains (sFLC) in patients after heart transplantation (HTX) and to use these biomarkers to predict the risk of infection in these patients. A total of 88 patients with an immunosuppressive regimen involving tacrolimus who underwent HTX were analyzed over 24 months of follow-up. sFLC and sHLC levels were determined before and at three time points after HTX. TAC concentrations were determined at several time points after HTX, and mean TAC concentrations and areas under the curve (AUCs) of TAC concentration were calculated. Relevant clinical data were obtained from patients' medical records. A larger AUC of TAC was associated with decreases in the concentrations of IgG total (p < 0.05); similarly, cumulative AUC of TAC during 18 post-transplant months correlated inversely with sHLC IgG kappa (r = -0.228, p < 0.05) and IgG total (r = -0.352, p < 0.05). Concentrations of sFLC kappa, sFLC lambda, sHLC IgG kappa, and sHLC IgG total were significantly lower in infected patients (in the 9th month after HTX, all p < 0.05). Combined criteria for increased AUC (greater than the median of 12.9 mg·d/l) and decreased sFLC kappa (less than the median of 12.5 mg/l) correlated with the presence of infection (p < 0.03) in the 9th month after HTX. Ratio of concentration of TAC to sFLC kappa or lambda was significantly higher in infected patients (both p < 0.05). Intensive treatment with tacrolimus after HTX is possibly reflected by decreases in sFLC and sHLC (mainly sHLC IgG). Patients with decreased concentrations of these biomarkers are at increased risk for infection, primarily in the 9th month after HTX, when the concentrations of tacrolimus were the highest., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking., Methods: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring., Results: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig V H ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig V H 4-34 IgM-RF decreased following immunosuppression and remission of MC., Conclusion: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones., (© 2018, American College of Rheumatology.)

We investigated the prognostic impact and clinical utility of serum free light chains (sFLC) and serum heavy-light chains (sHLC) in patients with multiple myeloma treated according to the GEM2005MENOS65, GEM2005MAS65, and GEM2010MAS65 PETHEMA/GEM phase III clinical trials. Serum samples collected at diagnosis were retrospectively analyzed for sFLC (n = 623) and sHLC (n = 183). After induction or autologous transplantation, 309 and 89 samples respectively were available for sFLC and sHLC assays. At diagnosis, a highly abnormal (HA) sFLC ratio (sFLCr) (<0.03 or >32) was not associated with higher risk of progression. After therapy, persistence of involved-sFLC levels >100 mg/L implied worse survival (overall survival [OS], P = 0.03; progression-free survival [PFS], P = 0.007). Among patients that achieved a complete response, sFLCr normalization did not necessarily indicate a higher quality response. We conducted sHLC investigations for IgG and IgA MM. Absolute sHLC values were correlated with monoclonal protein levels measured with serum protein electrophoresis. At diagnosis, HA-sHLCrs (<0.29 or >73) showed a higher risk of progression (P = 0.006). Additionally, involved-sHLC levels >5 g/L after treatment were associated with shorter survival (OS, P = 0.001; PFS, P = 0.018). The HA-sHLCr could have prognostic value at diagnosis; absolute values of involved-sFLC >100 mg/L and involved-sHLC >5 g/L could have prognostic value after treatment., Competing Interests: The authors have declared that no competing interests exist.

More than 35 years after the Binet classification, there is still a need for simple prognostic markers in chronic lymphocytic leukemia (CLL). Here, we studied the treatment-free survival (TFS) impact of normal serum protein electrophoresis (SPE) at diagnosis. One hundred twelve patients with CLL were analyzed. The main prognostic factors (Binet stage; lymphocytosis; IGHV mutation status; TP53, SF3B1, NOTCH1, and BIRC3 mutations; and cytogenetic abnormalities) were studied. The frequencies of IGHV mutation status, cytogenetic abnormalities, and TP53, SF3B1, NOTCH1, and BIRC3 mutations were not significantly different between normal and abnormal SPE. Normal SPE was associated with Binet stage A, nonprogressive disease for 6 months, lymphocytosis below 30 G/L, and the absence of the IGHV3-21 gene rearrangement which is associated with poor prognosis. The TFS of patients with normal SPE was significantly longer than that of patients with abnormal SPE (log-rank test: P = 0.0015, with 51% untreated patients at 5.6 years and a perfect plateau afterward vs. a median TFS at 2.64 years for abnormal SPE with no plateau). Multivariate analysis using two different Cox models and bootstrapping showed that normal SPE was an independent good prognostic marker for either Binet stage, lymphocytosis, or IGHV mutation status. TFS was further increased when both normal SPE and mutated IGHV were present (log-rank test: P = 0.008, median not reached, plateau at 5.6 years and 66% untreated patients). A comparison with other prognostic markers suggested that normal SPE could reflect slowly advancing CLL disease. Altogether, our results show that a combination of normal SPE and mutated IGHV genes defines a subgroup of patients with CLL who evolve very slowly and who might never need treatment., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Heavy/light chain (HLC) assay can quantify involved as well as uninvolved immunoglobulin pairs and is used to detect monoclonal proteins., Patients and Methods: We compared the sensitivity between HLC assay and serum protein electrophoresis, serum immunofixation electrophoresis (IFE), and free light chain (FLC) assay in patients with symptomatic multiple myeloma (n = 111) whose responses were stable disease or better., Results: Among patients with negative IFE and normal FLC ratios, 84.4% (38 of 45) and 80% (36 of 45) exhibited normal HLC ratios and no pair suppression, respectively (13.3% [6 of 45], moderate pair suppression and 6.7% [3 of 45], severe pair suppression). The lower the monoclonal protein levels, the more the possibility that the patients had normal HLC ratios and no matched pair suppression (both P < .000001). HLC ratios or pair suppression combined with IFE results and FLC ratios were more sensitive for detecting monoclonal proteins than were IFE results and FLC ratios alone (P = .016 and .0039, respectively). A combination of all 4 methods (IFE, FLC, HLC, and pair suppression) was far more sensitive than were IFE findings plus FLC ratios alone (P = .00024)., Conclusion: Abnormal HLC ratios and HLC-matched pair suppression can increase the sensitivity for detecting residual disease in patients with multiple myeloma with deep responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Automated serum heavy + light chain (HLC) immunoassays can measure the intact immunoglobulins of each light chain type separately. We though to compare HLC assays with electrophoretic techniques in determining International Myeloma Working Group (IMWG) response criteria. 114 myeloma patients from 2 trials were included. HLC measurements were made utilizing archived sera and response assessments compared with those based on electrophoretic analysis at the time of the trials. Assessments at ∼90 days and maximal response were compared as was the power of the 2 techniques for predicting later responses, overall survival, and progression. The kappa statistic indicated good agreement between the 2 methods for determining IMWG response criteria, although HLC measurements might give better predictions of subsequent responses and frequently gave an earlier indication of change. HLC measurements could represent an alternative to electrophoretic techniques in determining IMWG response. Validation with a greater range of patient responses is needed for confirmation.

Although high-throughput sequencing and associated bioinformatics technologies have enabled the in-depth, sequence-based characterization of human immune repertoires, only a few studies on a relatively small number of sequences explored the characteristics of antibody repertoires in neonates, with contradictory conclusions. To gain a more comprehensive understanding of the human IgM antibody repertoire, we performed Illumina sequencing and IMGT/HighV-QUEST analysis of IgM heavy chain repertoire of the B lymphocytes from the cord blood (CB) of neonates, as well as the repertoire from peripheral blood of healthy human adults (HH). The comparative study revealed unexpectedly high levels of similarity between the neonatal and adult repertoires. In both repertoires, the VDJ gene usage showed no significant difference, and the most frequently used VDJ gene was IGHV4-59, IGHD3-10, and IGHJ3. The average amino acid (aa) length of CDR1 (CB: 8.5, HH: 8.4) and CDR2 (CB: 7.6, HH: 7.5), as well as the aa composition and the average hydrophobicity of the CDR3 demonstrated no significant difference between the two repertories. However, the average aa length of CDR3 was longer in the HH repertoire than the CB repertoire (CB: 14.5, HH: 15.5). Besides, the frequencies of aa mutations in CDR1 (CB: 19.33%, HH: 25.84%) and CDR2 (CB: 9.26%, HH: 17.82%) were higher in the HH repertoire compared to the CB repertoire. Interestingly, the most prominent difference between the two repertoires was the occurrence of N2 addition (CB: 64.87%, HH: 85.69%), a process that occurs during V-D-J recombination for introducing random nucleotide additions between D- and J-gene segments. The antibody repertoire of healthy adults was more diverse than that of neonates largely due to the higher occurrence of N2 addition. These findings may lead to a better understanding of antibody development and evolution pathways and may have potential practical value for facilitating the generation of more effective antibody therapeutics and vaccines.

Objectives: To analyze the most common primary and secondary cytogenetic events in myelomas using a probe panel designed in our laboratory, and to associate those events with hematological and biochemical findings., Methods: Blood specimens from patients diagnosed with myeloma were processed to determine complete blood count and levels of albumin, creatinine, and beta-2 microglobulin. We evaluated bone-marrow specimens for plasma-cell percentage by light microscopy and for cytogenetic abnormalities by fluorescence in situ hybridization (FISH). The Mann-Whitney U test was used to compare hematological and biochemical parameters., Results: We observed immunoglobulin heavy chain (IgH) gene translocations in 43.3% and t(4;14) in 21% of specimens; t(11;14) was observed in 7.7% of specimens. Gain of chromosomes was observed in 67.2% and loss observed in 16.6% of specimens., Conclusions: Gains of chromosomes were observed in two-thirds of patients with myeloma. The most common IgH translocation was t(4;14); del13/monosomy13 was the most common secondary cytogenetic abnormality. Partial or complete tetrasomies were associated with higher beta-2 microglobulin levels., (© American Society for Clinical Pathology 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Objective: The heavy/light chain (HLC)-immunoassay quantifies light chain types of each immunoglobulin class in patients with monoclonal gammopathies., Methods: We assessed 147 consecutive patients with different forms and stages of plasma cell dyscrasias (PCD) who received standard tests (serum and urine protein electrophoresis [SPEP, UPEP], immunofixation [IFE], serum-free light chain [SFLC]), and HLC-immunoassay. Patients with multiple myeloma (MM, n = 102), smoldering MM (SMM, n = 5), monoclonal gammopathy of undetermined significance (MGUS, n = 28), and Waldenström's macroglobulinemia (WM, n = 12) were included., Results: We verified a significant correlation between HLC- and standard monoclonal protein (mp)-parameters, and HLC-increases with higher disease stage and unfavorable remission status. In patients with difficult to quantify mp, more abnormal HLC- than SPEP-, immunoglobulin-, or SFLC-results were found. In WM, a pathological HLC κ/λ-ratio and M-component were observed in 95% and 58%, respectively. In 21/28 MGUS and 5/5 SMM patients, HLC κ/λ-ratios were abnormal. Testing different HLC cutoffs, patients with extreme HLC values showed impaired progression-free survival (PFS)., Conclusions: Despite the fact that different PCD patients were included, the assessment of the HLC-immunoassay in MGUS, SMM, MM, and WM, our comparison with standard mp-assays, and relevant PFS differences may excite future applications, which should be confirmed in prospective multicenter trials., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cardiac involvement and presenting dFLC (difference between involved and uninvolved free light chains) are independent predictors of outcome in systemic AL amyloidosis. These markers have limited prognostic utility in patients surviving the initial months following diagnosis. Here we assessed immunoparesis, as determined by novel heavy+light chain (HLC) immunoassays, as a prognostic marker for survival in AL amyloidosis. HLC measurements identified immunoparesis of at least one immunoglobulin (Ig) isotype in 145 (85%) patients; and severe immunoparesis (≥2 Ig isotypes suppressed by >50% below normal levels) in 29 (17%) patients. Median overall survival (OS) on intention to treat (ITT) analysis was 26·2 months. In the ITT cohort, dFLC >180 mg/l was associated with shorter OS (P = 0·05); whereas HLC immunoparesis was not prognostic. On a landmark analysis of 127 patients alive at 6 months, presenting dFLC was not prognostic for OS (P = 0·33) and severe HLC immunoparesis trended towards poorer survival (20·2 vs. 42·8 months; P = 0·09). In the subset of patients with cardiac involvement, severe HLC immunoparesis conferred very poor outcome (median OS 8·8 vs. 29·9 months, P = 0·007). In conclusion, severe HLC immunoparesis is an independent marker of long-term poor prognosis in AL patients with cardiac involvement. The pathophysiological significance of this observation needs further study., (© 2017 John Wiley & Sons Ltd.)

Background: The management of IgM monoclonal gammopathies undetermined significance (IgM-MGUS) and Waldenstrom's macroglobulinemia (WM) may be challenging. Modern immunoassays that quantify specific monoclonal heavy and light chain immunoglobulins are promising for their use in these applications., Methods: Ninety consecutive patients (39 IgM-MGUS, 32 indolent WM [iWM], and 19 WM) seen between January 2007 and March 2014 were analyzed. Heavy/light chain (HLC) and serum free light chains assays (FLC) were determined at diagnosis to study their utility as biomarkers in IgM monoclonal gammopathies., Results: The HLC involved to uninvolved IgM ratios (iHLC/uHLC) showed a progressive increase when going from IgM-MGUS, to iWM and to WM (p=0.002). Furthermore, an iHLC/uHLC>62 identified a group of iWM patients with a shorter time-to-progression (TTP) (108 vs. 133 months, p=0.033). Separate analysis of the involved and uninvolved components showed that only the suppression of the uninvolvedimmunoglobulin was predictive of shorter TTP (HR=3.04, p=0.03) suggesting that it could be the majorcontributor to the prognostic value of the Hevylite assay. Additionally, a multivariate analysis showed that immunosuppression (either classical immunoparesis or Hevylite immunosuppression) was an independent prognostic factor (p=0.016) reinforcing its relevance in the disease mechanism. Finally, monoclonal sFLC levels were highest in WM patients, with 83% presenting values>60 mg/L., Conclusions: The results suggest that the levels of immunosuppression and/or the iHLC/uHLC ratio of IgM immunoglobulins measured by Hevylite are associated with greater disease activity which significantly impacts in the outcome of WM patients and may also help in the differentiation of IgMMGUS from iWM.

Background: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation., Methods: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components., Results and Conclusion: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care., (© 2016 Wiley Periodicals, Inc.)

Immunoglobulin (Ig) heavy/light chain (HLC) assays enable the separate quantification of the different light chain types of each Ig class. We retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome in 120 patients with multiple myeloma (MM). Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. Among the 85 patients who achieved ≥VGPR, the patients remained HLCR abnormal were showed significantly shorter overall survival (OS) compared to those achieving a normal HLCR (not reached vs 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs 30.1 months, P = 0.014), but not in IgG myeloma patients when patients were analyzed separately. Univariate and multivariate analysis of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 - 15.26; P = 0.012) for shorter OS in this subset of patients. This study highlighted the HLC assay as a prognostic predictor in patients with IgA myeloma., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Serum and urine immunofixation electrophoreses (SIFE/UIFE) are used for clonal detection in plasma cell dyscrasias, while serum free light chain (sFLC) testing provides quantitation of clonal disease. Up to 20% of patients with light chain (AL) amyloidosis may present with normal FLC ratio (FLCr)., Methods: We assessed the diagnostic, quantitative and prognostic potential of serum heavy light chain ratio (HLCr) in 199 untreated patients at initial evaluation., Results: An abnormal HLCr was found in 37.2%, abnormal FLCr in 81.9% and positivity by SIFE/UIFE in 94% of patients. HLCr together with SIFE/UIFE identified clonality in 94% patients; the combination with FLCr yielded 100% sensitivity. An HLCr abnormality was significantly over-represented in normal compared to abnormal FLCr group (63.9% versus 31.3%). HLCr did not predict overall survival (OS) (log rank, p = 0.09), while an abnormal FLCr was associated with decreased OS (log rank, p = 0.03). The combined use of both ratios trended toward increased OS in the abnormal HLCr/normal FLCr group (log rank, p = 0.11; Wilcoxon, p = 0.04). On multivariate analysis, HLCr was not predictive of OS, whereas an abnormal FLCr was associated with shorter OS (HR = 1.7, p = 0.04)., Conclusions: The HLC assay has potential as a supplemental test to quantify monoclonal protein in patients with normal FLCr results.

Purpose: Accurate quantification of monoclonal IgM immunoglobulins is essential for response assessment in patients with Waldenström's macroglobulinaemia (WM). The propensity of IgM to form multimers in serum makes sample evaluation by current laboratory methods particularly challenging., Experimental Design: We assessed the precision and linearity of IgMκ and IgMλ heavy/light chain (HLC, Hevylite) assays, and established reference intervals using 120 normal donor sera. We compared the quantitative performance of HLC assays with serum protein electrophoresis (SPE) and total IgM nephelometry for 78 diagnostic samples and follow-up samples from 25 patients with WM. Comparisons were made between the three methods for diagnostic sensitivity and response assessment., Results: IgMκ and IgMλ HLC assays showed low imprecision and good linearity. There was good agreement between summated HLC (IgMκ + IgMλ) and total IgM (measured nephelometrically; R 2 = 0.90), but only moderate agreement between involved IgM HLC and SPE densitometry (R 2 = 0.49). Analysis of 120 normal donor sera produced the following normal ranges: IgMκ: 0.29-1.82 g/L; IgMλ: 0.17-0.94 g/L; IgMκ/IgMλ ratio: 0.96-2.30. Using these ranges, IgM HLC ratios were abnormal in all WM presentation sera tested, including 15 with non-quantifiable SPE. Despite discordance in quantitation, responses assigned with HLC assays showed excellent agreement to those based on international guidelines using SPE or total IgM; although abnormal HLC ratios indicated residual disease in some patients with negative electrophoresis results., Conclusions: Nephelometric assessment of IgMκ and IgMλ HLC pairs offers a quantitative alternative to traditional laboratory techniques for the measurement of monoclonal IgM and may aid in the management of WM. Clin Cancer Res; 22(20); 5152-8. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objective: To compare the differences of immunological characteristics between newborn and adults, we performed high-throughput sequencing to reveal the diversity of umbilical cord blood and adult peripheral blood at both T-cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) levels., Study Design: High-throughput sequencing was performed to analyze the expression of TRB-CDR3 and IGH-CDR3 in circulating T and B cells isolated from 20 healthy adults, 56 pregnant women, and 40 newborns., Results: Our results revealed different immunological characteristics between newborn and adults, such as distinctive complementarity determining region 3 (CDR3) lengths, usage bias of variable and joining segments, random nucleotide addition, a large number of unique CDR3 peptides, and a greater repertoire diversity. Moreover, each newborn had a distinctive TRB-/IGH-CDR3 repertoire that was independent of the maternal immune status., Conclusions: This study presents comprehensive, unrestricted profiles of the TRB/IGH-CDR3 repertoire of newborns, pregnant women, and healthy adults at a sequence-level resolution. Our data may contribute to a better understanding of the immune system of newborns and benefit the efficient application of umbilical cord blood transplantation in future., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Idelalisib is a small-molecule inhibitor of PI3Kδ with demonstrated efficacy for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL). To evaluate idelalisib as front-line therapy, we enrolled 24 subjects in a phase 2 study consisting of 2 months of idelalisib monotherapy followed by 6 months of combination therapy with idelalisib and the anti-CD20 antibody ofatumumab. After a median follow-up period of 14.7 months, hepatotoxicity was found to be a frequent and often severe adverse event. A total of 19 subjects (79%) experienced either grade ≥1 ALT or AST elevation during the study, and 13 subjects (54%) experienced grade ≥3 transaminitis. The median time to development of transaminitis was 28 days, occurring before ofatumumab introduction. Younger age and mutated immunoglobulin heavy chain status were significant risk factors for the development of hepatotoxicity. Multiple lines of evidence suggest that this hepatotoxicity was immune mediated. A lymphocytic infiltrate was seen on liver biopsy specimens taken from 2 subjects with transaminitis, and levels of the proinflammatory cytokines CCL-3 and CCL-4 were higher in subjects experiencing hepatotoxicity. All cases of transaminitis resolved either by holding the drug, initiating immunosuppressants, or both, and rates of recurrent toxicity were lower in patients taking steroids when idelalisib was reinitiated. A decrease in peripheral blood regulatory T cells was seen in patients experiencing toxicity on therapy, which is consistent with an immune-mediated mechanism. These results suggest that caution should be taken as drugs within this class are developed for CLL, particularly in younger patients who have not received prior disease-specific therapy. This study was registered at www.clinicaltrials.gov as #NCT02135133., (© 2016 by The American Society of Hematology.)

POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Reduced uninvolved immunoglobulin (Ig) levels are a hallmark of multiple myeloma. We previously showed that B-cell maturation antigen (BCMA) is solubilized and at high levels in multiple myeloma patient serum. We hypothesize that soluble BCMA binds B-cell-activating factor (BAFF) preventing its function to stimulate late B cells, and would result in lower polyclonal antibody levels in these patients., Experimental Design: Mice were dosed with recombinant human BCMA (rhBCMA) and BCMA-BAFF complexes were analyzed in plasma, and its effects on antibody and Ig heavy chain mRNA levels determined. Using flow cytometry, BAFF binding to B cells was examined in the presence of rhBCMA and sera from multiple myeloma patients. In multiple myeloma sera, BCMA-BAFF complex formation and BCMA, IgA, IgG levels, and heavy-light chain isoform pair levels were determined., Results: rhBCMA-BAFF complexes formed in immune-competent and deficient mice. Mice with human multiple myeloma xenografts, which contain plasma hBCMA and hBCMA-BAFF complexes, showed reduced plasma-free BAFF levels. rhBCMA administered to immune competent mice markedly reduced plasma IgA, IgG, and IgM levels and splenic Ig heavy chain mRNA levels. In serum from multiple myeloma patients, BCMA-BAFF complexes were detected and BAFF levels were reduced. Multiple myeloma patient sera containing BCMA prevented binding of BAFF to B cells. There is an inverse correlation between serum BCMA and uninvolved polyclonal Ig level in multiple myeloma patients., Conclusions: Our results show that soluble BCMA sequesters circulating BAFF, thereby preventing it from performing its signaling to stimulate normal B-cell and plasma cell development, resulting in reduced polyclonal antibody levels in multiple myeloma patients. Clin Cancer Res; 22(13); 3383-97. ©2016 AACR., (©2016 American Association for Cancer Research.)

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., (© 2016 John Wiley & Sons Ltd.)

Background: Asymptomatic monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), are clinical conditions that usually precede symptomatic multiple myeloma. Therefore, risk stratification is crucial owing to the heterogeneous progression rate among these patients. In previous years, suppression of the uninvolved chain of specific heavy/light chain (HLC) pairs in serum has been identified as a new risk factor in MGUS. The aim of the present study was to investigate the prognostic effect of involved and uninvolved HLC pairs and HLC ratios on progression in a series of patients with MGUS and SMM., Patients and Methods: All specific serum HLC pairs were measured in 114 patients diagnosed with SMM (n = 27) and MGUS (n = 87) from 1983 to 2003. Also, the HLC ratios were calculated., Results: Progression to symptomatic multiple myeloma was observed in 13 patients (8 with SMM and 5 with MGUS). The risk of progression was 6 times greater in those with SMM (P = .001) and 4 times greater for those with the IgA isotype (P = .01). The suppression of any IgM isotypes (IgMκ or IgMλ) in patients with IgA or IgG gammopathy or any IgA isotypes (IgAκ or IgAλ) in patients with IgG or IgM gammopathy was associated with a shorter time to progression to symptomatic gammopathy (P = .001 and P = .03, respectively). On multivariate analysis, the evolving pattern and suppression of any IgM HLC pair remained significant., Conclusion: HLC ratios could be a valuable tool in the risk stratification of patients with SMM and MGUS, especially patients with IgG isotypes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The measurement of monoclonal protein (M-protein) is vital for stratifying risk and following individuals with a variety of monoclonal gammopathies. Direct measurement of the M-protein spike by electrophoresis and immunochemical measurements of specific isotypes or free light chains pairs has provided useful information about the quantity of M-protein. Nonetheless, both traditional electrophoresis and immunochemical methods give poor quantification with M-proteins smaller than 10 g/L (1 g/dL) when in the presence of polyclonal immunoglobulins that co-migrate with the M-protein. In addition, measurements by electrophoresis of M-proteins migrating in the β- and α-regions are contaminated by normal serum proteins in those regions. The most precise electrophoretic method to date for quantification involves exclusion of the polyclonal immunoglobulins by using the tangent skimming method on electropherograms, which provides a 10-fold improvement in precision. So far, however, tangent measurements are limited to γ migrating M-proteins. Another way to improve M-protein measurements is the use of capillary electrophoresis (CE). With CE, one can employ immunosubtraction to select a region of interest in the β region thereby excluding much of the normal proteins from the M-protein measurement. Recent development of an immunochemical method distinguishing heavy/light chain pairs (separately measuring IgGK and IgGL, IgAK and IgAL, and IgMK and IgML) provides measurements that could exclude polyclonal contaminants of the same heavy chain with the uninvolved light chain type. Yet, even heavy/light results contain an immeasurable quantity of polyclonal heavy/light chains of the involved isotype. Finally, use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) looms on the horizon as a means to provide more consistent and sensitive measurements of M-proteins.

Background: POEMS syndrome is defined by Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes. The vascular endothelial growth factor (VEGF) appears to play a key role in the pathogenesis of the syndrome, and its concentrations are deemed to correlate to disease activity. The aim of the present study was to verify whether other biochemical markers including serum free light chains (FLC) and heavy/light chains (HLC) would be of value in monitoring POEMS patients., Methods: Fifty-three serum samples were collected from seven POEMS patients at diagnosis and during a follow-up period (range 14-56 months). VEGF was measured using an ELISA method, while FLC and HLC concentrations were measured using Binding Site reagents on a BNII (Siemens) nephelometer., Results: At diagnosis all patients presented high VEGF concentrations, while the κ/λFLC ratio (FLCr) was within the reference range. Four patients had abnormal HLC, HLCκ/HLCλ (HLCr) and FLC values. The relationship between the trend of VEGF and both HLC and FLC during the follow-up was analysed by means of Cohen's κ coefficient. VEGF and HLC values displayed a significant κ-Cohen (0.537, p=0.002) in all chemotherapy-responder patients while in non-responders it did not. Conversely, in both responders and non-responders, VEGF and FLC values did not attain a significance on κ-Cohen analysis. In three out of four responders HLCr values increased, thus reflecting an improved clinical condition., Conclusions: The findings made in the present study indicate that HLC, either as intact immunoglobulin or as HLCr, may provide useful information, particularly in identifying responders and confirm that the role of FLC is unreliable in monitoring patients with POEMS syndrome.