Light Chain Isotype Research Articles

Phylogeny, genomic organization and expression of λ and κ immunoglobulin light chain genes in a reptile, Anolis carolinensis

The reptiles are the last major taxon of jawed vertebrates in which immunoglobulin light chain isotypes have not been well characterized. Using the recently released genome sequencing data, we show in this study that the reptile Anolis carolinensis expresses both lambda and kappa light chain genes. The genomic organization of both gene loci is structurally similar to their respective counterparts in mammals. The identified lambda locus contains three constant region genes each preceded by a joining gene segment, and a total of 37 variable gene segments. In contrast, the kappa locus contains only a single constant region gene, and two joining gene segments with a single family of 14 variable gene segments located upstream. Analysis of junctions of the recombined VJ transcripts reveals a paucity of N and P nucleotides in both expressed lambda and kappa sequences. These results help us to understand the generation of the immunoglobulin repertoire in reptiles and immunoglobulin evolution in vertebrates.

Eight Colors Flow Cytometry Phenotyping for Blood Minimal Residual Disease Monitoring in Hairy Cell Leukaemia Patients.

Abstract 1609Poster Board I-635Purine analogues (2-chlorodeoxyadenosine: 2-CdA and 2'-deoxycoformycin: dCF) significantly improved the complete response (CR) rate and the overall survival of hairy cell leukaemia (HCL) but are usually not curative with about 40% relapse rate. We developed an 8 colors flow cytometry (8-FC) technique (CANTO IITM, BD Biosciences) to follow the blood minimal residual disease (MRD) and evaluated its clinical relevance. 34 patients diagnosed with HCL at the Necker University Hospital (France) were retrospectively evaluated on frozen blood samples taken at diagnosis to select the relevant markers for MRD analysis. MRD follow-up (106 samples) using 8-FC was compared to IgH PCR clonality, using consensus qualitative IgH FR1 and FR2 PCR and Genscan revelation, performed on the same samples.Several antibodies were tested. The phenotype of hairy cells (HC) appeared to be heterogeneous from one patient to another. After a first identification of HC on the basis of CD45high, CD3 negativity, CD19/CD20/CD103/CD25 (moderate to high) positivity and light chain isotype restriction, we tested the expression of CD123 and CD305 (LAIR-1). They were expressed in all cases with high intensity as defined by the mean fluorescence intensity ratio (MFIR) calculated by dividing the MFI of each marker by that of the isotypic control. They could distinct HC from normal B cells in 82% and 78% whereas CD103 and CD25 were expressed with a lesser intensity and were not relevant for MRD analysis in 30% and 22%. Therefore, we have chosen the 8 antibodies combination as follows: CD103FITC/CD305PE/CD19Percp Cy5.5/CD123APC/CD25PC7/CD3Alexa700/CD45Amcyan/CD20Pacific Blue which discriminates HC in 100%. This combination was used to define in all 34 patients a “hairy cell associated phenotype” based on the expression of at least 2 HCL associated markers. The specificity was assessed on 10 normal blood samples and Our 8-FC combination appeared very specific with a robust sensitivity of at least 1.10-4. The comparative analysis of blood CF versus molecular MRD analyses showed an overall concordance of 81%: 52 samples (49%) being negative and 35 samples (33%) being positive by both techniques. 19 samples (18%) were discordant. In all these cases, CF MRD was positive (levels ranging from 10-4 to 10-2) but IgH-PCR appeared polyclonal. These data demonstrate the higher sensibility of CF as already shown by others.We then looked for a clinical relevance of FC MRD follow-up in a cohort of 18 patients with normal blood cell counts after 2-CdA therapy (5 in first line and 13 after 2 to 8 previous therapies including purine analogues, splenectomy, interferon-á and others). A median of 4 samples by patient were tested and the median follow-up time was 44 months [18-84]. Two groups could be distinguished. (A) Patients (n=13) with a sustained negative FC MRD (<10-4). Nine patients were negative from the first sample taken at around 6 months post 2-CdA. A FC MRD relapse was observed in three of them at 32, 46 and 72 months after therapy. Four patients were positive for the first sample but became negative at around 1 year post 2-CdA and remained negative after a follow-up of at least 18 months. (B) Patients (n=5) remaining FC MRD positive (median of 3 samples). Three were treated for haematological relapse at 40, 48 and 49 months from 2-CdA therapy. Two kept normal blood cell counts after a follow-up of 38 and 60 months.These data suggest that the risk of relapse after 2-CdA therapy is lower in case of negative blood FC MRD (<10-4) obtained in the 12 months following therapy than in patients with sustained positive MRD (≥10-4). This should lead, at least, to a closer haematological and FC follow-up of the positive patients. A prolonged follow-up is required to determine if such patients would benefit from a complementary therapy. Finally, we propose modified NCI criteria of response as follows in table 1.Table 1Modified NCI criteria of response. Abbreviations: N normal, PR partial response, VGPR very good PR. Progressive disease is the reappearance or exacerbation of disease-related signs (clinical +/- biological) after achieving any kind of response and relapse is the need for re-treatment.CRVGPRPRHCL-related symptoms000Organomegaly00Decrease ≥ 50%Blood cell countsNN- Correction ≥ 1 cytopenia - No decrease in any cell countHC on blood smear000Blood FC MRD-++ DisclosuresNo relevant conflicts of interest to declare.

Triple MEL100 Therapy in Multiple Myeloma

Background Tandem high-dose melphalan therapy with autologous peripheral stem cell support has emerged as the standard of care for patients without prohibitive comorbidities. Mucositis and gastrointestinal side effects are the most common extrahematologic side effects. Two previously published studies presented a triple transplant with a conditioning regimen of melphalan 100 mg/m 2 (MEL100) with peripheral stem cell support every 2 to 5 months for patients with prohibitive comorbidities for high-dose tandem transplantation. We present a novel approach that investigates the triple melphalan 100/m 2 approach on a dose-dense, every-3-weeks schedule in a patient population without significant comorbidities. Patients and methods Thirteen standard or high-risk patients with stage III multiple myeloma were prospectively treated. This population contained eight patients with immunoglobin G clonality, three immunoglobin A, one nonsecretory, and one light chain isotype. The induction regimens of the 13 patients were heterogenous and included five VAD, three DCIE, two Thal/Dex, two CIE, and one pulse decadron. Patients underwent peripheral blood leukopheresis, and these cells were divided into three equal sets and frozen. The patients were scheduled to receive melphalan at 100 mg/m 2 on days 1, 20, and 41, and then the autologous infusions occurred at days 0, 21, and 42. Results All patients were able to receive all three cycles of the MEL100 regimen. Seven patients (54%) received the treatments on the every-3-weeks schedule; three treatments (23%) during the second cycle and six treatments (46%) of the third cycle had to be delayed a median of 6 and 4 days, respectively. Three patients were managed completely in the outpatient setting, and the average total hospital stay for the three transplants was 18 days. Median progression-free survival was 854 days (range 73 to 1571), and the overall survival of this cohort has yet to be reached. No patient had worse than grade II mucositis, and no serious adverse events were recorded. Conclusion Our regimen of three consecutive autologous peripheral stem cell transplants with a reduced dose of melphalan at 100 mg/m 2 given every 3 weeks was very well tolerated. The progression-free survival and overall survival are similar and can be compared favorably with the standard tandem myeloma regimens. Our data is intriguing, and further studies with larger numbers need to be performed to confirm these results.

Proliferative glomerulonephritis with monoclonal IgG deposits.

Dysproteinemias that result in monoclonal glomerular deposits of IgG are relatively uncommon. Here, we report the largest series of proliferative glomerulonephritis with monoclonal IgG deposits, a form of renal involvement by monoclonal gammopathy that mimics immune-complex glomerulonephritis. We retrospectively identified 37 patients, most of whom were white (81%), female (62%), or older than 50 yr (65%). At presentation, 49% had nephrotic syndrome, 68% had renal insufficiency, and 77% had hematuria. In 30% of the patients, we identified a monoclonal serum protein with the same heavy- and light-chain isotypes as the glomerular deposits (mostly IgG1 or IgG2), but only one patient had myeloma. Histologic patterns were predominantly membranoproliferative (57%) or endocapillary proliferative (35%) with membranous features. Electron microscopy revealed granular, nonorganized deposits, and immunofluorescence demonstrated glomerular deposits that stained for a single light-chain isotype and a single heavy-chain subtype, most commonly IgG3kappa (53%). During an average of 30.3 mo of follow-up for 32 patients with available data, 38% had complete or partial recovery, 38% had persistent renal dysfunction, and 22% progressed to ESRD. Correlates of ESRD on univariate analysis were higher creatinine at biopsy, percentage of glomerulosclerosis, and degree of interstitial fibrosis but not immunomodulatory treatment or presence of a monoclonal spike. On multivariate analysis, higher percentage of glomerulosclerosis was the only independent predictor of ESRD. Only one patient lacking a monoclonal spike at presentation subsequently developed a monoclonal spike and no patient with a monoclonal spike at presentation subsequently developed a hematologic malignancy. We conclude that proliferative glomerulonephritis with monoclonal IgG deposits does not seem to be a precursor of myeloma in the vast majority of patients.

IgD+ cell populations in channel catfish, Ictalurus punctatus (81.8)

Abstract Only two immunoglobulin (Ig) molecules, IgM and IgD have been identified in the channel catfish. While IgM has been extensively studied and in many aspects resembles mammalian IgM, catfish IgD is quite different. It is a chimeric molecule consisting of a rearranged VDJ region spliced to the Cμ1 domain, followed by seven Cδ domains and either a transmembrane or secreted tail. Through the use of anti-IgM and anti-IgD monoclonal antibodies three different B cell populations were identified that differentially express IgM and IgD; IgM+/IgD-, IgM+/IgD+ and IgM-/IgD+. PCR and RACE protocols amplified appropriate Ig rearranged message from these sorted populations indicating that they contained functional B cells. Furthermore, co-immunoprecipitation, Western blot analyses, and RT-PCR showed that IgD in the IgM-/IgD+ sorted cell population preferentially associates with the catfish Ig light (L) chain isotype sigma (IgL σ?. In contrast, the IgM+/IgD-, IgM+/IgD+ B cell populations express all four of the catfish IgL isotypes, IgLσ, IgLλ??and the IgL¿? which includes F and G IgL chains. In addition a third IgD+ cell population was identified consisting of IgM-/IgD+ granular-like cells of unknown origin, which do not express IgD message, and thus appear to be armed with exogenous IgD via a putative IgD binding receptor. Supported by NIH grant R01 AI19530 and UMMC IRSP 59908.

Immunoglobulin isotypes in multiple myeloma: laboratory correlates and prognostic implications in total therapy protocols

Immunoglobulin isotypes in multiple myeloma: laboratory correlates and prognostic implications in total therapy protocols

N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-K1SV cells. The glycans detected on the Fc fragment were mainly of the core-fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non-core-fucosylation between the 105 different cell lines, suggesting clone-to-clone variation. These differences may change the Fc-mediated effector functions by such antibodies. Large variation was also observed in the oligomannose-5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed-batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone-to-clone glycosylation variation but batch-to-batch consistency provides a rationale for selection of optimal production cell lines for large-scale manufacturing of biopharmaceutical human IgG.

The T(14;20)(q32;q12) : A Rare Cytogenetic Change in Multiple Myeloma Associated with Poor Outcome

Introduction: Aberrations involving immunoglobulin genes (Ig) are recurrent in Multiple Myeloma (MM) and carry prognostic significance. One of them, the t(14;20) (q32;q12) leading to overexpression of the transcription factor MAFB has been identified in myeloma cell lines, rarely in MM, and never in MGUS. The clinical significance of this cryptic aberration remains poorly unknown. The aim of the present study was to assess the frequency and prognostic value of this chromosomal change.Materials and Methods: A series of 1200 samples from patients with MM or MGUS were assessed by interphase FISH either on immunologically identified kappa/lambda positive plasmacells (I-FISH) or on CD138 purified plasmacells (PCs). Cases selected were consecutive cases sent for routine cytogenetic analysis in which a translocation involving Ig was demonstrated by FISH (split signal with the Breakapart probe, Abbott) and was shown to be distinct from t(11;14), t(4;14), t(14;16) and t(6;14) (using specific probes), respectively. A double color interphase FISH assay was applied for the detection of the t(14;20), using a set of BAC probes located on 20q11-q12: RP4-616B8 and RP3-404H4 (centromeric to MAFB, labelled in Spectrum Orange) and RP4-644L1 and RP1-94E24 (telomeric to MAFB, labelled in Spectrum Green).Results: A characteristic split signal, was observed in 10 patients (0.8%), 8 females and 2 males, aged 49–83 years (median 69). All but 2 of them suffered from MM in advanced stage (Durie and Salmon stage II in 2 and stage III in 6 patients) and displayed multiple bone lesions. The light chain isotype was kappa in 4, lambda in 3, unknown in 1. Two cases were non secreting (but showed kappa intracytoplasmic expression). The heavy chain isotype was IgG in 6, and IgA in 1. Therapy was required in all but 1 case (1 line in 1, 2 lines in 3, and 3 lines in 5 patients, respectively). The front line consisted of VAD (n=5) followed by high dose Melphalan and autologous stem cell transplantation (ASCT)(n=4), among these, 1 had a double AST and one had a reduced intensity conditioning regimen allogeneic transplantation), Melphalan-Prednisone (MP) (n=1) and MP+Thalidomide or Lenalidomide or Bortezomib (n=3). All but 1 cases treated with VAD showed response (at least PR) which persisted at least transiently after high dose therapy (2 cases relapsed after achieving response). Two patients treated upfront with MP associated with one of the new drugs responded to this regimen. The patient treated with MP alone did not respond, was subsequently refractory to Thalidomide+Dexamethasone, but responded to Bortezomib. After a median follow-up period of 10,5 months (range 1 months – 10 years), 7 patients are alive (1 with progressive disease) and 3 died because of progressive disease, 2, 19 and 120 months after diagnosis, respectively. Conventional cytogenetic analysis showed clonal aberrations in 2 cases (hypodiploid karyotypes with complex changes). FISH analysis showed a balanced t(14;20) in all cases. The second IgH allele was intact in 9 patients and was involved (subclonally) in a t(7;14)(q31;q32) in 1 patient. Associated changes were: loss of 13q in 8, and loss of 17p13 in 1 cases. In conclusion, the t(14;20)(q32;q12) is a rare change in MM, and is associated with hypodiploidy (i.e. loss of 13q), advanced stage and poor outcome.

Staging multiple myeloma patients with active disease using serum levels of β2m-free HLA class I heavy chain together with IgM or platelet count

Purpose: In multiple myeloma (MM), serum beta-2-microglobulin (β2m)-free heavy chains (FHC) of HLA class I has been shown to reflect disease activity. We investigated the possibility of stratifying patients with active disease according to FHC and other clinical parameters. Experimental design: We studied 146 patients with MM, including 100 at diagnosis, 31 in relapse and 15 unresponsive to therapy. Univariate and multivariate analyses were used to assess the prognostic significance of FHC together with continuous variables (age, albumin, creatinine, hemoglobin, erythrocyte sedimentation rate, β2m, calcium, IgM, platelet count) and categorical variables (Durie–Salmon disease stage, gender, bone lesion burden, heavy and light chain isotypes of M-component, clinical status). Survival tree analysis on significant variables was used to develop an MM staging system. Results: FHC, IgM, platelet count and hemoglobin were independent predictors of prognosis. Survival tree analysis of these variables defined 2 three-risk-group staging systems involving FHC and either IgM or platelet count. Median survival for FHC/IgM stages II and III was 41.5 and 27.8 months, whereas it was not reached for stage I patients ( p < 0.0001). In the FHC/platelets system, median survival was 93.2 (stage I), 44.1 (stage II) or 27.8 (stage III) months ( p < 0.0001). Similar results were obtained for the 117 MM patients without renal insufficiency (FHC/IgM p < 0.0001; FHC/platelets p = 0.001). For the 100 patients at diagnosis, FHC/IgM ( p = 0.001) was more effective than FHC/platelets ( p = 0.04). Conclusions: The independent prognostic markers FHC, IgM and platelets provide two staging systems unaffected by renal insufficiency. Both are effective in evaluating MM patients with active disease.

First thalidomide clinical trial in multiple myeloma: a decade

AbstractThe clinical outcomes of 169 patients enrolled in the first clinical trial of thalidomide for advanced or refractory myeloma are updated. Seventeen patients remain alive and 10 are event-free, with a median follow-up of 9.2 years. According to multivariate analysis of pretreatment variables, cytogenetic abnormalities, present in 47% of patients within 3 months of enrollment, and λ light chain isotype both affected overall survival and event-free survival adversely. Forty percent of the 58 patients lacking these 2 unfavorable features, one-half of whom had no disease recurrence, survived at least 6 years, in contrast to fewer than 5% among those with 1 or 2 risk features (P < .001). Patients who had received cumulative thalidomide doses in excess of 42 g in the first 3 months enjoyed superior overall and event-free survival. The poor outcome associated with λ-type myeloma may relate to its overrepresentation in molecularly defined high-risk disease gleaned from studies in newly diagnosed myeloma.

Immunochemical characterization of human antibodies to lymphobiastoid interferon

Antibodies produced in recurrent respiratory papillomatosis (RRP) patients treated with lymphoblastoid interferon (lyIFN) may neutralize antiviral activity or may only bind to lyIFN. These antibodies were characterized for immunoglobulin class, IgG subclass, and light chain type by an indirect immunoassay. Serum dilutions were incubated on lyIFN-coated plates and the presence of antibody detected using peroxidase-conjugated goat antibodies to each human immunoglobulin class and light chain isotype, or using MoAbs to each human IgG subclass. Neutralizing activity was measured as the inhibition of lyIFN antiviral activity for Vervet monkey cells challenged with Semliki Forest Virus. Among antibody-positive patients, 12% produced IgM coincident with IgG, and 25% produced IgA coincident with IgG. Thus, antibody responses in patients treated with lyIFN are not exclusively of IgG class. The predominant lyIFN-specific subclasses were IgG1 and IgG3, which occurred in 70% and 83% of patients, respectively. An IgG4 response was detected in two patients who also had antibody of other isotypes; no IgG2 antibody was detected in any patient. Antibodies were not IgG subclass-restricted, a trend which was more pronounced in patients having neutralizing antibody than non-neutralizing antibody. Light chain molecules of lyIFN-specific antibody were of both kappa and lambda isotypes, with kappa chains occurring most frequently. Among patients having non-neutralizing antibodies, monotypic light chains occurred in 65% of the patients, whereas no patient with neutralizing antibody had monotypic light chain antibody. Sera from 599 normal human volunteers were assayed for antibody, and seven were found to be immunoreactive to lyIFN. Only one serum of the seven was positive for neutralizing activity.

Immunoglobulin light chain isotypes in the teleost Trematomus bernacchii

Three immunoglobulin light chain (IgL) isotypes TrbeL1, TrbeL2, and TrbeL3 were identified in the Antarctic teleost Trematomus bernacchii by immunoscreening a cDNA expression library, and using RT-PCR, and 5′ RACE. One of them was distinguished in two subisotypes TrbeL1A and TrbeL1B. Real-time PCR experiments showed that the different isotypes were expressed in similar ratios in the various tissues analyzed. Interestingly, the expression level of TrbeL1A isotype was very high in all tissues. Molecular models of the CH1–CL domain pairings were built and minimized for the different isotypes. Several differences were identified in the superimposable structures mainly in the loops. In addition, the isotype-specific residues determined a different distribution of the charges on the external CL domain surface. Phylogenetic trees of 43 isotype representative sequences of CL domain from teleost species, built by different methods, indicated that all teleost light chain isotypes are distributed into three groups. Furthermore, the split of the group IgL1 into two subgroups, one of them carrying a micro-satellite DNA insertion, may have occurred in the Acanthopterygean ancestor.

Syndrome POEMS

The POEMS syndrome combines a constant polyneuropathy (P), organomegaly (O), endocrinopathy (E), monoclonal gammopathy (M) (or other plasma cell disorder) and skin changes (S). Other manifestations may be observed: anasarca, fever, sweating, clubbed fingers, renal damage, arterial obstruction, pulmonary hypertension, thrombocytosis, and polycythemia. Its pathogenesis is not well elucidated but elevated levels of vascular endothelial growth factor (VEGF) appear to characterize it. Consistent plasma cell disorders include a monoclonal component, often in small quantities with a lambda light chain isotype, and plasmacytoma, often solitary lesions. Treatment depends on specific characteristics of the disease and the patient (radiation therapy for plasmocytoma, autologous bone marrow transplantation in young subjects, corticosteroid therapy or chemotherapy in the elderly). The usefulness of thalidomide and bevacuzimab in refractory POEMS syndromes remains to be seen.

Four primordial immunoglobulin light chain isotypes, including λ and κ, identified in the most primitive living jawed vertebrates

The discovery of a fourth immunoglobulin (Ig) light (L) chain isotype in sharks has revealed the origins and natural history of all vertebrate L chains. Phylogenetic comparisons have established orthology between this new shark L chain and the unique Xenopus L chain isotype sigma. More importantly, inclusion of this new L chain family in phylogenetic analyses showed that all vertebrate L chains can be categorized into four ancestral clans originating prior to the emergence of cartilaginous fish: one restricted to elasmobranchs (sigma-cart/type I), one found in all cold-blooded vertebrates (sigma/teleost type 2/elasmobranch type IV), one in all groups except bony fish (lambda/elasmobranch type II), and one in all groups except birds (kappa/elasmobranch type III/teleost type 1 and 3). All four of these primordial L chain isotypes (sigma, sigma-cart, lambda and kappa) have maintained separate V region identities since their emergence at least 450 million years ago, suggestive of an ancient physiological distinction of the L chains. We suggest that, based upon unique, discrete sizes of complementarity determining regions 1 and 2 and other features of the V region sequences, the different L chain isotypes arose to provide different functional conformations in the Ig binding site when they pair with heavy chains.

Effect of fiber type on postmortem proteolysis in longissimus muscle of Landrace and Korean native black pigs

The current study was conducted to characterize objective meat quality, fiber type and their relations to postmortem proteolysis in longissimus muscle of Landrace and Korean native black (KNP) pigs. Longissimus muscles from each 10 market-weighted male pigs were removed after conventional slaughtering and chilling procedures, and aged for 1 or 7 days at 4 °C to determine WB-shear force, objective meat color, proportion of myosin heavy chain I (MyHC I), intramuscular fat content and rate of proteolysis by a proteomics approach. KNP had a significantly ( p < 0.05) higher content of MyHC I, and that concurred with greatly ( p < 0.05) higher intramuscular fat content and Hunter a ∗ value, and significantly ( p < 0.05) lower drip loss than those seen in Landrace. One-dimension SDS-PAGE indicated that GAPDH, troponin I and creatine kinase were prominent proteolytic products during chiller ageing. By applying a gel-based proteome analysis, 26 proteins were identified, which showed different degradation properties during ageing between the breeds. Biopsied sample revealed that myosin regulatory light chain 2, myosin light chain isotype v/sb, fatty acid-binding protein and albumin were expressed at a greatly higher level for KNP, but their relation to fiber type (or genetic background) is unclear. It was particular noticeable that different actin isoforms showed various degradation behavior during ageing time.

The Treatment of Relapsed and Refractory Multiple Myeloma

Relapsed and refractory multiple myeloma (MM) constitutes a specific and unmet medical need. Median survival ranges from as little as 6 to 9 months, and responses to treatment are characteristically short. Patients with relapsed/refractory disease are defined as those who, having achieved minor response or better, relapse and then progress while on salvage therapy, or experience progression within 60 days of their last therapy. In the era prior to the development of novel biologically based therapies for MM, relapse from successive treatment regimens resulted in progressively shorter response durations, which typically reflected emerging drug resistance, as well as changes in disease biology within each patient, with tumor cells expressing a more aggressive phenotype, higher proliferative fraction and lower apoptotic rates. Both bortezomid- and lenalidomide-based therapies are expecially active, with bortezomib in particular being shown to provide a platform for combinations able to overcome resistance in this setting. The addition of novel and conventional agents to the treatment backbone of lenalidomide, thalidomide, and bortezomib are areas of active study, with participation in clincial trials a clear priority for such patients. Clinical challenges in the relapsed/refractory population include light chain and IgA isotype, renal failure, extramedullary disease, hyposecretory myeloma, and advanced bone disease.

Dose Dense Dreifach* Melphalan100 for Multiple Myeloma.

Background: Tandem high dose melphalan therapy with autologous peripheral stem cell support has emerged as standard of care for patients without prohibitive comorbidities. Mucositis and gastrointestinal side effects are the most common extrahematologic side effects. Two previously published studies presented a triple transplant with a conditioning regimen melphalan 100mg/m2 (MEL100) with peripheral stem cell support every two to five months schedule. This dose reduced regimen was implemented to limit those extrahematologic dose limiting side effects in the elderly or high risk population. We are presenting a novel approach that investigates the triple melphalan100/m2 approach on a dose dense, every three weeks schedule in a patient population without significant comorbidities for conventional high dose tandem transplantation.Patients and methods: Thirteen standard or high risk patients with stageIII multiple myeloma were prospectively treated This population contained eight patients with IgG clonality, 3 IgA, 1 nonsecretory and one light chain isotype. The induction regimens of the thirteen patients were heterogenous and included 5 VAD, 3 DCIE, 2 Thal/Dex, 2 CIE and 1 pulse decadron. Patients had stem cells harvested through peripheral blood leukopheresis, and these cells were divided into three equal sets and frozen. The patients were scheduled to receive Melphalan at 100mg/m2 on day -1, 20, and 41 and then the autologous infusions occurred at day 0, 21, and 42.Results: All patients were able to receive all three cycles of the MEL100 regimen. Seven patients (54%) received the treatements on the every three week schedule; three treatments (23%) during the second cycle and six treatments (46%) of the third cycle had to be delayed a median of 6 and 4 days respectively. Three patients were managed completely in the outpatient setting, and the average total hospital stay for the three transplants was 18 days. Median progression free survival was 761 days (range 73 – 1571) and the overall survival of this cohort has yet to be reached. No patient had worse than grade II mucositis, and no serious adverse events were recorded.Conclusions: Our regimen of three consecutive autologous peripheral stem cell transplants with a reduced dose of melphalan at 100mg/m2 given every three weeks is very well tolerated. The progression free survival and overall survival are similar and can be compared favorably with the standard tandem myeloma regimens. Although our data is intriguing, further studies with larger numbers need to be performed to confirm these results.

Comparison of Serum Free Light Chain Levels and 24 Hour Urinary Monoclonal Protein Secretion in Patients with Myeloma: Concordant Changes with Response to Therapy.

Background: The measurement of monoclonal (M) protein in the serum and urine is critical for response assessment and disease evaluation in patients with multiple myeloma (MM). The serum free light chain (FLC) assay offers a new and sensitive method of assessing response to therapy. An important question that has not been adequately addressed is the correlation between 24 hour urine M protein levels and serum FLC measurements, and the extent to which response to therapy estimated using the FLC assay correlates with that assessed using the 24 hour urine M protein level.Methods: A total of 2194 sets of data, with simultaneous UPEP and serum FLC measurement, were studied. These included 752 unique patients, with individual patients having 1–23 paired assessments over time. FLC estimation was carried out using the serum FLC assay (Freelite; The Binding Site Limited, UK) performed on a Dade-Behring Nephelometer. Based on the established reference range, kappa/lambda FLC ratio <0.26 or >1.65 were defined as abnormal indicating the presence of monoclonal lambda and kappa FLC, respectively. The monoclonal light chain isotype was considered the involved FLC isotype, and the opposite light chain type as the uninvolved FLC type. The Urine M protein by UPEP was compared to the serum levels of the involved light chain using Spearman Rank Correlation. For comparisons in individual patients over time, those with at least 10 measurements each were studied.Results: The median involved FLC level in patients with an undetectable urine M protein was 2.3 mg/dl compared to 32.2 mg/dL among those with a detectable urine M protein (P<0.001). Among the 1676 points with an abnormal FLC ratio, only 75% had an M protein detected in the urine, P < 0.001. Conversely, among patients with a positive urine M-protein, 91% had an abnormal FLC ratio. When all the 2194 data points were considered together, there was a significant correlation between the urine M protein level and the FLC levels (FLC level calculated as the difference between involved and uninvolved levels), rho=0.763, P < 0.001. The correlation did not change when patients with a serum creatinine of over 2.5 were excluded. The correlation between FLC levels and urinary M protein can be affected by several factors such as renal function that will differ across patients. Therefore, we examined whether the correlation between the two variables is stronger when the variations introduced by inter-patient differences in the relationship between the two variables are eliminated. In order to do this, we studied individual patients on whom multiple data points over time were available. One patient who had the maximum number of paired assessments (23 pairs) of serum FLC level and urinary M protein; the correlation between the two variables over time was highly significant, rho 0.981, p<0.001. Similarly 26 other patients who had measurable urine M protein levels in whom 10 nor more paired observations over time were available, also showed significant correlations, rho, range 0.726–0.981, p<0.01.Conclusion: There is a significant correlation between urine M-protein and serum free light chain across patients and the correlation is stronger in individual patients in whom the effect of inter-patient variation in other confounding factors can be eliminated. These data if confirmed in a clinical trial setting would support the use of serum FLC levels instead of urinary M protein measurements to assess response to therapy.

Evidence for Non-Random Immunoglobulin VH Gene and Light Chain Isotype Usage in Follicular Non-Hodgkin's Lymphoma.

Background: Antibody diversity is generated by recombination of individual immunoglobulin (Ig) gene segments and subsequent somatic diversification driven by antigen recognition. In the repertoire of expressed B cell receptors (BCR) among normal peripheral B cells, variable heavy (VH) gene segments are not equally represented. The ratio of kappa to lambda light chain usage is also skewed; the normal κ/λ is 1.5. Investigation of the BCR repertoire may provide clues to the genesis of B cell malignancies, as suggested in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). BCR V gene identification during the production of recombinant antibodies used in our ongoing PhII and PhIII FavId® (idiotype/KLH) immunotherapy studies has enabled us to analyze V gene usage from 475 B cell follicular lymphoma (FL) tissue samples. This study reports the results of VH gene and κ/λ gene expression in this FL sample collection.Methods: Ig heavy chain (HC) and light chain (LC) isotypes from B cell FL samples were identified by flow cytometry. VH and VL regions were sequenced from gene specific cDNA libraries prepared from these samples. VH gene usage and κ/λ ratios were compared to frequencies determined for normal peripheral B cells isolated from six healthy volunteers as well as published reports for normal peripheral B cells and other B cell malignancies.Results: Compared to VH gene family usage determined for normal B cells, VH3 usage is higher (68% vs. 42%), VH1 usage is lower (7.8% vs. 22%) and VH4 usage is equivalent (22% vs. 26%) in our cohort of FL patients while VH2, 5, 6 and 7 are infrequently used in both populations. Usage of the VH3 genes within FL derived sequences also depends upon isotype, in that this gene family is preferentially associated with the IgM HC isotype relative to IgG (76% and 57% respectively). Additionally, the combined usage of the specific genes VH3-23 and VH3-48 in our patient collection accounts for over 29% of all VH genes - compared to 9% among normal B cells. These VH gene usages also differ from reports of VH gene expression among CLL and MCL patients. With respect to LC usage, VH3 isolates are associated with a normal κ/λ ratio of 1.6 while VH4 gene isolates are preferentially associated with λ light chains with a κ/λ ratio of 0.9. Finally, FL B cells expressing the IgM HC isotype preferentially co-express κ light chains (κ/λ ratio of 2.4) while IgG expressing cells preferentially utilize λ chains (κ/λ ratio of 0.6).Conclusions: Non-random V gene and LC expression among patients with FL is noted. These distortions in Ig gene expression suggest that lymphomagenesis in FL may be associated with B cell stimulation by common antigens. A program to investigate the epitopes recognized by FL derived BCRs via binding of recombinant FL derived antibodies to protein arrays containing common auto-antigens is currently underway.

Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells

AbstractThe B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (κ or λ) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.