IGHV Region Research Articles

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

Novel Oncogenic Non-Coding RNA：circRIC8B Regulates Lipid Metabolism Via Mir-199b-5p /LPL Axis in Chronic Lymphocytic Leukemia

Objective:During tumor development, energy constraints caused by malnourished microenvironments could exert selective pressure on cancer cells. Tumor cells are driven to metabolic reprogramming to meet the increased demand for energy and metabolites for their rapid proliferation and survival. Chronic lymphocytic leukemia (CLL) is a disease with about 1% of CLL cells proliferating every day which is highly than commonly thought. CLL cells were reported to maintain high levels of proliferation through metabolic changes, but extensive studies did not clearly explain the underlying mechanism of driving genes in CLL metabolism. Circular RNA (circRNA) has recently been shown to play an important role in cell metabolism through lipid accumulation. The purpose of this study is to explore the role of circRNA in lipid metabolism of CLL and provide novel therapeutic targets for CLL.Methods:To analyze circRNAs expression profiles and metabolism map in CLL, peripheral blood mononuclear cells (PBMC) from 53 treatment-naïve CLL patients were collected for transcriptome sequencing. Candidate circRNA circRIC8B in a larger cohort of patients was validated and the clinical characteristics were analyzed. Overexpression and knockdown virus were constructed to infect CLL cells, and untargeted metabolomics was used to find the key lipid metabolic pathway modulating by circRIC8B. The oncogenic functions of circRIC8B were further measured in CLL cell lines (MEC-1 and JVM-3) by performing CCK8 assay, flow cytometry, nile red staining and triglyceride detection. Moreover, we explored the molecular mechanisms of circRIC8B and verified the interactions among circRIC8B, miR-199b-5p and LPL by performing RNA-FISH, RIP, dual-luciferase reporter assay and Western blotting. The killing effects of lipid metabolism inhibitors on CLL cells were detected by CCK8 and flow cytometry.ResultsTranscriptome analysis showed that abnormal lipid metabolism was significantly related to the survival and prognosis of patients with CLL, and circRNAs could be involved in the regulation of lipid metabolism. Kaplan-Meier survival analysis confirmed that patients with higher fatty acid biosynthesis had a significantly lower OS (Figure 1A-B). circRIC8B which is positively correlated with the expression of lipoprotein lipase (LPL) was finally selected for further investigation. qRT-PCR analysis showed that circRIC8B was significantly higher expressed in CLL compared with healthy donors. Moreover, consistent with the sequencing results, circRIC8B was positively correlated with LPL and highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL (Figure 1C). Patients with higher circRIC8B level are associated with worse survival and advanced disease progression (Figure 1D and E). LC-MS/MS results showed that circRIC8B are able to modulated lipid metabolism of CLL cells. Functional analysis demonstrated the promoting role of circRIC8B in cell proliferation. Nile red staining showed lipid accumulation in CLL cells with circRIC8B overexpression increased significantly, while lipid accumulation in circRIC8B knockdown cells decreased significantly, and the quantitative results of triglycerides were similar.Next, we unraveled an original mechanism in CLL that up-regulated circRIC8B was mainly enriched in the cytoplasm, acted as a “sponge” of miR-199b-5p. CCK8 assay, nile red staining showed that the cell viability and lipid accumulating of CLL cell lines were evidently decreased after RNAi of circRIC8B and this result could be reversed by miR-199b-5p inhibitor transfection (Figure 1F-H). In addition, ezetimibe, one of the inhibitors of lipid metabolism was found effectively inhibit the proliferation and promote apoptosis of CLL cells.ConclusionsIn conclusion, as an independent prognostic factor of CLL, circRIC8B was involved in the progress of CLL disease through the miR-199b-5p/LPL axis. In addition, circRIC8B is a key factor in regulating lipid accumulation in CLL, resulting in significant changes in cellular lipid storage, thus supporting the proliferation of CLL cells. Metabolic inhibitor Ezetimibe can effectively block this process and exert anti-tumor functions. This study provides new clues for the role of circRNA in abnormal lipid metabolism of CLL and novel therapeutic strategy for CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Association of an IGHV3-66 gene variant with Kawasaki disease.

In a meta-analysis of three GWAS for susceptibility to Kawasaki disease (KD) conducted in Japan, Korea, and Taiwan and follow-up studies with a total of 11,265 subjects (3428 cases and 7837 controls), a significantly associated SNV in the immunoglobulin heavy variable gene (IGHV) cluster in 14q33.32 was identified (rs4774175; OR = 1.20, P = 6.0 × 10−9). Investigation of nonsynonymous SNVs of the IGHV cluster in 9335 Japanese subjects identified the C allele of rs6423677, located in IGHV3-66, as the most significant reproducible association (OR = 1.25, P = 6.8 × 10−10 in 3603 cases and 5731 controls). We observed highly skewed allelic usage of IGHV3-66, wherein the rs6423677 A allele was nearly abolished in the transcripts in peripheral blood mononuclear cells of both KD patients and healthy adults. Association of the high-expression allele with KD strongly indicates some active roles of B-cells or endogenous immunoglobulins in the disease pathogenesis. Considering that significant association of SNVs in the IGHV region with disease susceptibility was previously known only for rheumatic heart disease (RHD), a complication of acute rheumatic fever (ARF), these observations suggest that common B-cell related mechanisms may mediate the symptomology of KD and ARF as well as RHD.

Circ-RPL15: A Plasma Circular RNA As Novel Oncogenic Driver to Promote Progression of Chronic Lymphocytic Leukemia

Objective Chronic lymphocytic leukemia (CLL) is a heterogeneous disease and characterized by the accumulation of monoclonal B lymphocytes in blood, bone marrow and lymphoid tissues. Although diagnosis and therapy have been developing, several high-risk features for CLL patients have been identified, only few of them are well-characterized and clinically actionable. IGHV mutation status has been confirmed as a valuable independent prognostic factor. However, it is of vital importance to explore alternative markers because the detection method of IGHV mutation is expensive and time consuming, which limits its application in the routine laboratory diagnosis process. Thus, further exploration of effective diagnostic biomarkers for CLL are worthy and important. Circular RNAs (circRNAs), a novel family of non-coding RNAs, have been described as critical regulators of gene expression and are stable in exosomes. However, the compelling role of circRNAs in mediating CLL progression was still poorly understood. In this study, we attempted to identify a novel circRNA which could serve as a plasma biomarker and explore its molecular mechanism and clinical significance in CLL. Methods To analyse circRNAs expression profiles in CLL, plasma samples from three untreated CLL patients and five normal donors were collected for circRNA microarray. Then, we validated and analysed the clinical characteristics of candidate circRNAs in a larger cohort of patients (137 CLL patients vs. 50 healthy donors, age- and sex-matched). The receiver-operating characteristic (ROC) curve analysis was performed to evaluate the potential diagnostic value of circ-RPL15 as a biomarker for CLL, plasma and PBMC samples from multiple lymphomas were collected for qRT-PCR detection as well. The oncogenic functions of circ-RPL15 were further measured in CLL cell lines (MEC-1 and JVM-3) by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. Moreover, we explored the molecular mechanisms of circ-RPL15 and verified the interactions among POLR2A, circ-RPL15, miR-146b-3p and RAF1 by performing RNA-FISH, ChIP, RIP, dual-luciferase reporter assay and Western blotting. Results A total of 3,656 circRNAs were identified, among which 71 circRNAs were dysregulated in CLL patients ( p≤0.05) and circ-RPL15 showed the most significantly high expression level and was finally selected for further investigation. Circ-RPL15 was abundantly present in exosomes and was significantly higher expressed in CLL compared with normal and other lymphomas. Moreover, we found that circ-RPL15 was highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL. Patients without IGHV mutation presented a higher circ-RPL15 levels and the ROC curve showed that circ-RPL15 could be a good alternative to IGHV status with AUC values of 0.86. Kaplan-Meier analysis showed that CLL patients with circ-RPL15 high expression had a significantly poorer OS than those with low expression of circ-RPL15 (Figure A-F). Next, we unraveled an original mechanism in CLL that up-regulated circ-RPL15 was mainly enriched in the cytoplasm, acted as a "sponge" of miR-146b-3p. CCK8 assay and flow cytometry analysis showed that the cell viability of CLL cell lines were evidently decreased after RNAi of circ-RPL15 and this result could be reversed by miR-146b-3p inhibitor transfection. Western blot suggested a critical role of circRPL15- sponged miR-146b-3p in the regulation of RAS/RAF1/MEK/ERK pathway (Figure G-L). Furthermore, we identified circ-RPL15, a circRNA derived from exon 2 and partial exon 3 of RPL15 pre-mRNA which is located on chromosome 3p24.2, was negatively regulated by transcription factor POLR2A and delivered by exosomes into CLL patient plasma (Figure M,N). Conclusion In this study, we uncovered a novel oncogenic biomarker for CLL patients, which contributes to promoting CLL progression and is associated with poor outcome. The higher expression of circ-RPL15 was positively correlated with IGHV mutation status which is crucial in the evaluation of CLL prognosis. The miR-146b-3p mediated suppression of RAS/RAF1/MEK/ERK pathway could be alleviated by circ-RPL15 up-regulation in CLL. Our results revealed that circ-RPL15 may represent a promising plasma biomarker for the diagnosis and prognosis of CLL as well as a reliable surrogate of IGHV mutation status. Figure Disclosures No relevant conflicts of interest to declare.

RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

PS1122 RECURRENT XPO1 MUTATIONS IDENTIFIED AS PREDICTIVE MARKER FOR ADVANCED CLL PROGRESSION

Background:Exportin 1 (XPO1/CRM1) is responsible for nuclear‐to‐cytosol export of RNAs and protein cargos, including various tumor suppressors (e.g. p53, IκB) and growth regulators (e.g. RB, p21). Our previous work has shown that XPO1 is upregulated in chronic lymphocytic leukemia (CLL), leading to mislocalization of critical cargos, which may contribute to activation of pathways involved in leukemogenesis and disease progression. Whole exome sequencing has revealed recurrent mutations to a highly conserved amino acid (E571G/E571K) near the XPO1 cargo binding groove in ∼5% of CLL cases and in ∼25% of CLL patients that develop a clinical complication of their disease with a very abysmal prognosis known as Richter's transformation. Despite inherent association with advanced hematologic malignancies, conflicting reports describe the merit of XPO1 mutations as a predictive marker for poor clinical outcome in CLL.Aims:We aimed to assess the significance of recurrent XPO1 E571 mutations on CLL progression and overall outcome by concurrent analysis of a human CLL clinical cohort and a large scale evaluation of a novel XPO1 transgenic mouse model.Methods:Retrospective analysis of an independent sample set of CLL cases (N = 466) was used to correlate XPO1 mutation status (N = 18, >30% clonal population CLL cells) with clinical data. These included time to first treatment, overall survival, IgHV mutation status, ZAP‐70 expression (%), and status of epigenetic programming. Differential gene expression analysis in newly diagnosed untreated CLL patient lymphocytes was constructed from RNA‐sequencing, with downstream pathway analysis performed by Ingenuity Pathway Analysis software. A novel XPO1 transgenic mouse model (Eμ‐XPO1) was developed with human XPO1 (WT/G/K) gene under the heavy chain promoter/enhancer of the B cell expression, and was then crossed with the Eμ‐TCL1 mouse to assess contribution of XPO1 mutations in concordance with murine CLL. Disease onset and progression was determined via flow cytometry (% CD19/CD5 double positive cells) analysis of peripheral blood.Results:In the CLL cohort we analyzed, presence of XPO1 E571 mutations (18/466, 4%) confers disease characteristics consistent with poor clinical prognosis; including association with low epigenetic programming (18/18), unmutated IgHV region (17/17), elevated ZAP‐70 expression (p = 0.012), and reduction in treatment free progression (p = 0.002) and overall survival (p = 0.068) compared with XPO1 un‐mutated CLL. Similarly, overexpression of E571WT, K or G as the sole genetic abnormality (Eμ‐XPO1) resulted in the development of lymphoid neoplasms at advanced ages (>20 mo) compared to age‐matched B6 mice. Furthermore, overexpression of E571G (N = 174, p = 0.001) and E571K (N = 188, p = 0.093), but not E571WT (N = 151, p = 0.713) XPO1 showed a trend of accelerated disease onset in the Eμ‐XPO1xTCL1 model consistent with a role of mutant XPO1 in disease progression. Lastly, RNA‐seq in XPO1 E571 or wild‐type CLL cells revealed unique clustering of differentially expressed genes specific to lymphocyte activation, signaling, and regulation, suggesting amplification of pro‐survival and anti‐apoptotic intracellular pathways.Summary/Conclusion:We report evidence for XPO1 E571 mutations as a predictive marker for poor CLL clinical prognosis warranting further investigation for selective targeting of mutated XPO1 as a personalized cancer therapy in CLL.

Mantle cell lymphoma pathology update in the 2016 WHO classification

Mantle cell lymphoma (MCL) is an aggressive mature B-cell neoplasm genetically characterized by the presence of the t(11;14)(q13;q32) that leads to the constitutive overexpression of cyclin D1. The pathological and biological spectrum of this neoplasm has been expanded in recent years. This improvement in the knowledge of the disease has provided a better understanding of the diverse clinical evolution of the patients. The characterization of cyclin D1-negative MCL has led to the identification of cyclin D2 and D3 translocations as alternative mechanisms in this variant. Two major biological and clinical subtypes of the disease have been recognized, conventional and leukemic non-nodal MCL (nnMCL). MCL derives from CD5+ mature B-cells that have bypassed or experienced the germinal center microenvironment, retain a naive or memory-like epigenetic signature and carry a variable load of somatic mutations in the IGHV region; from truly unmutated to highly mutated, respectively. These two subtypes of tumors also differ in their genomic alterations, and clinical behavior, the conventional MCL (cMCL) being more aggressive than the leukemic nnMCL. This review will focus on the new aspects of the pathology of MCL in the updated 2016 WHO classification and its relevance for the clinical practice.

The Correlation of APOBEC Gene Family Member Expression with Worse CLL Patient Outcome Suggests a Role in CLL Mutational Evolution

A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL.CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System.CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome.To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented.In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. DisclosuresBarrientos:Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation

During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy chain variable (Igh-V) region compared to the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increases in both SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM.

Molecular Interrogation of Biclonal Multiple Myeloma for Clonal Relatedness.

AbstractAbstract 2928Multiple myeloma (MM) is caused by the clonal expansion of malignant plasma cells (PCs) producing high levels of monoclonal immunoglobulin (Ig) or M-protein. However, ∼2% of all patients with monoclonal gammopathies (MG) have more than 1 M-protein, a condition termed biclonal gammopathy or double monoclonal gammopathy (DMG). These patients can express M-proteins with different heavy chain (HC) isotypes and different light chains (LCs) or different HCs that are LC-matched. Reports suggest that ∼87% of DMGs with different HCs have matched LCs, but without molecular analysis at the nucleotide level, clonal relationships are unknown. Thus, DMG patients may have either two independent malignant PC clones that coincidentally express the same type of LC or the two clones are related and reflect previous or ongoing class switch recombination (CSR). This latter variant would offer novel insight into the origin and differentiation of the malignant clone. Of note, prior reports of HC-dissimilar/LC-matched DMGs are limited to the analysis of single patients and with few exceptions; do not include molecular analysis of the variable regions in Ig HC or LC genes at the nucleotide level. In this study, we interrogate a cohort of MM patients with known HC-dissimilar/LC-matched DMGs for the frequency of clonal relatedness. The cohort was identified by searching our MG database for patients with: 1) HC-dissimilar/LC-matched Igs as determined by immunofixation; 2) >10% bone marrow (BM) PCs; and 3) no transplant prior to BM collection. Eight patients (7 MM and 1 smoldering MM/primary amyloid) were identified for further molecular evaluation. All 8 cases expressed IgG and IgA HCs. Three patients expressed kappa LC (K-LC) and 5 expressed lambda LC (L-LC). To determine if the IgG and IgA M-proteins were clonally related in each patient, we performed Ig HC variable gene (IGHV) PCR with sense primers to all IGHV genes and an antisense primer specific to IgG or IgA. In all 8 patients, a single productive IGHV gene was determined with IgG and IgA. In 2/8 patients, the IgG and IgA HC variable regions were clearly distinct and therefore genuinely biclonal. Of note, PCs from 6/8 patients used the same IGH V-D-J genes in both IgG and IgA transcripts. In 5 of these 6 patients, the entire IgG and IgA variable region and HCDR3 were identical at nucleotide and amino acid (AA) levels. Of interest, one patient had common, as well as distinguishing IGHV region somatic mutations (SHM) between the IgG and IgA transcripts. The HCDR3 regions (including N nucleotides) were highly homologous but differed by 7 nucleotides which resulted in 3 AA differences between the 2 clonally related sequences. While these are compelling data, it remained possible that the malignant PCs in these patients simultaneously expressed IgG and IgA as a result of long nuclear transcripts. To test this possibility, we used immunofluorescence (IF) and verified the presence of both IgG and IgA expressing PCs in all cases. Parallel analysis of both K-LC and L-LC variable gene by PCR was done on the 6 patients with the same IGHV gene in both IgG and IgA transcripts. In 3/6 patients, there was a single somatically mutated LC identified corresponding to the LC detected by immunofixation. These results were also confirmed by IF. The remaining 3 patients appeared to express more than 1 LC rearrangement. Taken together these results confirm that HC-dissimilar/LC-matched DMG can present with various phenotypes at the molecular level. We describe the largest cohort to date documenting the frequency of DMG patients that can be classified as expressing 2 identical HCs with either a single LC or >1 LC based on molecular analysis. The former case may reflect an original progenitor cell that divided with one daughter cell subsequently experiencing further CSR yet the LC remained untouched. In the case where the HCs were slightly different, there may have been CSR with additional SHM of a daughter cell, or the molecular differences may reflect random somatic mutations occurring independent of germinal center reactions. These results suggest that HC-dissimilar/LC-matched DMG may occur more often than thought. Larger studies tracking M-protein levels over time accompanied by cytogenetic analyses are needed to determine if the two clonally-related class switch variants in DMG patients exhibit similar biological properties. This analysis would offer new insight into the genesis of this unique subtype of MM. Disclosures:No relevant conflicts of interest to declare.

Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1(-/-) pro-B cells are greatly impaired in distal V(H) gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V(H) genes close to the DJ(H) rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V(H) genes to undergo efficient rearrangement.

Assessment of Multiple Myeloma IGHV Intraclonal Variation by Massively Parallel Pyrosequencing

Abstract 1814Multiple myeloma (MM) is an incurable hematologic disorder characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Like normal B cells and PCs, MM cells express a distinctive immunoglobulin (Ig) molecular fingerprint resulting from heavy and light chain gene rearrangements and unique mutations introduced by the somatic hypermutation (SHM) process. Detailed examination of the Ig molecule may offer insight into the etiology of B cell malignancies as well as the ontogeny of malignant transformation and ongoing genetic evolution. Because prior studies of the Ig fingerprint of MM cells have been limited in size and scope, our current understanding of MM Ig repertoire is minimal. We used massively parallel sequencing of normal and malignant PCs to interrogate: 1) DNA versus RNA (cDNA) as starting material for studies of Ig repertoire; 2) the Ig repertoire of normal and malignant PCs; and 3) intraclonal heterogeneity in MM. Bone marrow PCs (BMPCs) from an untreated MM patient and from a normal control subject were isolated by magnetic bead separation. DNA and RNA were isolated from purified cells and amplified in a multiplex PCR reaction with primers designed to capture the entire IGHV region including the heavy chain complementarity determining region 3 (HCDR3) necessary for defining clonal cells. Using Roche 454 GS-FLX Titanium chemistry and a GS FLX Titanium Genome Sequencer, over 30,000 sequence reads were obtained from each sample. Following a detailed algorithm to minimize Taq and sequencing related errors and removal of non-Ig or non-productive Ig sequences, the resulting sequences were grouped based on HCDR3 amino acid identity. Each unique HCDR3 represented one member of the repertoire and all the sequences with the same HCDR3 defined the clone size. This measurement showed that there was nearly a five-fold higher level of unique HCDR3s in normal BMPCs when the starting PCR template was DNA vs. cDNA. Thus, DNA is clearly a better starting template for repertoire analysis using deep sequencing methods. Thorough analysis of the MM DNA sample identified PCs expressing 37 different IGHV genes, and predictably, the vast majority of sequence reads (83%) had exact nucleotide identity in the IGHV and HCDR3 region (IGHV3-74 with 18 mutations in the IGHV region) thereby defining the dominant MM clone. There were also ∼5000 additional sequences that either shared an identical HCDR3 region but varied within the IGHV3-74 gene sequence (n=3975) or were identical within the IGHV3-74 sequence yet varied within the HCDR3 (n=862). These sequences were further analyzed, with sequences not present 10 or more times or not found in both the 5’ and 3’ directions eliminated from further scrutiny. This process revealed a total of 64 putative subclones with single point mutations in the IGHV or HCDR3 region (10–56 copies of each unique sequence representing a total of 1583 clonally related sequences). When we used a more stringent subclone frequency of ≥0.1%, 22 subclones remained. Of note, conventional Ig repertoire analysis would require sequencing ≥1000 colonies to detect MM subclones present at this frequency. Finally, 11 subclones displayed a significant number of IGHV gene nucleotide differences from the dominant MM clone despite exhibiting an identical HCDR3 region. Notably, a number of these distinguishing IGHV gene mutations are silent and thus may preserve the antigenic specificity of these clonally related cells. These subclones failed our frequency criteria but the type and quality of deviations from the clonal sequence suggest that they are not artifact and warrant further investigation. Notably, all of the putative subclones were also found in the MM cDNA cohort providing further evidence as genuine subclones. The very large number of identical MM clonal sequences is consistent with the idea that MM is a post germinal center malignancy that can no longer undergo SHM. However, we present clear evidence of MM-related subclones albeit relatively small in number. The subclone sequence deviations discovered may reflect on-going genetic evolution independent of the formal SHM mechanism. Alternatively, these subclones may indeed reflect “sister” cells generated during the original germinal center reaction that resulted in the malignant clone. In summary, our studies demonstrate the extraordinary potential of this methodology to track clonal evolution in MM and its precursor conditions over time. Disclosures:No relevant conflicts of interest to declare.

MUTATIONAL STATUS of REARRANGED IMMUNOGLOBULIN HEAVY CHAIN VARIABLE Genes in a COHORT of SLOVENIAN PATIENTS with CHRONIC LYMPHOCYTIC LEUKAEMIA.

Abstract 4399 IntroductionThe aims of the study were to determine the mutational status and IGHV gene usage in our cohort of patients with B-CLL and compare the results to those published in the literature. Patients and Methods57 B-CLL patients (22 female, 35 male), median age 65 (range from 37 to 88 years) in various Binet stages (47 Binet A, 6 Binet B, 4 Binet C) were included in the study. All the patients were untreated at the time of blood collection. Leukaemia cells were isolated from venous blood samples of patients by Ficoll density gradient centrifugation. Total cellular RNA was isolated from leukaemia cells and used to the prepare cDNA by reverse transcription. After heteroduplex analysis, clonal PCR products were identified with polyacrylamide gel electrophoresis. The PCR products were sequenced and mutational status was determined by comparing the sequence of the IGHV region of the patient sample to the most homologous germ line V sequence. Sequence that was homologous more than 98 % with their corresponding germ line sequence was considered unmutated. ResultsUnmutated and mutated IGHV mutational status in our cohort of patients with B-CLL was found in 42 % (N=24) and 58 % (N=33), respectively.At the IGH subgroup level, the most frequently used subgroup was IGHV3 (38,55 %), followed by IGHV1 and IGHV4, found in 28,10 % and 22,85 %, respectively. Subgroups IGHV2 and IGHV5 were found out in 2 patients each (3,5 %). The other subgroups, IGHV6 and IGHV2, were determined in one patient each. At the specific gene level, the most frequently used gene IGHV was IGHV1-69, found out in 15,85 %. Genes V4-34, V4-39 in V3-23 were determined in 7,05 %, followed by genes V3-07, V3-11, V3-15, V3-30 and V3-48 identified in 5,25 % each. The others were identified at the lower frequency.In patients with unmutated IGHV genes, the most frequently observed gene was V1-69 (33,3 %). The most frequently used gene in mutated rearrangements was IGHV4-34 (12,13 %). The gene V3-21 was not found out in our cohort of patients. ConclusionsIn the study performed, we have found out that the portion of unmutated IGHV genes of the clonal B lymphocytes, the gene usage and somatic mutations frequency were similar to those published in the literature. Interestingly, the gene V3-21 was not found out in our cohort of patients, the results that is similar to the Mediterranean-based study. In the future, the geographic comparison and more definitive conclusions could be made on the population based investigations of IGHV gene use in large series of CLL patients in Slovenian region. Disclosures:No relevant conflicts of interest to declare.

Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis

AbstractSomatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

Telomeric IGH Losses Detectable by Fluorescence in Situ Hybridization in Chronic Lymphocytic Leukemia Reflect Somatic VH Recombination Events

Routine interphase fluorescence in situ hybridization (FISH) analysis of chronic lymphocytic leukemia (CLL) with LSI IGH/CCND1 assay, applied to differentiate CLL from leukemic mantle cell lymphoma, identified a subset of cases (42/174) with translocation-like IGH signal pattern. To unravel the underlying 14q32/IGH aberrations, 14 of these cases were subjected to cytogenetic, detailed FISH, and V(H) mutation analyses. FISH identified cryptic losses of various portions of the IGHV region in all 14 cases. Fine mapping of these V(H) deletions revealed a strict correlation between their distal border and localization of the used VH gene, suggesting that they are not oncogenic but reflect physiological events accompanying somatic V-D-J assembly. This hypothesis was further supported by FISH analysis of 20 CLL and hairy cell leukemia cases with the known V(H) usage showing a constant loss of sequences proximal to the used gene, identification of V(H) deletions in normal B cells, and their exclusive demonstration in B cell malignancies, but not of T cell and myeloid linage. Given that these cryptic physiological VH losses in B cells may seriously complicate analysis of B cell leukemia/lymphoma and lead to false conclusions, FISH users should take them into consideration when interpreting IGH aberrations in these malignancies.

Antibody repertoire development in teleosts—a review with emphasis on salmonids and Gadus morhua L

The group of teleosts is highly diverse, comprising more than 23000 extant species. Studies of the teleost antibody repertoire have been conducted in many different species within different orders, though some species and families have been better characterised than others. The Atlantic cod ( Gadus morhua L.) and several species within the Salmoninae (e.g. Salmo salar and Oncorynchus mykiss) are among the best-studied teleosts in terms of the antibody repertoire. The estimated size of the repertoire, the organisation of immunoglobulin (IG) gene segments, the expressed IG repertoire, the IgM serum concentration, and the serum antibody responses reveal some fundamental differences between these species. The serum IgM concentration of G. morhua is some ten times higher than that of S. salar, though G. morhua is characterised as a ‘low’ (or ‘non’) responder in terms of specific antibody production. In contrast, an antibody response is readily induced in S. salar, although the response is strongly regulated by antigen induced suppression. The IGHD gene of G. morhua has a unique structure, while the IGHM and IGHD genes of S. salar have a characteristic genomic organisation in two parallel loci. In addition, salmonids, express a broad repertoire of IGH and IGI V-region gene segments, while a single V gene family dominates the expressed heavy and light chain repertoire of G. morhua. Little is known about the developing antibody repertoire during ontogeny, in different stages of B-cell maturation, or in separate B-cell subsets. Information on the establishment of the preimmune repertoire, and the possible role of environmental antigens is also sparse.

Determination of gene organization in the human IGHV region on single chromosomes

Organization of the IGHV genes (n=108) on single human chromosomes has been determined by detecting these sequences in single sperm using multiplex PCR amplification followed by microarray detection. A total of 374 single sperm samples from five Caucasian males were studied. Three deletion/insertion polymorphisms (Del I-Del III) with deletion allele frequencies ranging from 0.1 to 0.3 were identified. Del I is a previously reported polymorphism affecting three IGHV genes (IGHV1-8, IGHV3-9, and IGHV2-10). Del II affects a region 2-18 kb containing two pseudogenes IGHV(II)-28.1 and IGHV3-29, and Del III spans approximately 21-53 kb involving genes IGHV4-39, IGHV7-40, IGHV(II)-40-1, and IGHV3-41. Deletion alleles of both Dels II and III were found in a heterozygous state, and therefore, could not be easily detected if haploid samples were not used in the study. Results of the present study indicate that deletions/insertions together with other possible chromosomal rearrangements may play an important role in forming the genetic structure of the IGHV region, and may significantly contribute to antibody diversity. Since these three polymorphisms are located within or next to the 3' half of the IGHV region, they may have an important role in the expressed IGHV gene repertoire during immune response.

Murine interstitial nephritis. VIII. Characterization of Igh-V restriction determinants in the development of anti-idiotypic immunity using blocking antibodies

We have prepared antisera specific for Igh-V-linked determinants (αIgh-V) in order to study Igh-V restriction during the development of protective, T cell-mediated anti-idiotypic immunity in autoimmune interstitial nephritis. Suppressor T cells in this network use an antigen-binding (RE-Id +) factor (TsF 1) secreted by first-order suppressor cells (Ts-1) to induce an anti-idiotypic (RE-αId +) soluble factor (TsF 2) released by effectorphase, Ts-2 suppressor cells. Each of these soluble suppressor factors requires homology in the Igh-V region to complete its regulatory functions. Our αIgh-V antisera, adsorbed against network idiotypes, can interfere with the Igh-V restriction used in the induction of Ts-2 suppression by TsF 1. The antisera bind both TsF 1 and TsF 2 in an allele-specific manner and are cytotoxic to induced Ts-2 cells, but not their precursors. These Igh-V determinants appear to behave like activation molecules. They lie outside of the ligand-binding site, do not map with the serologic binding of idiotype, and thus act as distinct associative-recognition elements in the maturation of anti-idiotypic immunity.

Growth inhibition of a B cell leukemia: evidence implicating an anti-idiotype immune response for protective tumor immunity.

BCL1, a spontaneous surface IgM (mu lambda)-positive (sIgM+) B cell leukemia of BALB/c (Igha) origin rarely grows in the Ig heavy chain (Igh) congenic mouse C.B-20 (Ighb) but is highly metastatic and lethal in the host strain of origin. Previous studies indicated that BCL1 tumor immunity in C.B-20 mice was associated with a T cell-mediated immune response against H-40, a minor histocompatibility (H) antigen controlled by a gene linked to the Igh locus. However, we observed that BCL1 leukemia grew progressively in BAB-14 (Igha/b) mice, a strain capable of generating an anti-H-40 immune response. This suggested that anti-H-40 immunity was insufficient for protection and implied that an Igh-V (variable) region gene product was also important for BCL1 growth inhibition. We therefore evaluated the role of two possible Igh-V region-linked gene products in BCL1 growth inhibition; namely, an Igh-V region-linked minor H antigen or alternatively the BCL1 IgM idiotype (Id). We could find no evidence for an Igh-V region-linked minor H antigen because immunosuppressed (500 R) CB-20 mice reconstituted with C.B-20 anti-BAB-14 splenocytes were susceptible to BCL1 growth, whereas recipients reconstituted with C.B-20 anti-BALB/c splenocytes were resistant to BCL1 challenge. In contrast, C.B-20 mice immunized against purified BCL1 IgM protein could adoptively confer BCL1 tumor immunity. C.B-20 mice immunized against other BALB/c IgM myeloma proteins containing either lambda or kappa light chains failed to protect C.B-20 mice suggesting that recognition of a unique determinant (Id) and not an allotype was crucial for tumor immunity. The BCL1 mu-chain appeared to make the major contribution to the idiotypic determinant because a hybridoma product composed of BCL1 mu-chains and BALB/c kappa-chains still elicited BCL1 immunity. Adoptive transfer of C.B-20 anti-BCL1 Id splenocytes into irradiated recipients that prevented an anti-H-40 response due to H-40 tissue expression failed to adoptively confer BCL1 immunity. Thus, these data suggest that BCL1 growth inhibition requires a T cell-mediated response against both H-40 and the BCL1 Id; these responses must be elicited concurrently in the tumor-bearing host to achieve protective BCL1 immunity.

Genetic control of the murine IgM plaque-forming cell response to type III pneumococcal polysaccharide.

The direct 5-day plaque-forming cell response of different inbred mouse strains to pneumococcal polysaccharide type III (SSS-III) varied from more than 10,000 per spleen, in BALB/c mice, to less than 2000 in C57BL mice. Responses of Igh congenic and recombinant inbred lines bearing different combinations of BALB/c and C57BL genes indicate that two or more gene loci are involved in controlling high or low responses. At least one is in the Igh-V region since BALB/c, BAB-14, and CB-8 KN mice (Igh-Va) had two to four times higher responses than CB-20 and CB-16 KN mice (Igh-Vb). Other gene loci must be involved, but nothing can be said about them at present.