Ig Light Research Articles

Chemotherapy-Based Stem Cell Mobilization Does Not Result in Significant Paraprotein Reduction in Myeloma Patients in the Era of Novel Induction Regimens

Novel induction agents markedly improved remission rates in multiple myeloma (MM), and the continued use of chemotherapy for CD34+ stem cell mobilization (SCM) has been questioned. We examined the additional effect of chemotherapy in SCM regarding remission status/morbidity. We reviewed 236 consecutive MM patients (aged 36 to 75 years) with first autologous stem cell transplantation from January 2009 to March 2016 after chemotherapy-based SCM. Responses were measured by changes in intact Ig and free light chain levels before and after chemomobilization (International Myeloma Working Group [IMWG] criteria). Most patients (225/236, 95.3%) received novel induction regimens, which were bortezomib-based (n = 223) and/or lenalidomide-based (n = 19). Most patients (170/190, 89.5%) achieved at least partial remission postinduction and pre-SCM. Stem cells were mobilized with granulocyte colony-stimulating factor and cyclophosphamide-based (212/227, 93.4%) or etoposide-based (15/227, 6.6%) regimens. There were insignificant changes in serum Ig and free light chain levels before and after chemomobilization either in the whole cohort or subgroups. Significant improvements of the IMWG remission status were documented in only 7 of 236 patients (3.0%). Sixty-seven patients (28.4%) developed chemotherapy-related complications (neutropenic fever, sepsis, and others), resulting in 9 hospitalizations (3.8%). Our study suggests that although causing significant morbidity, chemotherapy-based mobilization fails to improve remission status. The value of incorporating additional chemotherapy for SCM is thus not evident.

A murine Ig light chain transgene reveals IGKV3 gene contributions to anti-collagen types IV and II specificities

A subset of autoimmune diseases result from autoantibodies targeting epitopes on matrix collagen. The most extensively studied are anti-glomerular basement membrane glomerulonephritis (or its systemic counterpart Goodpasture’s disease) that destroys kidneys and lungs, and rheumatoid arthritis that leads to disabling arthritis. Autoantibodies in these disorders bind evolutionarily conserved conformational epitopes on the noncollagenous domain 1 (NC1) of the alpha3 chain of type IV [alpha3(IV)NC1] collagen in glomerular and alveolar basement membranes, and on native or citrullinated type II collagen (CII) in joint cartilage, respectively. The genetic origins of pathogenic anti-collagen B cells in these diseases is unknown, but observations from murine models raise the possibility that they overlap despite distinct in vivo immunopathologies. Monoclonal autoantibodies isolated from mice immunized with alpha3(IV)NC1 collagen or CII show a biased use of Ig light chains (LC) encoded by genes of the IGKV3 subgroup (previously Vk21 family), paired with diverse Ig heavy chains. To further explore this relationship and determine if a single murine IGKV3 LC independently predisposes to both anti-collagen responses, we generated a novel transgenic (Tg) C57BL/6 mouse that expresses a productively rearranged IGKV3-encoded LC, termed mLCV3-Tg, in conjunction with endogenously rearranged Ig heavy chains. Tg mice are also genetically deficient in endogenous kappa chains to permit tracking of the mLCV3 transgene. We show that mLCV3-Tg mice are susceptible to humoral autoimmunity against both collagen chains. Anti-alpha3(IV)NC1 collagen, but not anti-CII, mLCV3-encoded Ig are detected in serum of unmanipulated Tg mice, while Toll-like receptor ligands induce secretion of mLCV3-Tg autoantibodies of both collagen specificities from splenocytes ex vivo. This indicates developmental survival of mLCV3-Tg B cells reactive with each antigen, and is consistent with production of the two anti-collagen autoIg from distinct B cell populations. Reduced B cell numbers, low serum Ig kappa levels, low cell surface Ig kappa density, and abundant endogenous lambda chain expression suggest that subsets of IGKV3-encoded B cells are regulated in vivo by mechanisms that include deletion, anergy, and LC editing. These results support the notion that murine IGKV3 LCs contribute structural fitness to antigen binding sites that support diverse anti-collagen autoimmune responses, that these responses are regulated in vivo, and that these cells can nonetheless readily escape immune regulation.

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms.

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ-expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM+ cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ+ splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ+ cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ.

Ig Light Chain Precedes Heavy Chain Gene Rearrangement during Development of B Cells in Swine.

The current mammalian paradigm states that 1) rearrangements in the IgH locus precede those in IgL loci, 2) IgLλ genes rearrange only when IgLκ genes are consumed, and 3) the surrogate L chain is necessary for selection of productive IgH gene rearrangements. We show in swine that IgL rearrangements precede IgH gene rearrangements, resulting in the expression of naked IgL on a surface of precursor B cells. Findings also suggest that there is no dependency on the surrogate L chain, and thus the authentic IgL proteins may be used for selection of the IgH repertoire. Although rearrangement starts with IgLκ genes, it is rapidly replaced by IgLλ rearrangement. Fast replacement is characterized by occurrence of IgLλloIgLκlo dual-expressing precursors in which IgLκ expression is a remnant of a previous translation. Most IgLκ+ B cells are then generated later, indicating that there are two waves of IgLκ synthesis in different developmental stages with IgLλ gene rearrangements in between. In the absence of stromal cells, the stepwise order of rearrangements is blocked so that IgLλ gene rearrangements predominate in early B cell development. To our knowledge, this is the first evidence that some mammals can use an inverted order of Ig loci rearrangement. Moreover, a situation in which the generation of BCR-bearing IgLκ is delayed until after IgLλ becomes the dominant isotype may help explain the extreme deviations in the IgLκ/IgLλ ratios among mammals.

PU.1 Regulates Ig Light Chain Transcription and Rearrangement in Pre-B Cells during B Cell Development.

B cell development and Ig rearrangement are governed by cell type- and developmental stage-specific transcription factors. PU.1 and Spi-B are E26-transformation-specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)-deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B-deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation-sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igκ locus, including Vκ promoters and regions located downstream of Vκ second exons. Induction of PU.1 induced Igκ transcription and rearrangement. Upregulation of Igκ transcription was impaired in small pre-B cells from PU.1/Spi-B-deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igκ transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development.

The Prognostic Significance of Polyclonal Bone Marrow Plasma Cells in Patients with Actively Relapsing Multiple Myeloma

Background: Prior studies suggest that the presence of >5% polyclonal plasma cells (pPCs) among total plasma cells (PCs) within the bone marrow (BM) is associated with a longer progression-free survival, higher response rates, and lower frequency of high-risk cytogenetic abnormalities in patients with newly diagnosed multiple myeloma (MM). However, the incidence and prognostic utility of this factor in patients with relapsed and/or refractory MM has not been previously evaluated. Thus, we evaluated the prognostic value of quantifying the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM.Methods: We evaluated all MM patients with actively relapsing disease (biochemical and/or symptomatic) seen at the Mayo Clinic, Rochester, from 2012 to 2013, who had BM samples evaluated by seven-color multiparametric flow cytometry. All patients had at least 24 months of follow-up from the date of flow evaluation. Cell surface antigens were assessed by direct immunofluorescence antibodies for CD45, CD19, CD38, CD138, cytoplasmic Kappa and Lambda Ig light chains, and DAPI nuclear stain. The flow cytometry data was collected using the Becton Dickinson FACSCanto II instruments that analyzed 150,000 events (cells); this data was then analyzed by multi-parameter analysis using the BD FACS DIVA Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Clonal PCs were separated from pPCs based on the differential expression of CD45, CD19, DAPI (in non-diploid cases), and immunoglobulin light chains. The percentage of pPCs was calculated in total PCs detected. Survival analysis was performed by the Kaplan-Meier method and differences were assessed using the log rank test.Results: There were 180 consecutive patients with actively relapsing MM who had BM biopsies analyzed via flow cytometry as part of their routine clinical evaluation. The median age of this group was 65 years (range: 40 - 87); 52% were male. At the time of this analysis, 104 patients had died, and the 2-year overall survival (OS) rate for the cohort was 58%. The median number of therapies received was 4 (range: 1 - 15). Of these patients, 61% received a prior ASCT, and almost all (99%) received prior regimens containing either immunomodulators or proteasome inhibitors. There were 55 (30%) patients with >5% pPCs among the total PCs in their BM. The median percentage of pPCs among total PCs in these 55 patients was 33% (range: 5 - 99). The median OS for those with >5% pPCs was not reached compared with 22 months for those with <5% pPCs (P = 0.028; Figure 1). Patients with <5% pPCs PCs had a higher likelihood of high-risk FISH cytogenetics compared with the rest of the patients. In a univariate analysis, increasing number of pPCs was associated with an improved OS, while higher labeling index, number of prior therapies, and the presence of high-risk FISH cytogenetics were associated with a worse OS. In a multivariate analysis, only the increasing number of pPCs (P = 0.006), higher labeling index (P = 0.0002) and number of prior therapies (P = 0.003) retained statistical significance.Conclusion: Quantitative estimation of the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM was determined to be a predictor of worse OS. As such, this parameter is able to identify a group of patients with MM with actively relapsing disease who have a particularly poor outcome. Further studies evaluating its biological significance are warranted. [Display omitted] DisclosuresKapoor:Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Gertz:Prothena Therapeutics: Research Funding; Novartis: Research Funding; Alnylam Pharmaceuticals: Research Funding; Research to Practice: Honoraria, Speakers Bureau; Med Learning Group: Honoraria, Speakers Bureau; Celgene: Honoraria; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Ionis: Research Funding; Annexon Biosciences: Research Funding. Kumar:AbbVie: Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy.

Oxidative post-translational modifications of an amyloidogenic immunoglobulin light chain protein

Immunoglobulin light chain amyloidosis (AL) is a plasma cell disorder characterized by overproduction and deposition of monoclonal immunoglobulin (Ig) light chains (LC) or variable region fragments as amyloid fibrils in various organs and tissues. Much clinical evidence indicates that patients with AL amyloidosis sustain cardiomyocyte impairment and suffer from oxidative stress. We seek to understand the underlying biochemical pathways whose disruption or amplification during sporadic or sustained disease states leads to harmful physiological consequences and to determine the detailed structures of intermediates and products that serve as signposts for the biochemical changes and represent potential biomarkers. In this study, matrix-assisted laser desorption/ionization mass spectrometry provided extensive evidence for oxidative post-translational modifications (PTMs) of an amyloidogenic Ig LC protein from a patient with AL amyloidosis. Some of the tyrosine residues were heavily mono- or di-chlorinated. In addition, a novel oxidative conversion to a nitrile moiety was observed for many of the terminal aminomethyl groups on lysine side chains. In vitro experiments using model peptides, in-solution oxidation, and click chemistry demonstrated that hypochlorous acid produced by the myeloperoxidase – hydrogen peroxide – chloride system could be responsible for these and other, more commonly observed modifications.

B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

Molecular Methods and Bioinformatic Tools for Adjuvant Characterization by High-Throughput Sequencing.

Adjuvants in vaccine formulations are designed to enhance immune responses against a target antigen or pathogen. The ability of these vaccines to induce activation and differentiation of mature naïve B cells to produce pathogen-specific antibodies (immunoglobulins; Ig) helps guarantee long-lived humoral immunity. This process involves clonal expansion of antigen-specific B cells, genomic rearrangement of Ig heavy (IgH) and light (IgL) loci, somatic hypermutation (SHM), and clonal selection for affinity-matured antibody, resulting in a vast but directed repertoire of B cells expressing highly specific antibody proteins. High-throughput sequencing of the IgH and IgL complementary determining regions (CDRs) derived from various B cell populations provides an unprecedented way to observe dynamic responses of the humoral immune repertoire in response to vaccination. However, applying high-throughput sequencing (HTS) methodologies to multi-armed in vivo experiments requires careful coordination of sample preparation with downstream bioinformatics, particularly with regard to issues of quantitation, sequence fidelity, bar-coding, and multiplexing strategies. Here, we overview strategies of high-throughput sequencing and analysis of the adaptive immune complex loci applied to multi-armed, multiplexed experiments.

A possible serologic biomarker for maternal immune activation-associated neurodevelopmental disorders found in the rat models

Epidemiological studies have shown that maternal infection during early pregnancy increases the risk of neurodevelopmental disorders (i.e., schizophrenia or autism) in offspring. Recently, diagnostic/stratification biomarkers for the maternal immune activation background in patients with neurodevelopmental disorders have been energetically searched for in the patient blood. Here, we report a novel serologic marker candidate for the disorders found in the maternal immune activation (MIA) rat model. Serum proteome analysis of the MIA rat showed that the immunoglobulin (Ig) light chain is reproducibly augmented. The Ig light chain in sera takes two forms — free form or bound to the Ig heavy chain. Only the former is an inflammatory disease marker, but pro-inflammatory cytokine levels in the sera of the MIA rats were below detectable limits of the ELISA protocol we used. We thereby carried out serum assays of Ig light chains and pro-inflammatory cytokines of commercially available schizophrenia patient sera for research. Although the number of samples was limited, we found augmentation of free Ig light chains but not pro-inflammatory cytokines in sporadic schizophrenia patient sera. Our findings suggest that Ig light chain assay of the schizophrenia/autism patient sera would be worthy to be validated in larger scale.

Highly sensitive and unbiased approach for elucidating antibody repertoires

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice.

Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used μMT mice, which have a disruption of the first transmembrane exon of the μ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (μ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of μMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the μ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig.

Flow cytometry remission by Ig light chains ratio is a powerful marker of outcome in multiple myeloma after tandem autologous transplant: a real-life study.

BackgroundThe achievement of complete response (CR) significantly correlates with a better clinical outcome in multiple myeloma (MM) patients treated with autologous stem cell transplant (ASCT). The depth of response is one of the most relevant factors to predict patient’s outcome, however the definition of CR through standard criteria has shown several limitations.MethodsIn this study we evaluated the minimal residual disease (MRD) in 50 consecutive MM patients who underwent an up-front tandem ASCT in our center, using a single-tube six-colors flow cytometry assay (FC) based on intra-cytoplasmic immunoglobulin (cy-Ig) light chains ratio evaluated on patient-specific plasma cells (PC) immune profile, in a real-life setting.ResultsWith a sensitivity up to 10−5, clonal-PC were documented by FC in 36.4 % (12/33) of patients in conventional CR after second transplant. The number of flow MRD-negative patients significantly increased after induction and first ASCT, but not between first and second transplant. The 5-years progression-free survival (5ys-PFS) of flow MRD-negative patients after second transplant was significantly better than patients who remained MRD-positive considering both all patients (5ys-PFS: 70 % vs 5 %) and patients in CR according to standard criteria (5ys-PFS: 67 % vs 0 %).ConclusionsFC remission through cy-Ig light ratio on PC sub-populations is a sensitive, highly informative, low-cost and routinely applicable MRD assay, a powerful tool in treatment response evaluation and a crucial marker of outcome in MM.

Proteasome inhibitors: situation and prospects (literature review and own data)

Multiple myeloma is a clonal B-cell malignancy characterized by proliferation of plasma cells that accumulate mainly in bone marrow and usually secrete monoclonal Ig and/or Ig light chains. The history of therapy development in this disease has more than 50 years. After ubiquitin-proteasome system of apoptosis become apparent bortezomib has been included in the mains therapy regimes. Simultaneously, the group of proteasome-inhibitor drugs is continually developing and opening more therapeutic options in refractory or relapse forms.

Clinical Significance of Circulating Plasma Cells in Myeloma: Multiparameter Flow Cytometric Analysis

[Introduction] It has been reported that clonal circulating plasma cells (cPC) were detected in patients with multiple myeloma (MM) and other plasma cell dyscrasias (PCD), and their detection has been shown to be a risk in newly diagnosed MM and shortened progression free survival in patients smoldering MM (sMM). It is unclear that the detection of cPCs in peripheral blood may be a simple reflection of tumor burden of bone marrow plasma cells or may represent different. This study was performed to evaluate the clinical utility of quantifying clonal cPC using 6-color FCM in patients with MGUS, SMM, and symptomatic MM (symMM) at diagnosis (Dx). They were compared with bone marrow samples (BM PCs) simulatneously analyzed in the same way. Besides we evaluated the transition of quantification of clonal cPC of PB samples at various stages after treatment.[Materials and Methods] We analyzed 280 peripheral blood (PB) samples from 194 patients with PCD (MGUS, SMM, and symMM; n = 34/39/121, respectively) diagnosed at the Department of Hematology/Oncology, Kameda Medical Center and Kanazawa University Hospital from January 2012 to July 2015. Similarly, we analyzed 30 PB samples from 30 healthy controls. Mononuclear cells (MNC) were isolated from PB by density gradient centrifugation and stained with antibodies to CD45, CD38, CD19, CD138, CD56, and cytoplasmic kappa and lambda immunoglobulin (Ig) light chains. The cells were collected using a Beckman-Coulter NAVIOS and analyzed using Kaluza software. The gating strategy employed was expression of CD38, CD45, CD138, and cytoplasmic Ig light chains. Clonal cPC was defined as the population of CD138+, CD38+ CD45-~±, and CD19-. To confirm the clonality of these cells, light chain restriction of cytoplasmic Ig was used (κ/λ>4 or <0.5) using the 2nd tube. Quantification was performed by acquiring > 2×106 total events. At least ≥ 20 clonal cPC were required for the analysis.[Results] We analyzed clonal cPC of 194 PB samples with MGUS, SMM, and symMM (n = 34/39/121, respectively) at diagnosis (Dx). The mean values of clonal cPC were: MGUS, 2.67×10-5/MNC (range, 0 - 3.0×10-4); SMM, 2.43×10-4/MNC (range, 0 - 3.6×10-3); symMM, 6.99×10-3/MNC (range, 0 - 1.5×10-1). The number of clonal cPC was significantly higher in SMM than MGUS, and in symMM than SMM (P < 0.001). In healthy controls, the mean value of cPC without CD19 expression was 3.84×10-6/MNC (range, 0 - 1.3×10-5). Although these cPC lacked the expression of CD19, none of the samples did not show the light chain restriction of cytoplasmic Ig. Next, we analyzed clonal cPC of 185 PB samples (Dx, n = 85; partial response [PR], n = 31; very good partial response [VGPR], n = 25; complete response [CR], n = 22; progressive disease [PD], n = 23) in various treatment responses of symMM. The mean values of clonal cPC were: at Dx, 6.99×10-3/MNC (range, 0 - 1.5×10-1); at PR, 5.19×10-5/MNC (range, 0 - 3.5×10-4); at VGPR, 9.79×10-5/MNC (range, 0 - 9.7×10-4); at CR, 1.90×10-5/MNC (range, 0 - 1.2×10-4); at PD, 1.94×10-3/MNC (range, 0 - 3.9×10-2). The number of clonal cPC was significantly higher at Dx than from PR to CR, and significantly lower in CR than from PR to VGPR (P < 0.01, P = 0.23, respectively). However, the number of clonal cPC was not significantly different between PR and VGPR (P = 0.61). When patients with PCD at Dx were divided into high and low clonal cPC groups (high cPC ≥ 1.0x10-4/MNC and low cPC < 1.0x10-3/MNC), the number of the high cPC group was as follows: (MGUS 2/35 (5.7%), SMM 9/38 (23.7%), symMM 35/85 (41.2%)) (P<0.01). In symMM patients, clinical characteristics were comparable between the two groups except for bone marrow PCs. A total of 133 bone marrow samples (BM PCs) were measured simultaneously with PB samples at Dx. The number of clonal cPC tended to be related to the percentage of clonal plasma cells/total plasma cells in BM in symMM (R = 0.579), but no such relation was observed in MGUS and SMM (R = 0.112, -0.03).[Conclusions] In conclusion, quantification of clonal cPC by 6-color FCM is feasible and appears to reflect the tumor burden in patients with newly diagnosed MM and sMM, but not in patients after treatment. Further studies using quantification of clonal cPC combined with other hematological parameters will identify patients at high risk of early progression and poor prognosis. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MYC/BCL2 Double Hit Lymphoma: What Really Matters

Introduction: MYC/BCL2 double hit lymphoma (DHL), defined as a large B-cell lymphoma with concurrent MYC and BCL2 translocations, is the most common type of DHL. Although multiple studies focused on DHL have been published, several issues regarding impact on prognosis remain controversial including: 1) history of low grade B cell lymphoma; 2) morphology (diffuse large B-cell lymphoma [DLBCL] versus B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma [BCLU)]; 3) Absence or low expression of MYC or BCL2; 4) MYC translocation partner gene; and especially 5) most effective therapy. The aim of this study was to attempt clarify the prognostic importance of these factors in DHL.Methods: 157 patientsdiagnosed with MYC/BCL2 DHL between 2003 and April 2015 at two institutions were included in this study. MYC and BCL2 gene rearrangement were confirmed by FISH using a MYC breakapart probe and BCL2 and IGH dual color dual fusion probes. BCL6/3q27gene status was tested either by FISH using breakapart probe or by karyotype. MYC partner gene was identified by karyotype. MYC/BCL2 DHL cases were identified if they had rearrangements of MYC and BCL2 but not BCL6. Positive for MYC or BCL2 by immunohistochemistry was defined by >40% and >50% of lymphoma cells showed positive expression, respectively. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Fisher's exact test was used to compare the clinicopathologic features. Statistical analysis was performed using SPSS 23 software.Results: There were 103 men and 54 women with a median age of 61 years (range, 18-87). 110 patients had de novo disease and 47 patients had a history of low-grade B-cell NHL, mostly follicular lymphoma. The clinicopathologic features were similar (P>0.05) between patients with a history of low-grade B-cell NHL and patients with de novo NHL, and therefore analysis was performed on all 157 DHL cases. Using the 2008 WHO classification, there were 91 DLBCL, 61 BCLU, and 5 composite lymphoma (4 DLBCL + follicular lymphoma and 1 DLBCL + B-lymphoblastic lymphoma). 99% of cases had a germinal center B-cell immunophenotype by the Hans algorithm. MYC expression was observed in 39/47 (83%) and BCL2 in 129/141(91%) of cases. MYC and BCL2 dual expression was present in 34/46(74%) cases. Of the 39 cases assessed, the MYC translocation partner was IGH in 13, IG light chain in 19, and a non-IG gene in 7 cases. 144 patients had complete treatment information: 61 received the R-CHOP regimen initially, 31 R-EPOCH, 29 R-HCVAD, and 23 various other chemotherapy regimens. 39 patients also received stem cell transplant (SCT) including 31 autologous and 8 allogeneic. 62 patients reached complete remission (CR) after initial chemotherapy. The median overall survival was 19 months. In a univariate analysis that evaluated 17 clinicopathologic parameters including those mentioned in introduction, extranodal sites of disease, bone marrow involvement, CNS involvement, advanced stage, and high/high-intermediate International Prognostic Index score were associated with a worse overall survival (OS, P<0.05), whereas CR and SCT were associated with a better OS (P<0.05). By multivariate analysis, stage, IPI, SCT, and CR predicted OS (Table 1 and Figure 1).Conclusion: MYC/BCL2 DHL are clinically aggressive B cell lymphomas with a germinal center phenotype and patients have a poor prognosis. In this study, a history of low grade B cell lymphoma, morphology (DLBCL vs BCLU), MYC expression, BCL2 expression, MYC/BCL2 dual expression (double expresser lymphoma), MYC translocation partner gene, and initial chemotherapy regimens were not associated with overall survival. The independent prognostic factors shown by multivariate analysis were IPI score, stage, achievement of CR, and a therapeutic regimen including SCT.Table 1Multivariate analysisFeaturesHR95% CIPBM involvement0.920.48 - 1.800.817Extranodal sites ≥ 21.120.48 - 2.650.793Stage III /IV vs I/II34.454.92 - 241.31<0.001IPI (H/H-I vs L/L-I)3.001.14 - 7.770.026Initial ChemotherapyR-EPOCH vs R-CHOP1.170.49 - 2.840.723R-HCVAD vs R-CHOP0.950.47 - 1.930.894Stem cell transplant0.220.10 - 0.46<0.001CR after initial chemotherapy0.140.07 - 0.30<0.001 [Display omitted] DisclosuresReddy:ImmunoGen: Consultancy; PCYC: Consultancy; Gilead: Other: Speaker; Seattle Genetics: Consultancy; Celgene: Consultancy, Research Funding.

Crystal-storing histiocytosis: a clinicopathological study of 13 cases.

Crystal-storing histiocytosis (CSH) is a rare lesion composed of histiocytes with abnormal intralysosomal accumulation of immunoglobulin (Ig) as crystals, reported in patients with plasmacytic/lymphoplasmacytic neoplasms. The aims of this study were to report the clinicopathological features of 13 patients with CSH, and to describe the proteomic composition of the crystals in three cases analysed by mass spectrometry (MS). There were seven men and six women, with a median age of 60years (range, 33-79years). CSH was generalized in one patient (8%) and localized in 12 (92%) patients, involving various sites. CSH was associated with a low-grade B-cell lymphoma with plasmacytoid differentiation or a plasma cell neoplasm in all cases. In 10 (77%) cases, CSH represented >50% of the neoplastic infiltrate. According to immunohistochemical studies, histiocytes were positive for monotypic kappa in 5 (50%) cases, and for monotypic lambda in 4 (40%) cases; in 1 (10%) case, the results were equivocal. MS analysis of the histiocyte contents in all three tested cases showed a predominance of variable-region fragments of Ig light and/or heavy chains. CSH is frequently associated with an underlying lymphoplasmacytic neoplasm. MS findings suggest that Ig alterations and/or possibly defects in the ability of histiocytes to process Ig play a role in pathogenesis.

DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF INTRATHECAL SYNTHESIS OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN MULTIPLE SCLEROSIS

Increased intrathecal synthesis and oligoclonal banding of immunoglobulins (Ig) in cerebrospinal fluid (CSF) are major immunological findings in multiple sclerosis. Free light chains are fragments of Ig molecules that are produced in parallel to oligoclonal Ig, and their production reflects total Ig synthesis within central nervous system. Assessment of intrathecal kappaand lambda free light chain production within CNS compartment will help to improve diagnosis and prediction of outcomes in multiple sclerosis. Concentrations of kappa and lambda free light chains and clonality of immunoglobulin pattern synthesis were studied in paired CSF and serum samples of 151 patients, including 92 patients with multiple sclerosis, and 33 patients with clinically isolated syndrome which later was transformed into the definite multiple sclerosis. A control group consisted of 26 patients with other inflammatory diseases of central nervous system. Several Ig parameters have been determined in the patients, i.e., Ig clonality in CSF; concentrations of free light chains (both kappaand lambda-) in CSF; as well as their indexes and ratios. It was established that synthesis of kappa free light chains were significantly elevated in patients with multiple sclerosis. Moreover, the amounts of free light Ig chains in patients with positive oligoclonality test were significantly higher than in cases without intrathecal oligoclonal synthesis. With respect to diagnostic significance, the kappa quotient proved the best available option for diagnosis of multiple sclerosis. Its combination with Ig oligoclonality assays caused a decrease in false-negative diagnostics by 42%. Diagnostic benefit of the kappa quotient could be also confirmed by the data of ROC analysis. Also concentration of lambda free light chains in cerebrospinal fluid showed a negative correlation with conversion terms of clinically isolated syndrome to evident multiple sclerosis. Therefore, the indexes of free Ig light chains enables more precise diagnostics, like as more efficient severity evaluation of multiple sclerosis.

The Reversal of Renal Impairment and its Impact on Survival in Newly Diagnosed Multiple Myeloma Patients

Background: Proliferation rate of plasma cells (PC) is a powerful tool in evaluating all types of monoclonal gammopathies, predicting for progression as well as survival. A flow cytometry (FC)-based method to delineate the cell cycle of clonotypic PCs and determine the proportion in S-phase was developed. This method replaces a labor-intensive slide based methodology, but the prognostic power of this FC approach has not been clearly elucidated. Methods: A total of 1061 patients who had bone marrow (BM) FC PC proliferation analysis at Mayo Clinic, Rochester from May 2012 to December 2013 were studied of who 257 had relapsed disease (RMM). PC proliferation was measured by 8-color FC using antibodies to CD19, CD38, CD138, CD45, and cytoplasmic kappa and lambda Ig light chains, and DAPI DNA staining. The DNA content of the abnormal, clonotypic PCs was specifically analyzed and the %S-phase was determined by measuring the proportion of cells between G0/G1 and G2/M. Patients with inadequate plasma cells for assessment of %SP were designated as 0%. Results: The median follow up from testing for the entire cohort was 13 months (95% CI; 12.7, 13.3); 156 (15%) had died. We specifically examined the clinical features and outcomes based on two cutoffs, %SP 1 and 2. Among relapsed disease, %SP was prognostic for OS using both cutoffs (Table). In univariate analysis, %SP 2, B2microglobulin >5.5, HR FISH (t4; 14, t14;16, t14;20 & del17p) and LDH>221 were all significant, but only %SP 2 was significant in multivariable analysis. Conclusion: The %S-phase of clonal PCs determined by FC is of prognostic value in patients with RMM. This is particularly relevant because it is independent of some of the other known poor prognostic factors such as high risk FISH.

Isolation of Dengue neutralizing antibodies from single cell sorted human antigen specific memory B cell cultures (VAC11P.1107)

Abstract Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here we describe a method to develop a direct flow cytometric assay for the detection of Dengue envelope-specific memory B cells using a Dengue envelope specific mouse hybridoma line. We used this flow cytometry assay and a cultured B ELISPOT assay to enumerate Dengue-envelope specific B memory cells from a cohort of Dengue seropositive donors. Furthermore, we were able to culture the single sorted Dengue-envelope specific memory B-cells and differentiate them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them recombinantly as full length antibodies to reproduce the activity seen in culture supernatants. In conclusion, we have established a robust methodology to enumerate antigen-specific memory B-cells and assay their encoded antibodies for functional characterization.