Immunoglobulin Kappa Locus Research Articles

Routine Evaluation of Minimal Residual Disease in Myeloma Using Next-Generation Sequencing Clonality Testing: Feasibility, Challenges, and Direct Comparison with High-Sensitivity Flow Cytometry

The 2016 International Myeloma Working Group consensus recommendations emphasize high-sensitivity methods for minimal residual disease (MRD) detection, treatment response assessment, and prognostication. Next-generation sequencing (NGS) of IGH gene rearrangements is highly specific and sensitive, but its description in routine clinical practice and performance comparison with high-sensitivity flow cytometry (hsFC) remain limited. In this large, single-institution study including 438 samples from 251 patients, the use of NGS targeting the IGH and IGK genes for clonal characterization and monitoring, with comparison to hsFC, is described. The index clone characterization success rate was 93.6% (235/251), which depended on plasma cell (PC) cellularity, reaching 98% when PC ≥10% and below 80% when PC <5%. A total of 85% of cases were successfully characterized using leader and FR1 primer sets, and most clones showed high somatic hypermutation rates (median, 8.1%). Among monitoring samples from 124 patients, 78.6% (147/187) had detectable disease by NGS. Concordance with hsFC was 92.9% (170/183). Discordant cases encompassed 8 of 124 hsFC MRD+/NGS MRD− patients (6.5%) and 4 of 124 hsFC MRD−/NGS MRD+ patients (3.2%), all with low-level disease near detection limits for both assays. Among concordant hsFC MRD−/NGS MRD− cases, only 5 of 24 patients (20.8%) showed subsequent overt relapse at 3-year follow-up. HsFC and NGS showed similar operational sensitivity, and the choice of test may depend on practical, rather than test performance, considerations.

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Real World Observational Study Using Clonoseq® Next Generation Sequencing in Hematologic Malignancies: The ’Watch’ Registry

Real World Observational Study Using Clonoseq® Next Generation Sequencing in Hematologic Malignancies: The ’Watch’ Registry

Extraosseous Plasmacytoma of Thyroid Arising in Hashimoto’s Thyroiditis

Plasma cell neoplasms of the thyroid gland are uncommon. They may occur either as a primary extraosseous (extramedullary) plasmacytoma or as secondary involvement by multiple myeloma (MM). Here, we report the case of a 62-year-old female, presenting with goiter and Hashimoto’s thyroiditis, in whom the histologic diagnosis of extraosseous plasmacytoma was unexpected. Histology of the total thyroidectomy specimen showed a diffuse infiltration of well-differentiated plasma cells against a background of Hashimoto’s thyroiditis. By immunohistochemistry, the majority of the plasma cells are positive for IgG heavy chain and kappa light chain (kappa:lambda ratio was about 6-7:1). PCR analysis of the immunoglobulin heavy and kappa chain (IGH, IGK) gene rearrangements showed clonal IGH and IGK gene rearrangements. MM was ruled out by lack of MM-related end organ damage and negative serum protein electrophoresis, immunofixation, and bone marrow biopsy. Although rare, plasmacytoma should be considered in patients presenting with enlarging thyroid gland and autoimmune thyroiditis. Histologic diagnosis and differential diagnoses are comprehensively discussed.

Immunoglobulin gene rearrangement and BAFF responsive maturation defines a novel B cell population undergoing extra-BM development

Abstract Transitional type-1 (T1) cells are peripheral immature B cells known to populate the spleen (spl) after completing their BCR assembly in the bone marrow (BM). To advance the understanding of splenic T1 (CD19posCD24hiCD21neg) B cells, we addressed the heterogeneity and biology of these cells using flow cytometry combined with genetically modified mice. Most recent emigrant T1 cells were selected by excluding CD23pos and including CD93high (AA4.1) B cells termed T12123DN. Transcriptomic analysis identified RAG1 and 2 as signature genes for this B cell population. Further separation of T12123DN cells based on surface IgM expression revealed a previously undescribed cell subset with undetectable cell surface IgM (-IgMneg). The spl-IgMneg subsets expresses RAG1/2 and actively undergoes Igk gene rearrangement at levels comparable to BM pre-B cells. Upon in vitro exposure to BAFF or transplantation into immunodeficient hosts, spl-IgMneg cells can give rise to fully mature IgMposIgDpos B cells. Furthermore, BAFF-R and NF-kB pathways are required for their efficient maturation. Our findings suggest that the spl-T1 population encompasses a subset of B cells that resemble but are distinct from the developing B cells in the BM. These spl-IgMneg B cells may represent receptor editing B cells, and/or precursor B cells undergoing BCR assembly and selection in the periphery, possibly providing an opportunity for tolerance induction to tissue restricted self-antigens and microbiota-derived antigens.

B-cell acute lymphoblastic leukemia with t(2;9)(p11;p13) involving the immunoglobulin kappa locus (IGK) and PAX-5

B-cell acute lymphoblastic leukemia with t(2;9)(p11;p13) involving the immunoglobulin kappa locus (IGK) and PAX-5

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Igcaller: Reconstructing the Rearranged Immunoglobulin Gene in Lymphoid Neoplasms from Whole-Genome Sequencing Data

Introduction: Mature normal and tumor B cells express a unique rearranged immunoglobulin (IG) gene that can be used as a marker of the clonal expansion of the cell. Somatic hypermutation (SHM) in the V(D)J region of IG genes are acquired in the germinal center and are a surrogate imprint of the cell of origin of lymphoid neoplasms. In chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the identification of SHM distinguishes subtypes of tumors with different clinical and biological behavior. Although still less studied, cases carrying highly similar IG sequences (i.e. stereotyped IG), specific light chain (LC) rearrangements, and the presence of class switch recombination (CSR) of the constant region of the heavy chains further sub-classify patients into potentially distinct clinico-biological subgroups. The analysis of the rearranged IG gene is currently performed by specific Sanger sequencing (SSeq) or next-generation sequencing protocols. Whole-genome sequencing (WGS) of B-cell neoplasms should store the information to reconstruct the entire rearranged IG gene [heavy (IGH) and kappa or lambda (IGK, IGL) LC]. However, the high genomic complexity and homology of these regions have prevented the analysis of the rearranged IG genes in WGS using standard bioinformatics pipelines. Aim: To assess the use of WGS data to fully characterize the rearranged IG gene in B-cell neoplasms. Methods: We developed IgCaller, a fast, easy-to-run program that uses already aligned WGS data to dissect the rearranged IGH V(D)J genes, IGK and IGL VJ genes, and the presence of constant heavy chain CSR. IgCaller also determines the homology of the rearranged sequences compared to the patient's germ line or reference genome. We demonstrated the accuracy of IgCaller using WGS data of 331 B-cell neoplasms [240 CLL (152 cohort 1 (C1)-CLL; 88 independent cohort 2 (C2)-CLL), 61 MCL, and 30 multiple myeloma (MM)] and compared with SSeq of the IGH V(D)J and/or LC and isotype expression. Results: IgCaller identified a complete IGH productive rearrangement [V(D)J] in 133 (88%) C1-CLL, 80 (91%) C2-CLL, 61 (100%) MCL, and 21 (70%) MM. A partial (VJ) rearrangement was detected in 8 (5%) C1-CLL and 1 (3%) MM. Available SSeq of the V(D)J or at least V gene for 131 C1-CLL, 10 C2-CLL, and 60 MCL successfully characterized by WGS highlighted only one discordant V(D)J rearrangement. Small discrepancies (only J or V disagreement) were found when the J (n=4) or V (n=1) gene identified by SSeq based on homology (IMGT/V-QUEST tool) did not correspond to the rearranged gene detected by WGS, which was the second scoring gene in IMGT/V-QUEST suggesting that our non-homology WGS-based approach might be more accurate. Of note, IgCaller identified the presence of two distinct IGH subclones in 1 case. Next, the comparison of the percentage of homology of the rearranged sequence to the germ line in 131 C1-CLL and 60 MCL with complete V gene both by SSeq and WGS showed a high correlation and concordance in both cohorts [R&gt;0.95, p&lt;1e-30; Passing Bablok regression: 0.05+1*SSeq (CLL), -0.19+1*SSeq (MCL)]. Only 1 CLL patient was differentially classified as mutated or unmutated between SSeq (complete rearrangement) and WGS (partial rearrangement) using the cut off of 98%. A productive IGK or IGL was found in 147 (97%) C1-CLL, 85 (97%) C2-CLL, 61 (100%) MCL, and 26 (87%) MM. These results fully agreed with the LC expression observed by flow cytometry (FC). Of clinical relevance, a total of 26/232 (11%) CLL carried IGLV3-21. The presence of IGLV3-21 was associated with a shorter time to first treatment (TTFT) independently of the IGHV status (p=0.045). Finally, IgCaller identified the CSR matching the isotype expressed by FC analysis in 26/30 (87%) MM. One of the 4 discordant cases with IgM by WGS expressed only LC by FC. Overall, IgCaller could not identify the isotype switch in 3 (10%) MM expressing IgG or IgA. In CLL, CSR was observed in 47/240 (20%) cases. Noteworthy, the presence of CSR identified 24% of M-CLL patients with a shorter TTFT than non-switched M-CLL (p=0.004), and similar to that of unmutated IGHV cases (p=0.15). Conclusions: IgCaller successfully characterized the entire IG gene of &gt;90% B-cell neoplasms studied. The complete characterization of the rearranged IG gene based on WGS data, when available, could facilitate the analysis of LC rearrangements and CSR, and replace the traditional SSeq of the IG loci both in research and clinical settings. Disclosures No relevant conflicts of interest to declare.

Moffitt Cancer Center 2-Year Single-Institution Experience with Next-Generation Sequencing Minimal Residual Disease Detection: Clinical Utility, Application, and Correlation with Outcomes in Plasma Cell and Lymphoid Malignancies

Moffitt Cancer Center 2-Year Single-Institution Experience with Next-Generation Sequencing Minimal Residual Disease Detection: Clinical Utility, Application, and Correlation with Outcomes in Plasma Cell and Lymphoid Malignancies

The application of antigen receptor gene rearrangement of BIOMED-2 in the pathologic diagnosis of 348 cases with non-Hodgkin lymphoma in a single institution in Southwest of China

ObjectiveTo explore the clinical value of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangement in the diagnosis of non-Hodgkin lymphoma. MethodsUsing the standardized BIOMED-2 multiplex PCR strategy to detect IgH, IgK and TCR in 272 cases of mature B-cell lymphoma, 55 cases of mature T-cell lymphoma, 21 cases of extranodal NK/ T-cell lymphoma, nasal type, and 20 cases of lymphoid tissue reactive hyperplasia. ResultsAmong all mature B-cell lymphomas, the sensitivity of Ig gene rearrangement was 91.18% (248/272), IgH and IgK gene rearrangement was 76.47% (208/272) and 75.00% (204/272), respectively, meanwhile the sensitivity of TCRγ rearrangement was 3.68% (10/272). In the 55 cases of mature T-cell lymphoma, the sensitivity of the detection of TCRγ was 76.36% (44/55), at the same time the sensitivity of Ig gene rearrangement was 14.55% (8/55), IgH and IgK gene rearrangement was 7.27% (4/55) and 12.73% (7/55), respectively. In 21 cases of extranodal NK/T cell lymphoma, nasal type, and 20 cases of reactive lymphoid hyperplasia, no gene rearrangement was found in the samples of IgH, IgK and TCR. The sensitivity of gene rearrangement in Ig/TCR in B and T-cell lymphoma was significantly different from that in the control group (P < 0.05). ConclusionThe Ig/TCR gene rearrangement of BIOMED-2 multiplex PCR strategy has important auxiliary value in the diagnosis of B/T-cell non-Hodgkin lymphoma respectively, however, a few B-cell lymphomas may company TCR gene rearrangement as well as a few T-cell lymphomas may accompany Ig gene rearrangement, it must be comprehensively judged with the combination of morphology, immunohistochemistry and clinical features.

Influence of metabolic status and genetic merit for fertility on proteomic composition of bovine oviduct fluid†.

The oviduct plays a crucial role in fertilization and early embryo development providing the microenvironment for oocyte, spermatozoa, and early embryo. Since dairy cow fertility declined steadily over the last decades, reasons for early embryonic loss have gained increasing interest. Analyzing two animal models, this study aimed to investigate the impact of genetic predisposition for fertility and of metabolic stress on the protein composition of oviduct fluid. A metabolic model comprised maiden Holstein heifers and postpartum lactating (Lact) and non-lactating (Dry) cows, while a genetic model consisted of heifers from the Montbéliarde breed and Holstein heifers with low- and high-fertility index. In a holistic proteomic analysis of oviduct fluid from all groups using nano-liquid chromatography tandem-mass spectrometry analysis and label-free quantification, we were able to identify 1976 proteins, among which 143 showed abundance alterations in the pairwise comparisons within both models. Most differentially abundant proteins were revealed between low fertility Holstein and Montbéliarde (52) in the genetic model and between lactating and maiden Holstein (19) in the metabolic model, demonstrating a substantial effect of genetic predisposition for fertility and metabolic stress on the oviduct fluid proteome. Functional classification of affected proteins revealed actin binding, translation, and immune system processes as prominent gene ontology (GO) clusters. Notably, Actin-related protein 2/3 complex subunit 1B and the three immune system-related proteins SERPIND1 protein, immunoglobulin kappa locus protein, and Alpha-1-acid glycoprotein were affected in both models, suggesting that abundance changes of immune-related proteins in oviduct fluid play an important role for early embryonic loss.

Improved clonality analysis based on immunoglobulin kappa locus for canine cutaneous plasmacytoma

Sensitivity of clonality analysis based on immunoglobulin heavy chain (IGH) in canine cutaneous plasmacytoma is lower than that in diffuse large B cell lymphoma (DLBCL) because of somatic hypermutation occurring at the IGH locus. Therefore, this study aimed to improve the sensitivity of clonality analysis for canine cutaneous plasmacytoma. To achieve this, clonality analysis based on the immunoglobulin kappa chain (IGK) locus was established. Sensitivity and specificity were examined in genomic DNA extracted from formalin-fixed paraffin-embedded sections of cutaneous plasmacytomas, DLBCLs, and lymph nodes without lymphoma. Forward primers were designed based on the IGKV genes, and reverse primers were designed based on the IGKJ genes and kappa deleting element (Kde). Analysis using IGKV and IGKJ primers demonstrated clonality in 24 of 29 cutaneous plasmacytomas (82.8%), while analysis with primers for IGKV and Kde showed clonality in 16 of 29 cases (55.2%). In DLBCL, the IGKV and IGKJ primer set yielded clonality in 18 of 23 cases (78.3%), and the IGKV and Kde primer set yielded 9 of 23 cases (39.1%). No clonal results were obtained from 23 lymph nodes without lymphoma. Sensitivity of the IGKV and IGKJ primer set was significantly higher than that of the IGH primers reported previously. Thus, clonality analysis based on the IGK locus can be utilized for canine B cell tumors. In conclusion, clonality testing based on IGH and IGK may be beneficial as an adjunct tool for diagnosis of canine B cell tumors including cutaneous plasmacytoma.

PB1684 THE FIRST‐REPORTED PATIENT WITH NATURAL KILLER‐LYMPHOBLASTIC LEUKEMIA/LYMPHOMA IN KOREA

Background:Natural killer (NK)‐lymphoblastic leukemia/lymphoma is a rare hemopoietic neoplasm that was established as an independent entity by the WHO in 2008 and in 2016, it was recategorized as a precursor lymphoid neoplasm. Its identification remains challenging because its features overlap with those of other hematological malignancies, and markers specific to NK‐cells such as CD94 and CD161 are not commonly tested. Owing to the disease's rarity, only 3 patients who meet the 2008 WHO criteria have been reported.Aims:We report the first Korean patient with NK‐lymphoblastic leukemia/lymphoma including her NGS‐based genetic profile and clinical course.Methods:.Results:A 51‐year‐old woman was referred to our institution with her WBC count 1.6 × 109/L, hemoglobin 11.9 g/dL, and platelet count 176 × 109/L. Peripheral blood smear showed a few abnormal‐looking large lymphocytes, while BM aspirate showed decreased normal hemopoietic precursors and increased blasts (63.0%). The blasts were large and had a high nuclear/cytoplasmic ratio with agranular cytoplasm, nuclei with indentation, and conspicuous nucleoli. All were negative for peroxidase and Sudan Black B but some were positive for acid alpha‐naphthyl acetate esterase. BM biopsy showed hypocellularity (10%) but increased immature cells that were positive for CD34 and terminal deoxynucleotidyl transferase (TdT) on immunohistochemistry (IHC). Flow cytometry showed that the majority of blasts were positive for HLA‐DR, CD7, CD34, and CD56 but negative for myeloid antigens (CD13, CD33, CD14, CD117, myeloperoxidase), B‐cell antigens (CD10, CD19, CD20, CD79a), and other T‐cell antigens (CD2, cytoplasmic CD3, surface CD3, CD4, CD5, CD8). The BM karyotype was 46,XX,‐7,del(7)(q35),add(11)(q24),‐12,+1mar,+2mar[10]/46,XX[10]. Multiplex reverse‐transcriptase PCR revealed negativity for all 28 tested leukemia‐causing chromosomal translocations. Targeted next‐generation sequencing of 54 genes, including BRAF, IKZF1, KRAS, MYD88, NRAS, and TP53, showed no oncogenic mutations; tests for TCRB, IGH, and IGK gene rearrangement showed no dominant clonotypes. Hence, she was diagnosed with NK‐lymphoblastic leukemia/lymphoma. Physical and imaging examinations revealed no organomegaly, lymph node enlargement, or skin lesions. The patient underwent 3 cycles of fludarabine and cyclophosphamide for 2 months and has been stable for 6 months since her diagnosis.Summary/Conclusion:The blasts of our patient was positive for CD34, TdT, HLA‐DR, CD7, and CD56 but not for other T‐cell, B‐cell, or myeloid markers; this ruled out T‐acute lymphoblastic leukemia/lymphoma (T‐ALL) and mixed‐phenotype acute leukemia. The lack of specific TCR and IG clonotypes also ruled out T‐ALL. The morphology of blasts and negativity for CD4 ruled out the possibility of blastic plasmacytoid dendritic cell neoplasm (BPDCN). Further analysis for CD4, CD56 and CD123 using IHC will be helpful to verify the differential diagnosis of NK‐lymphoblastic leukemia/lymphoma from BPDCN.

Abstract B099: Elucidating the mechanisms that underpin RAG chromatin scanning in V(D)J recombination at antigen receptor gene loci

Abstract Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons are assembled from V, D and J segments by V(D)J recombination, which is initiated by RAG1/RAG2 endonuclease (RAG) that introduces DNA double-stranded breaks (DSBs) between a pair of V, D, and J coding segments and flanking recombination signal sequences (RSSs). IgH V(D)J recombination is ordered, with D-JH joining occurring before appendage of a VH to a DJH intermediate in pro-B cells. In pre-B cells, a single-step VL-to-JL recombination event is invoked at IgL loci, with Ig kappa rearrangement usually preceding that of Ig lambda. The mechanisms of Ig V(D)J recombination, especially long-range VH-to-DJH and Vkappa-to-Jkappa recombination are still not fully understood. Recently, our work showed CTCF-binding elements (CBEs) immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSS alone. This CBE activity becomes particularly striking upon the inactivation of IgH IGCR1 control element, which promotes DJH intermediate formation by insulting Ds, JHs, and recombination center (RC) from upstream VHs in a CBE-anchored chromatin loop. By developing and applying 3C-HTGTS, a new high-throughput chromosome conformation capture method that shows great advantages over classical 4C-seq in sensitivity and specificity, we found VH-associated CBEs promotes interaction with the RC. Based on these, we proposed a RAG chromatin scanning model for V(D)J recombination, in which VH CBEs stabilize interactions of D-proximal VHs first encountered by the DJH RC during linear RAG scanning on chromatin and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function. However, it is still not known if such chromatin scanning also happens on other antigen receptor loci and what the underlying mechanism is. Here, we will show our new findings on the roles of RAG chromatin scanning in Vkappa-to-Jkappa recombination at Ig kappa locus by multiple comprehensive approaches. Moreover, we will also discuss our most updated understanding of the mechanisms underlying RAG chromatin scanning using a novel and sophisticated approach. The potent mutagenic and oncogenic effects of aberrant RAG chromatin scanning at RAG “off-target” sites genome-wide will be also discussed. Citation Format: Zhaoqing Ba, Suvi Jain, Jiazhi Hu, Frederick Alt. Elucidating the mechanisms that underpin RAG chromatin scanning in V(D)J recombination at antigen receptor gene loci [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B099.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B‐cell neoplasms

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

Improved clonality detection in Hodgkin lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH and IGK rearrangements: A paraffin-embedded tissue study.

The BIOMED-2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin-embedded tumor sections. We previously described the usefulness of the semi-nested BIOMED-2 IGH assay in B-cell malignancies. In this study, we devised a novel semi-nested BIOMED-2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED-2, BIOMED-2 followed by BIOMED-2 re-amplification, and BIOMED-2 followed by semi-nested BIOMED-2, all targeting IGH and IGK, using paraffin-embedded tissues. In both IGH and IGK assays, semi-nested assays yielded significantly higher clonality detection rates than the standard assays and re-amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi-nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30-positive cell density, CD20-positive cell density, or Epstein-Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two-thirds of classical HL cases using semi-nested BIOMED-2 IGH/IGK assays using paraffin tumor sections. These semi-nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs.

Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci

Dogs are an excellent model for human disease. For example, the treatment of canine lymphoma has been predictive of the human response to that treatment. However, an incomplete picture of canine (Canis lupus familiaris) immunoglobulin (IG) and T cell receptor (TR)—or antigen receptor (AR)—gene loci has restricted their utility. This work advances the annotation of the canine AR loci and looks into breed-specific features of the loci. Bioinformatic analysis of unbiased RNA sequence data was used to complete the annotation of the canine AR genes. This annotation was used to query 107 whole genome sequences from 19 breeds and identified over 5500 alleles across the 550 genes of the seven AR loci: the IG heavy, kappa, and lambda loci; and the TR alpha, beta, gamma, and delta loci. Of note was the discovery that half of the IGK variable (V) genes were located downstream of, and inverted with respect to, the rest of the locus. Analysis of the germline sequences of all the AR V genes identified greater conservation between dog and human than mouse with either. This work brings our understanding of the genetic diversity and expression of AR in dogs to the same completeness as that of mice and men, making it the third species to have all AR loci comprehensively and accurately annotated. The large number of germline sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci.

The value of detecting immunoglobulin gene rearrangements in the diagnosis of B-cell lymphoma.

ObjectiveTo discuss the clinical value of immunoglobulin gene rearrangements in the diagnosis of B-cell lymphoma.MethodsA total of 209 cases of B-cell lymphomas and 35 cases of reactive lymphoid hyperplasia were selected for DNA extraction and PCR amplification using the BIOMED-2 primer system. Gel electrophoresis of heteroduplexes was used to analyze immunoglobulin gene rearrangements.ResultsA total of 209 cases of B-cell lymphoma, including 69 extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue, 63 diffuse large B-cell lymphomas, 39 follicular lymphomas, 15 small lymphocytic lymphomas, 6 plasmacytomas, 6 mantle cell lymphomas, 7 nodal marginal zone B-cell lymphomas, and 4 lymphoplasmacytoid lymphomas, were examined. Immunoglobulin gene rearrangements were found in all 209 cases, with 93 IGHA, 122 IGHB, 98 IGHC, 167 IGK, 100 IGL, 167 IGHA/B/C, 204 IGH/IGK, 209 IGH/IGK/IGL, 129 IGH+IGK, 81 IGH+IGL, 83 IGK+IGL and 68 IGH+IGK+IGL gene rearrangements. Immunoglobulin gene rearrangements were not found in the 35 cases of reactive lymphoid hyperplasia. IGH and IGK gene rearrangements were mainly found in mantle cell lymphomas, small lymphocytic lymphomas, extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue and diffuse large B-cell lymphomas. The IGH gene rearrangement was mainly found in lymphoplasmacytoid lymphomas and follicular lymphomas. IGK and IGL gene rearrangements were mainly found in plasmocytoma, and the IGK gene rearrangement was mainly found in nodal marginal zone B-cell lymphomas.ConclusionsThe BIOMED-2 standardized immunoglobulin gene rearrangement detection system is an important tool in B-cell lymphoma diagnosis. Analysis of IGH, IGK and IGL gene rearrangements is valuable in confirming the classification of B-cell NHL.