Heavy Chain Variable Region Research Articles

Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).

BackgroundDeletion of 13q14 is the most common cytogenetic change in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is detected in about 50 % of patients by fluorescence in situ hybridization (FISH), which can reveal presence of del(13)(q14) and mono- or biallelic deletion status without information about the size of the lost region. Array-comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) can detect submicroscopic copy number changes, loss of heterozygosity (LOH) and uniparental disomy (UPD) regions. The purpose of this study was detection of the size of del(13)(q14) deletion in our group of patients, comparing the size of the monoallelic and biallelic deletions, detection of LOH and UPD regions.ResultsWe have investigated 40 CLL/SLL patients by karyotype, FISH and CGH and SNP array. Mutational status was of immunoglobulin heavy-chain variable-region (IGVH) was also examined. The size of deletion ranged from 348,12 Kb to 38.97 Mb. Detected minimal deleted region comprised genes: TRIM13, miR-3613, KCNRG, DLEU2, miR-16-1, miR-15a, DLEU1. The RB1 deletions were detected in 41 % of cases. The average size in monoallelic 13q14 deletion group was 7,2 Mb while in biallelic group was 4,8 Mb. In two cases 13q14 deletions were located in the bigger UPD regions.ConclusionsOur results indicate that bigger deletion including RB1 or presence of biallelic 13q14 deletion is not sufficient to be considered as adverse prognostic factor in CLL/SLL. CytoSure Haematological Cancer and SNP array (8x60k) can precisely detect recurrent copy number changes with known prognostic significance in CLL/SLL as well as other chromosomal imbalances. The big advantage of this array is simultaneous detection of LOH and UPD regions during the same test.

Study on the relationship between the structure and functions of anti-human cervical cancer single-chain antibody and the lengths of linkers.

This study aimed to find the linker with minimal impact among chains to fight against the structure and function of cervical cancer (CC) single-chain antibody. The original variable region of heavy chain (VH) and variable region of light chain (VL), and the single-chain antibody with linkers of different lengths (n = 1-8) were modeling by homologous modeling, while the peptide chain structure of (Gly4Ser)n was utilized by the linkers. Comparison of the similarity of original VH/VL and VHn/VLn was carried out by applying the algorithm of spatial hierarchical alignment based on the spherical coordinates. The fore and aft distance and diffusion radius of alpha (α) were also calculated. The stability of antibody with different linker length was then compared. ELISA method was adopted to evaluate the immunological activity of single-chain antibody with different linkers. MTT assay was used to analyze the inhibition effect of ScFv-n on CC cells. When n = 4, the structures were the most similar between ScFv and the original VH/VL. When n = 3, the influence of adding connecting peptide on the stability of single-chain antibody was the least. The result of ELISA and MTT methods indicated that when n = 3, single-chain antibody gained the highest activity. The optimum length of linker of anti-human CC single-chain antibody was n = 3 from the point of mathematical modeling and biology experiments. This study provided new ideas for the design and constructions of single-chain antibody, and theoretical basis for the treatment of CC.

Heightened BTK-dependent cell proliferation in unmutated chronic lymphocytic leukemia confers increased sensitivity to ibrutinib.

In chronic lymphocytic leukemia (CLL), patients with unmutated immunoglobulin heavy chain variable region gene (UM-CLL) have worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was correlated with higher phospho-BTK and greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, heightened BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling.

Characterization of recombinant human monoclonal antibodies against the Bile Salt Export Pump

Background: Inhibitory, Bile Salt Export Pump (BSEP)-reactive IgG-class antibodies cause phenotypic recurrence of BSEP deficiency in some patients after liver transplantation for Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC-2). A detailed characterization of these disease-causing antibodies is key to elucidate the molecular mechanism of this emerging disease. Methods: BSEP-reactive B cells were isolated from peripheral blood of a patient with a high anti-BSEP titer by fluorescence-activated single cell sorting. Corresponding full-length immunoglobulin heavy and light chain variable region gene transcripts were amplified by single cell RT-PCR and cloned into eukaryotic expression vectors to enable the in vitro production of the respective monoclonal antibodies. These monoclonal antibodies were characterized by immunofluorescence studies of rat liver slices and transiently transfected cells, Western blot, FACS analysis, in situ rat liver perfusion and in vitro BSEP transport assays. Results: Immunofluorescent staining of fixed, transiently transfected human embryonal kidney (HEK293) cells identified several BSEP-reactive antibodies that also stained canalicular patterns on fixed rat liver slices. In contrast, only a few recognized denatured rat BSEP on Western blots, while all antibodies detected its denatured human homologue. FACS analysis of antibody-treated, unpermeabilized cells transiently expressing BSEP revealed BSEP-specific staining that was absent on empty control cells. BSEP-reactive antibodies reached the canalicular space of in situ perfused rat livers within two hours. Finally, in vitro vesicular transport assays showed clone-dependent influence on transport activity. Conclusion: We provide characterization of a novel set of patient-derived, monoclonal antibodies directed against extracellular features of BSEP. These include species that are BSEP-inhibitory, -neutral, and, strikingly, -stimulatory in vitro. The antibodies and their monovalent Fab derivatives may have great potential in applied research as well as in future clinical applications.

Next-Generation Deep Sequencing Reveals Multiple Ighv Clones in One Third of CLL Patients Defining New Prognostic Subgroups and Improving Previous Classification

Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by a heterogeneous clinical course. Currently, the mutational status of the immunoglobulin heavy chain variable (IgHV) region defines 2 risk groups: patients with ≥ 2% difference from the germline are considered as unmutated (UM) and have a poor prognosis while the opposite is observed for mutated (M) patients (Hamblin et al, 1999). Until now, clonality has been determined using PCR/gel electrophoresis and the percentage of IgHV mutations by Sanger sequencing using the Biomed-2 method (van Dongen, Leukemia 2003) which only allows for analysis of the major clone and assumes that only one clone per patient should be considered. Methods: in the present study, we sequenced the IgHV gene for 200 CLL patients with a median follow-up of 70 months (range, 1-309) by next-generation (NGS) and Sanger sequencing (SS) and investigated the impact on prognosis for the different subgroups. Briefly, 100ng of cDNA generated from RNA extracted from highly purified CD19+ cells obtained at diagnosis was amplified using IGH LEADER master mixes. For SS, clonality of PCR product was determined and monoclonal samples were directly sequenced. For NGS, PCR products were purified using magnetic beads, normalized and pooled to create a library for sequencing using the MiSeq v3 Reagent kit (600 cycles). Sequencing data was analyzed using LymphoTrack™ bioinformatics software and percentage of clone was based on the first 200 most abundant clones detected. Clones with an abundancy of < 2.5% were not considered. The IMGT database was used to calculate the percentage of mutation compared to germline. Results: Using SS, 49% (98/200) of patients were IgHV M, 38% (76/200) UM, while 13% (26/200) were polyclonal and further Sanger sequencing was inconclusive. Concordance between SS and NGS was 98.3%: for 90.2%, the major clone detected by NGS was the same by SS, for 8.5% the clone was the same but the percentage mutated compared to germ-line differed slightly with a median error of 0.6%, while for 1.7%, SS detected another clone which was less abundant by NGS. Interestingly, among patients considered as monoclonal by SS, 25.3% (44/174) were found to be polyclonal by NGS. NGS revealed that 35% (70/200) of patients display different IgHV re-arrangements in the same patient: the median frequency of the first two most abundant clones were 78.2% (range, 28.5-94.9) and 16.4% (range, 2.5-49.0) respectively. This implies that at least in these patients, leukemia-initiating events must occur prior to IgHV re-arrangement in a pro-B cell. Further in-depth studies of the HSC and early B cell progenitor compartments in these patients will be required to confirm these observations. In terms of prognosis, M, UM and polyclonal patients determined by SS had a median treatment-free survival (TFS) of 178, 29 and 129 months respectively (P<0.0001) and a median overall survival (OS) of >309, 183 and >309 months (P<0.0001). Since NGS was able to highlight different clones, we were able to create 5 different categories: patients with (a) multiple M clones, (b) 1 M clone, (c) a mix of M-UM clones, (d) 1 UM clone, (e) multiple UM clones. Using this new NGS classification, we found patients with different prognosis among patients already classified by SS. For example, SS-M patients were classified in 3 subgroups with a median TFS of >251 (a), 178 (b), 56 (c) months (P=0.0014). Similar results were observed for SS-UM patients who were divided in 3 subgroups with a median TFS of 94 (c), 24 (d), and 19 (e) month (P=0.0296). When all patients where considered, IgHV-NGS classification stratified patients in 5 different subgroups with median TFS of >251 (a), 178 (b), 57 (c), 24 (d), 19 (e) months (P<0.0001) and a median OS of 270, >309, >309, 183, 88 months (P<0.0001). Conclusions: determination of IgHV mutational status by NGS has excellent concordance with classical SS and in addition enables the detection of small clones which has a significant impact on prognosis. This allows the analysis of all clones without labor intensive manipulations especially for polyclonal patients. Here we showed for the first time that one third of CLL patients present with multiple IgHV subclones that impact on prognosis and refine the previous SS-IgHV classification. Figure 1. Figure 1. Disclosures Schuh: Acerta Pharma BV: Research Funding.

MYD88 L265P Mutations Influence Clinical Outcome and Identify a Pathway for Targeted Inhibition in Chronic Lymphocytic Leukemia

The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is recurrently observed in CLL; although this mutation has been demonstrated to have functional effects in multiple hematologic malignancies, its role in CLL is largely unknown. To address this gap in knowledge, we examined the clinical and biological impact of MYD88 L265P mutations in CLL by analysis of gene expression, cell viability and Toll-like Receptor 9 (TLR9)-induced signaling and cytokine production.Out of 160 CLL patient samples subjected to whole-exome sequencing and previously reported by our group, 10 were found to harbor MYD88 L265P mutations, all of which possessed mutated immunoglobulin heavy chain variable (IGHV) regions (p = 0.006). While IGHV mutated patients are generally known to exhibit better prognosis compared to IGHV unmutated patients, the presence of MYD88 L265P within the IGHV mutated subset was associated with earlier age of disease onset (p = 0.04) and worse overall survival (OS; p = 0.00017), comparable to IGHV unmutated samples with wild-type (WT) MYD88. No association with the presence of chromosome 13q deletions (p = 0.26) or prior treatment at the time of sampling (p = 0.10) was observed.Gene expression microarray analysis restricted to the IGHV mutated subset (MYD88 WT: n = 76; MYD88 L265P: n=10) and conducted using a PAM-based approach demonstrated that MYD88 L265P mutation was associated with differential expression of 28 genes, whose expression was then examined across all CLL samples with available gene expression data (n = 150). Using Cox modeling, a composite gene signature score was determined for each patient, who were subsequently dichotomized based on median signature. This method was able to predict both OS and event free survival (EFS) in a univariable analysis (OS: p = 1.2E-06; EFS: p = 7.6E-13). Statistical significance was maintained when a multivariable analysis was conducted, adjusting for known CLL risk factors including age, IGHV status, ZAP70 expression, cytogenetics and prior treatment (p < 0.0001 for OS and EFS). The univariable (OS: p = 1.6E-05; EFS: p = 5.7E-10) and multivariable findings (p < 0.003 for OS and EFS) were further confirmed in an independent validation cohort (n = 87). To identify a more parsimonious gene set, we applied a L1 penalized proportional hazards model to the discovery and validation cohorts, separately. This approach identified 5 overlapping genes (BCAT1, BMP6, CHAD, IKZF2, and TRIO) between the two cohorts that appear to be the main drivers of the predictive signature.To inhibit MYD88 signaling in CLL cells, we treated MYD88 L265P and WT cells (n = 6/group, matched for clinical characteristics: IGHV, ZAP70, cytogenetic, and treatment status) in vitro with a highly-selective small molecule IRAK4 inhibitor, ND-2158 (Nimbus Therapeutics). ND-2158 significantly reduced cell viability in a dose dependent manner in both MYD88 WT and L265P primary CLL cells, either alone or in combination with a fixed concentration of the B-cell receptor (BCR) pathway inhibitor, ibrutinib.The TLR9 agonist CpG was used to stimulate signaling through the MYD88 pathway in vitro. ND-2158 inhibition of CpG-induced IRAK4 activation in CLL cells (n = 3/group, matched for clinical characteristics) blocked IRAK1 and IκBα degradation and led to a dose-dependent decrease in the ratio of phospho/total proteins. No significant differences were noted between MYD88 WT and L265P samples, consistent with our cell viability results.CLL-secreted levels of IL-6, IL-10 and CCL3 were measured in culture supernatants treated with ND-2158+/- CpG stimulation (n = 4/group, matched for clinical characteristics). CpG stimulated cytokine levels (p < 0.0001 for all cytokines+/- CpG) were significantly inhibited in a dose-dependent manner by ND-2158. Again, no significant differences were observed between MYD88 WT and L265P CLL with respect to cytokine production, either at baseline or in CpG-stimulated DMSO treated control cells.In conclusion, the differences in clinical outcome and gene expression observed between MYD88 WT and L265P IGHV mutated CLLs indicate a functional role for MYD88 L265P in CLL. The inferior clinical outcome in IGHV mutated CLL with L265P mutation suggests that MYD88 signaling may be a relevant target in CLL. ND-2158 inhibits signaling in the MYD88 pathway, suggesting potential therapeutic utility of IRAK4 inhibitors in CLL. DisclosuresChaudhary:Nimbus Therapeutics: Equity Ownership. Miao:Nimbus Therapeutics: Employment. Westlin:Nimbus Therapeutics: Employment.

Development of a Novel Virus-like Particle (VLP) Vaccine for Personalized B-Cell Lymphoma and Chronic Lymphocytic Leukemia Therapy

BackgroundFor decades since its discovery, the idiotype protein (Id) expressed on the surface of B-cell malignancies has been pursued as a tumor-specific target in therapeutic antibody and vaccine development programs. The challenge in vaccine development has been to present the Id antigen so as to stimulate an effective anti-tumor response. Advances in our understanding of the immune system have revealed the importance of innate receptors, particularly the toll-like receptor (TLR) family, in stimulating adaptive immunity. Further understanding of virus-induced immunity reveals features such as viral trafficking to lymph nodes and presentation of antigens in an ordered array that facilitate immune response generation. Our strategy combines this knowledge with advanced protein engineering in order to produce a stabilized virus-like particle (sVLP) that displays Id in the context of innate immune stimulants. Multiple studies using the 38C13 lymphoma model were performed to evaluate and maximize the efficacy of Id-VLP vaccines using the historical "gold standard" Id-KLH as a reference.MethodsVLP vaccines were constructed by conjugating the tumor-associated Id antigen and immune stimulants, GM-CSF, CpG DNA, and flagellin, to the exterior surface of either the MS2 or Hepatitis B core (HBc) VLP. The bioconjugates were engineered to maintain biologic activity of the immune stimulants and present unperturbed heavy and light chain variable regions. A total of 14 different vaccines displaying 3 different 38C13 Id formats, IgM, F(ab')2 and scFv, with or without one or more of the three immune stimulant were produced. For example, with the scFv Id format VLP vaccines, 7 of the 8 possible combinations of immune stimulants were made.Three in vivo experiments were performed to evaluate vaccine compositions. C3H mice were vaccinated three times within a one-month period with control or experimental vaccines. Following vaccination, the mice were challenged with 38C13 Id-bearing tumor cells administered by intraperitoneal or subcutaneous routes. Sera samples were collected prior to, during, and after vaccination for analysis of total and isotype-specific anti-Id antibody titer.ResultsThe first study compared MS2- and HBc-based VLP vaccines. While both viral structures exhibited immune inhibition on their own, the HBc VLP vaccine conjugated with GM-CSF and CpG elicited a strong and protective humoral immune response as compared to experimental controls. This was confirmed in a second study where different HBc VLP vaccines explored additional combinations of immune stimulants and the different Id formats. While all HBc VLP vaccines elicited strong and protective immune responses, the VLP with scFv Id format and CpG provided better protection against tumor challenge than the Id-KLH conjugate. (70% vs 30% event-free survival). The third study further examined the scFv Id format and immune stimulants on stabilized HBc-based vaccines where disulfide bonds were introduced between dimers within the axes of symmetry to create a VLP structure in which every atom is indirectly covalently bonded to every other atom. Once again, the scFv Id-CpG-sVLP vaccine elicited a strong and protective response.ConclusionsCollectively, these studies provide strong rationale for the selected BB-001 composition (scFv Id-CpG-HBc sVLP) as a vaccine for Id-bearing lymphomas, as well as solid evidence that HBc sVLPs are a versatile and effective platform for further vaccine development. While all HBc-based Id vaccines with immune stimulants were statistically significantly superior to placebo groups in terms of efficacy and/or immune response, the simpler VLP vaccine with the TLR9 agonist CpG outperformed the combinations with GM-CSF and/or flagellin. The subcutaneous tumor challenge was more demanding, and showed a statistically significant survival advantage for BB-001(70%) versus Id-KLH (30%). Consistent with previous 38C13 studies, humoral immune response was critical for protection from challenge. Evidence of antibody class switching in the humoral immune response was also observed. DisclosuresTheriault:Bullet Biotechnology, Inc.: Employment, Equity Ownership. Levy:Bullet Biotechnology, Inc.: Consultancy. Swartz:Bullet Biotechnology, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Unraveling the Efficacy and Safety Profiles of Anti-CD123 Chimeric Antigen Receptors (CARs) in a Model of Acute Myeloid Leukemia Immunotherapy By Investigating CAR Binding Affinity and Density Variables

In the last years, adoptive cellular immunotherapy employing T lymphocytes genetically modified with Chimeric Antigen Receptors (CARs) has demonstrated impressive clinical results, particularly in the treatment of acute/chronic lymphoblastic leukemia and B-cell lymphoma, paving the way towards the possibility to translate this approach also to other hematological malignancies, such as Acute Myeloid Leukemia (AML).In the AML context, the CD123 antigen (IL-3 receptor alpha subunit) represents a good target antigen, being a poor prognosis over-expressed marker on AML cells and leukemic stem cells (LSCs), a rare population that plays a key role in perpetuating leukemia. However, CD123 is also expressed on the surface of healthy cells such as monocytes and endothelial cells, although at lower levels as compared to leukemic cells. The potential recognition of low antigen positive healthy tissues by CAR-redirected T cells, through the so called "on-target-off-organ" effect, limits a safe clinical employment of this immunotherapeutic approach. CARs are artificial receptors generated by joining the cytoplasmic TCR (T Cell Receptor) signaling modules to the heavy and light chain variable regions of a monoclonal antibody, whose affinity toward a target antigen is a variable capable of influencing the CAR-mediated functional responses.Therefore, in our study we investigated how the CAR affinity variable in the context of CD123 targeting, together with the CAR and CD123 target antigen density, could impact anti-CD123. CAR-redirected effector cells efficacy against leukemic cells and safety towards the healthy cells.To this aim, Cytokine-Induced Killer (CIK) effector cells have been genetically modified with four Chimeric Affinity Mutants (CAMs), CAM-1, CAM-2, CAM-3 and CAM-4, identified by means of a computational docking technique.In vitro cytotoxic assays, cytokine production and proliferation experiments have been performed in order to evaluate both the efficacy and safety profile of the CAR-redirected CIK cells, using un-manipulated CIK cells (NO DNA) and wild-type anti-CD123.CAR condition as controls.The functional characterization of all the CAMs revealed both the specificity and the effectiveness of CIK-CAR+ cells against the CD123+ THP-1 cell line and primary AML cells. However we observed that, at least in the context of CD123 targeting, a good CAR expression level is necessary for inducing effective later functions, such as proliferation and cytokine production, towards a high CD123+ target.When introducing leukemic cell lines with different CD123 density on their surface we observed different effector properties minimally influenced by the CAR affinity. In particular, in terms of killing activity, we noticed that a number of ≈1600 CD123 molecules is sufficient to induce a good cytotoxic response of all the CARs tested, with the CAM-2 (2-magnitude log lower in affinity) being less powerful. At the same time, this antigen density is not enough to determine a good proliferative capability which instead occurs with leukemic target cells expressing 5000 or more CD123 molecules. Considering the cytokine production (IL-2 and IFN-gamma), we observed that all CIK-CAR+ cells showed a cytokine release that is directly proportional to the target antigen density, with CAM-2 showing a reduced response towards low-CD123 expressing leukemic targets.When analyzing the safety profile of the CAMs against low-CD123+ endothelial target cells (≈1600 molecule/cell), we observed a lower functional activity of the CAMs as compared to the leukemic cell lines expressing the same level of CD123 molecules on their surface, with the low-affinity CAM-2 showing a major sparing capability in terms of killing activity (being the only one not statistically different from NO DNA).In conclusion, exploiting our model of affinity mutants we were able to in vitro characterize the role of the CAR density balanced with the affinity of the anti-CD123.CAR towards AML cells expressing different CD123 levels and CD123-low expressing normal tissues. In particular, the results obtained with CAM-2 suggested a potential threshold of affinity below which, even if the safety profile is preserved, the anti-leukemic efficacy would be impaired. DisclosuresNo relevant conflicts of interest to declare.

LRPAP1 Is a Frequent B-Cell Receptor Antigen in Mantle Cell Lymphoma (MCL)

Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens.Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens.Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1.Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36)Supported by Wilhelm-Sander-Stiftung DisclosuresNo relevant conflicts of interest to declare.

BARs (B -cell receptor a ntigens for r everse targeting): A Novel and Ultimately Specific Treatment Concept for B-Cell Neoplasms

Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms.Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA lymphoma biopsies. Whenever possible, "natural" Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to proteins expressed on macroarrays derived from human cDNA expression libraries and identical binding pattern of nFabs and rFabs was demonstrated by an antigen competition assay.Results: Two antigens (paratarg-7 and sumoylated HSP-90 which are hyperphosphorylated and sumoylated, respectively, in patients compared to healthy controls) are the targets of paraproteins from (depending on ethnicity) 30-50% of all multiple myeloma patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all mantle cell BCR target LRPAP1; optineurin is the BCR target of 12% follicular lymphomas and various autoantigens have been identified as the targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated, indicating partial dependence on antigenic stimulation even in cell lines that had been in culture for years. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with BCR specific for ARS2 (OCI-Ly3) was demonstrated in vitro and in vivo after establishment of OCI-Ly3 lymphomas in SCID beige mice.Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells. Supported by Wilhelm-Sander-Stiftung DisclosuresPfreundschuh:Roche, Janssen, Celgene: Honoraria, Research Funding.

IL-10 Production By CLL Cells Is Enhanced in the Anergic IGHV Mutated Subset and Associates with Reduced DNA Methylation of the IL-10 Locus

The B-cell receptor (BCR) of chronic lymphocytic leukemia (CLL) cells with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable (IGHV) regions shows a variable degree of anergy, associated with low surface IgM (sIgM) levels and signaling capacity, more evident in M-CLL. DNA methylation is also different between M-CLL and U-CLL. Patients from both subsets are immunosuppressed from the early stages, possibly influenced by the ability of activated CLL cells to produce IL-10, a property of B10 cells.The aim of this study was to investigate associations between capacity of circulating CLL cells to differentiate into B10 cells and features of anergy. In parallel, the methylation status of the IL-10 gene locus was probed in order to investigate associations between IL-10 gene methylation and production. CLL cells were isolated and cultured +/- TLR9 activation with CpG (ODN-2006). Cytokine secretion was measured in supernatants (Luminex) and IL10 transcript was measured (RT-qPCR) from cell pellets. IL-10 production by CLL cells was measured by intracellular flow cytometry following stimulation with CpG for 24 hours. Epigenetic profiling was performed using MassARRAY and the 450K Array.We found that activation of CLL cells by CpG consistently induced production and secretion of IL-10 protein, while no or low amounts of pro-inflammatory cytokines were detected. Production of IL-10 was significantly higher in M-CLL than in U-CLL. Levels also correlated with lower sIgM levels and signaling capacity, both features of anergy. A linear correlation was present between secretion, intracellular production and transcript levels of IL-10, suggesting no aberrant post-transcriptional controls.In the absence of CpG activation, IL-10 transcript levels were also detected at low levels in M-CLL, while they were even lower in U-CLL. To identify a potential basis for the differential expression, DNA methylation analysis of IL-10 gene locus was performed. While the promoter region displayed similarly low levels of methylation in both U-CLL and M-CLL, methylation differences were detected immediately downstream of the promoter within the first intron (approximately +200 to +500 bp, differentially methylated region 1, DMR1) and in the gene body (approximately +1300 to +1800 bp, DMR2). Both DMR1 and DMR2 were markedly more hypomethylated in M-CLL than U-CLL, with the greatest difference detected at cg17067005 within DMR1. Each of these regions display histone H3 lysine 27 acetylation (H3K27ac) in B cells (GM12878 cells), thus are likely to represent functional DNA elements. Analysis of IL-10 transcript levels with methylation demonstrated a mutually exclusive pattern between expression and methylation of both DMR1 and DMR2.These data document a strong link between capacity to differentiate into B10-like cells and anergy, and suggest an epigenetic component in the regulation of IL-10 production in CLL cells. This capacity may contribute to immunosuppression. While U-CLL appears less able to produce IL-10 on a per cell basis, higher tumor load may compensate, accounting for clinical immunosuppression in both subsets. DisclosuresNo relevant conflicts of interest to declare.

Idelalisib Plus Bendamustine and Rituximab (BR) Is Superior to BR Alone in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Results of a Phase 3 Randomized Double-Blind Placebo-Controlled Study

Introduction: Idelalisib (IDELA) is a 1st-in-class targeted PI3k delta inhibitor approved in combination with rituximab for the treatment of patients (pts) with relapsed CLL and as first-line treatment where 17p deletion or TP53 mutation exists in pts unsuitable for chemo-immunotherapy. Here, we present the results of a phase III, randomized, placebo-controlled study that evaluated the efficacy of IDELA added to BR, a common regimen for relapsed/refractory (R/R) CLL. A pre-specified interim analysis (IA) was performed demonstrating that PFS, the primary endpoint, and OS, a secondary endpoint, were superior in the investigational vs control arm. The IDMC recommended unblinding the study based on “overwhelming efficacy.”Methods: Between June 2012 and August 2014, 416 patients (pts) with R/R CLL were enrolled. The IA occurred when 75% of the total number of 260 planned events of CLL progression or death from any cause had occurred with a data cutoff of 15 June 2015. Key eligibility requirements included prior therapy containing a purine analog or bendamustine (ineligible if refractory to bendamustine); prior treatment with an anti-CD20 antibody; CLL progression within 36 months of the completion of the last prior therapy; and fitness to receive cytotoxic therapy. Patients were randomized to BR for 6 cycles Q 28 days (B = 70 mg/m2 D1, D2 of each cycle; R = 375 mg/m2 C1 and 500 mg/m2 C2-6) and IDELA 150 mg BID or placebo (administered continuously). Treatment with IDELA or placebo was administered until IRC-confirmed disease progression (PD), death, intolerable toxicity, or withdrawal of consent. Stratification was based on presence/absence of del(17p) and/or p53 mutation (mut), immunoglobulin heavy chain variable region (IGHV) mutated/unmutated (analysis performed by a central lab) and disease status refractory (CLL progression <6 months from completion of prior therapy) vs relapsed (CLL progression ≥ 6 months from completion of prior therapy). Imaging by CT was performed every 12 weeks and evaluated by an independent review committee (IRC) for evidence of PD. Crossover was not permitted at the time of PD.Results: The ITT analysis includes 207 pts in the IDELA + BR arm and 209 pts in the BR + placebo arm: gender 76% male; age 58%/42% (<65yrs/≥65 years); Rai stage III/IV 46%; median time since completion of last prior therapy 16 months; patients with high-risk features (del(17p)/p53mut 32.9%, unmutated IGHV 83.2%, refractory 29.8%); median number of prior therapies: 2 (range: 1-13); and median follow up 12 months. Median number of cycles of BR completed in the two arms was equal (6). Median PFS (mo) of IDELA + BR vs BR + placebo: 23 vs 11 (hazard ratio 0.33; p-value 2.8 x 10-14; 95% CI 0.24, 0.45) (Fig 1), corresponding HRs in pts with high-risk features (Fig 2); mOS (mo): IDELA + BR vs BR + placebo not reached for either arm (HR = 0.55; p-value 0.008; 95% CI 0.36, 0.86). The most common all-grade AEs with IDELA + BR were neutropenia and pyrexia (63.3% vs 41.5%), and with BR + placebo were neutropenia and nausea (53.6% vs 34.4%). The most common grades ≥3 AEs respectively were neutropenia (59.9%) and febrile neutropenia (20.3%); neutropenia (45.9%) and anemia (12%). Grade ≥3 diarrhea with IDELA + BR was 7.2% and BR + placebo was 1.9%. Incidence of SAE pneumonitis was 1.4% vs 0%. Transaminase abnormalities were observed more frequently in the IDELA + BR vs BR + placebo arm: all grades ALT 59.9%/30.6%, AST 52.2%/27.8%, Grade ≥3 ALT 21.3%/2.9%, AST 15.5%/3.3%. [Display omitted] [Display omitted] Conclusions: Idelalisib in combination with BR is superior to BR alone, reducing the risk of both PD and death, increasing PFS and OS, and with a safety profile consistent with prior reported studies. These results were consistent across pts with high-risk features. Addition of IDELA to BR was also beneficial in pts without del(17p)/TP53mut. This regimen represents an important new option for pts with R/R CLL. ClinicalTrials.gov ID: NCT01569295. Disclosures:Zelenetz:Gilead Sciences: Research Funding. Off Label Use: Idelalisib for relapsed/refractory CLL. Robak:MorphoSys AG: Consultancy , Honoraria , Research Funding ; Janssen: Consultancy , Research Funding ; Gilead Sciences: Research Funding. Coiffier:CELLTRION, Inc.: Consultancy , Honoraria ; Gilead Sciences: Research Funding. Delgado:Gilead Sciences: Research Funding. Marlton:Gilead Sciences: Research Funding. Adewoye:Gilead Sciences: Employment , Equity Ownership. Kim:Gilead Sciences: Employment , Equity Ownership. Dreiling:Gilead Sciences: Employment , Equity Ownership. Hillmen:Gilead Sciences: Honoraria , Research Funding ; Abbvie: Honoraria , Research Funding ; Janssen: Honoraria , Research Funding ; Roche: Honoraria , Research Funding ; Novartis: Honoraria , Research Funding ; Pharmacyclics: Honoraria , Research Funding ; Acerta Pharma BV: Research Funding.

Patterns of Lymphocytosis in Patients with Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL) Treated with Idelalisib

Patterns of Lymphocytosis in Patients with Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL) Treated with Idelalisib

Higher BTK-Dependent Cell Proliferation in Unmutated Chronic Lymphocytic Leukemia Confers Increased Sensitivity to Ibrutinib

In chronic lymphocytic leukemia (CLL), mutation status of the immunoglobulin heavy chain variable region gene (IGHV) has prognostic significance, with the unmutated phenotype (UM-CLL) having worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK. is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was associated with greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, higher BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling. DisclosuresNabhan:Celgene Corporation: Honoraria, Research Funding. Smith:Celgene: Consultancy; Pharmacyclics: Consultancy.

High-Level Expression of ROR1 Associates with Early Disease Progression in Patients with Chronic Lymphocytic Leukemia

ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells. We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment. On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort. However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort.We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC). CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core. We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expressionthat could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1- High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years. We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups. We found that high-level expression of ROR1retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1- High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years. The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%). A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001). Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL.Table 1Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohortTraining DatasetValidation DatasetROR1 MFI distribution (Median)0~475 (35.5)0~424(34.6)ROR1Low with Mutated IGHV139132ROR1Low with Unmutated IGHV6484ROR1High with Mutated IGHV181156ROR1High with Unmutated IGHV209214Total With Known IGHV Mutation Status593586 DisclosuresKeating:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

ROR1 Negative Chronic Lymphocytic Leukemia (CLL) Have a Distinctive Gene Expression Signature and May Represent an Indolent-Disease Subtype

ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). We identified patients with CLL cells that had negligible expression of ROR1, despite otherwise satisfying all standard criteria for diagnosis of CLL by iwCLL criteria.We performed next-generation-sequencing on the transcriptomes of 12 CLL cases that had negligible expression of ROR1 and 12 cases that expressed levels of ROR1 comparable to that typically observed in CLL. Eight of the 12 ROR1-negative cases expressed unmutated immunoglobulin heavy-chain variable region genes (IGHV) and 4 used mutated IGHV. Similarly, 7 of the 12 ROR1-positive cases used unmutated IGHV and 5 had mutated IGHV.We identified 3,094 genes that were differentially expressed between the ROR1-positive and ROR1-negative samples out of 14,761 protein-coding genes tested (DESeq2, BH-adjusted p < 0.05). Subnetwork analyses revealed 55 subnetworks that were differentially expressed between ROR1-positive and ROR1-negative cases. ROR1-positive CLL cells had higher-level expression of subnetworks associated with protein-kinase activation or proliferation of tumor cells, but lower-level expression of subnetworks associated with induction of apoptosis or RNA degradation and/or processing, than did ROR1-negative CLL cells. ROR1 and AKT1 were included in 7 subnetworks associated with proliferation, hematologic cancer, or inhibition of cell death. Fourteen (25%) of the 55 differentially expressed subnetworks previously were identified as being differentially expressed between ROR1-positve leukemias of ROR1xTCL1 transgenic mice and ROR1-negative leukemias of Eµ-TCL1-transgenic mice (see Widhopf et al, Proc Natl Acad Sci USA, 2014, PMC3896194).Gene-set enrichment analysis (GSEA) of genes encoding proteins involved in targeted signaling pathways in the BIOCARTA and Reactome database revealed that the ROR1+ leukemias had higher expression levels of genes encoding proteins in the AKT pathway than did the ROR1-negative cases. Immunoblot analysis revealed higher levels of activated pAKT relative to total AKT in representative cases of ROR1-positive CLL (8.8 ± 2.8, N = 7) than that detected in ROR1-negative CLL samples (1.0 ± 0.4, N = 4, P<0.01) (the ratios of pAKT/AKT were normalized to the mean ratio observed for ROR1-negative CLL samples); this is comparable to what we observed for ROR1-positive leukemias of ROR1xTCL1 mice, which had higher levels of activated AKT than the ROR1-negative leukemias of Eµ-TCL1 transgenic mice (Widhopf et al, Proc Natl Acad Sci USA, 2014, PMC3896194).Despite the small size of these two cohorts, it is noteworthy that the median time from diagnosis to initial therapy of the 12 patients with ROR1-negative CLL (9.4 years) was significantly longer than that noted for the 12 ROR1-positive CLL cases (2.5 years, (p < 0.01) used in this comparative analysis.In summary, this study describes a potentially new subtype of ROR1-negative CLL that has a distinctive gene expression signature and apparently indolent clinical course. DisclosuresKipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Plasma B-Cell Maturation Antigen Levels Are Elevated and Correlate with Disease Activity in Patients with Chronic Lymphocytic Leukemia

Background: B-cell maturation antigen (BCMA) serves as one of the receptors for B-cell activating factor (BAFF) or a proliferation-inducing ligand (APRIL), which are members of the tumor necrosis factor (TNF) family that promote survival of B-cells, including neoplastic B cells from chronic lymphocytic leukemia (CLL) patients (pts). We have found that BCMA is increased in plasma or serum from patients with multiple myeloma (MM). The level of serum BCMA found among MM pts associates with clinical status and predicts overall survival (OS). However, it is not known whether plasma (p) BCMA levels are elevated in pts with other B-cell malignancies, such as CLL. We examined plasma from healthy adults and pts with CLL for pBCMA and associated the observed levels in CLL pts with disease characteristics and/or known prognostic markers, such as white blood cell counts (WBC), serum β2-M, CLL-cell expression of mutated (M) or unmutated (U) immunoglobulin heavy-chain variable region (IGHV) or ZAP-70, or CLL-cytogenetic markers. We also examined the relationship between pBCMA and time from diagnosis (DX) or sample collection (SC) to initial treatment (treatment-free survival (TFS)) or OS.Methods: We examined the archived, plasma samples of 171 pts with CLL that were collected upon initial presentation to UCSD for studies on factors that could associate with disease outcome. Pts received therapy if they satisfied International Workshop on CLL (IWCLL) criteria for treatment. We examined serial plasma samples from a subset of these pts (n = 21) collected soon after DX, prior to first treatment, after treatment, and at relapse after therapy. We also assessed plasma samples of 76 healthy adults. We determined the pBCMA levels using a polyclonal anti-BCMA antibody in an ELISA available from R&D Systems (Minneapolis, MN). We used Mann-Whitney analysis to assess for associations between the relative pBCMA levels and disease characteristics assessed at the time of SC and Kaplan-Meier analysis to assess the relationship between measured pBCMA and TFS (median 4.1 years) and OS (median follow-up 7.8 years).Results: The median level of pBCMA in pts with CLL obtained from pts that were untreated at the time of first determination of pBCMA levels, regardless of whether they subsequently received therapy (n = 166; 58.54 ng/mL), was higher than that of healthy adults (n = 76; 6.32 ng/mL, P < 0.0001). The median pBCMA levels of samples from pts with CLL-cells that used M-IGHV (n = 87; median 42.13 ng/mL), or that lacked expression of ZAP-70 (n = 88; median 42.59 ng/mL), was lower than that of pts with CLL-cells that used U-IGHV (n = 84; 87.46 ng/mL, P < 0.0001), or that expressed ZAP-70 (n = 83; median 87.38 ng/mL, P < 0.0001). The median pBCMA level of pts with CLL cells that had del(13q) (n = 83; 46.57 ng/mL) was lower than that of pts with CLL cells that did not have detectable del(13q) (n = 77; 73.8 ng/mL; P = 0.0002). The median pBCMA of pts with CLL cells with del(17p) (n = 16; 77.23 ng/mL) appeared lower than that of pts without detectable del(17p) (n = 144; 51.77 ng/mL), but did not reach significance (P = 0.09). The TFS was longer (overall median time from SC to treatment: 2.36 years) among pts with pBCMA levels in the lowest three quartiles (median 3.72 years; range of pBCMA 13.21 - 104.69 ng/mL) when compared to patients with pBCMA in the highest quartile (median 0.67 years; range of pBCMA 110.1 - 782.97 ng/mL; P < 0.0001). Serial pBCMA levels consistently correlated with changes in clinical status among pts (n = 21) who had a pBCMA sample taken within three months of the determination of their clinical status. CLL pts with a pBCMA in the highest quartile had a significantly shorter OS when compared to pts with pBCMA levels in the lower three quartiles (P = 0.023).Conclusions: This is the first study to show that BCMA levels are increased in the plasma of CLL pts. We also demonstrate that the pBCMA levels correlate with other known prognostic indicators of CLL, such as IGHV mutational status, ZAP-70 expression and chromosomal abnormalities. CLL pts with higher pBCMA levels have a shorter TFS and OS. Furthermore, changes in pBCMA levels show consistent correlation with changes in the pts' clinical status during treatment. In summary, pBCMA appears to be a new plasma biomarker to monitor the disease course of CLL patients and determine their outcome. DisclosuresKipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.

A novel diagnostic biosensor for distinguishing immunoglobulin mutated and unmutated types of chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the immunoglobulin heavy-chain variable (IgVH) region may be mutated (Ig-mutated CLL) or unmutated (Ig-unmutated CLL); and the presence or absence of mutations in this region of CLL cells distinguishes two clinically distinct forms. It is important for physicians to distinguish between patients with Ig-unmutated CLL, where typically have more indolent disease with median survivals close to 25 years, and Ig-mutated CLL, where have more aggressive disease with median survivals around eight years. In this work, a biosensor capable of diagnosis and distinguishing between these two types of CLL was reported. The biosensor was fabricated by modifying a gold electrode with gold nanoparticles (AuNPS) followed by coating of ZAP70 oligonucleotide probe on the surface to detect specific sequence of ZAP70 gene. ZAP70 could predict the IgVH mutation status and is a good marker for differentiating Ig-mutated and Ig-unmutated CLL and serve as prognostic marker. First, we focused on achieving hybridization between probe and its complementary sequence. Hybridization between probe and target was determined with electrochemical impedance spectroscopy (EIS). Then, our efforts turned to optimize the conditions for the detection of any point mutation and also to maximize the selectivity. Under optimal conditions, the biosensor has a good calibration range between 2.0 × 10(-14) and 1.0 × 10(-9)molL(-1), with ZAP70 DNA sequence detection limit of 4.0 × 10(-15)molL(-1). We successfully detect hybridization first in synthetic samples, and ultimately in blood samples from patients. Experimental results illustrated that the nanostructured biosensor clearly discriminates between mutated and non-mutated CLL and predict the IgVH mutation status, which it has been considered as the single most informative stage independent prognostic factor in CLL.

Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses.

Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease.