An Exploratory Approach of Clinically Useful Biomarkers of Cvid by Logistic Regression.

Despite advancements in genetic and functional studies, the timely diagnosis of common variable immunodeficiency (CVID) remains a significant challenge. This exploratory study was designed to assess the diagnostic performance of a novel panel of biomarkers for CVID, incorporating the sum of κ+λ light chains, soluble B-cell maturation antigen (sBCMA) levels, switched memory B cells (smB) and the VISUAL score. Comparative analyses utilizing logistic regression were performed against established gold-standard tests, specifically antibody responses. Our research encompassed 88 subjects, comprising 27 CVID, 23 selective IgA deficiency (SIgAD), 20 secondary immunodeficiency (SID) patients and 18 healthy controls. We established the diagnostic accuracy of sBCMA and the sum κ+λ, achieving sensitivity (Se) and specificity (Spe) of 89% and 89%, and 90% and 99%, respectively. Importantly, sBCMA showed strong correlations with all evaluated biomarkers (sum κ+λ, smB cell and VISUAL), whereas the sum κ+λ was uniquely independent from smB cells or VISUAL, suggesting its additional diagnostic value. Through a multivariate tree decision model, specific antibody responses and the sum κ+λ emerged as independent, signature biomarkers for CVID, with the model showcasing an area under the curve (AUC) of 0.946, Se 0.85, and Spe 0.95. This tree-decision model promises to enhance diagnostic efficiency for CVID, underscoring the sum κ+λ as a superior CVID classifier and potential diagnostic criterion within the panel.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry.

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

Molecular basis for differential Igk versus Igh V(D)J joining mechanisms.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1-3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.

Synchronous multiple myeloma and lung adenocarcinoma: A clinical series.

BACKGROUND: Multiple myeloma (MM) is characterised by an increased number of monoclonal immunoglobulin-producing plasma cells that malignantly grow in the bone marrow. Lung cancer is one of the most common malignancies and at the advanced stage may become metastatic to the bone. Rarely, MM and lung cancer are synchronously present in the same patient. RESULTS: In this report, we describe five cases of MM synchronous with lung adenocarcinoma including λ light chain in three cases and Ï° light chain in two cases. Two patients achieved complete remission, and no progression was seen in two patients. CONCLUSION: In conclusion, synchronous MM and lung adenocarcinoma are clinically rare, and diagnosis should be made scrupulously based on morphology, immunology, cytogenetics, molecular biology and biopsy pathology.

Sequence diversity of kappa light chains from patients with AL amyloidosis and multiple myeloma.

BACKGROUND: AL amyloidosis (AL) results from the misfolding of immunoglobulin light chains (IG LCs). Aim of this study was to comprehensively analyse kappa LC sequences from AL patients in comparison with multiple myeloma (MM). OBJECTIVE: We analysed IGKV/IGKJ usage and associated organ tropism and IGKV1/D-33 in terms of mutational analysis and theoretical biochemical properties. MATERIAL AND METHODS: cDNA and bulk RNA sequencing of the LCs of AL and MM patients. RESULTS: We studied 41 AL and 83 MM patients showing that IGKV1 was most expressed among kappa AL and MM, with higher frequency in AL (80% vs. 53%, p = .002). IGKV3 was underrepresented in AL (10% vs. 30%, p = .014). IGKJ2 was more commonly used in AL than in MM (39% vs. 29%). Patients with IGKV1/D-33 were associated with heart involvement (75%, p = .024). IGKV1/D-33-segments of AL had a higher mutation count (AL = 12.0 vs. MM = 10.0). FR3 and CDR3 were most frequently mutated in both, with a median mutation count in FR3 being the highest (AL = 4.0; MM = 3.5) and one mutation hotspot (FR3 (83I)) for IGKV1/D-33/IGKJ2 was associated with cardiac involvement. CONCLUSION: This study confirmed that germline usage has an influence on AL amyloidosis risk and organ involvement.

Spi-C and PU.1 Counterregulate Rag1 and Igκ Transcription to Effect Vκ-Jκ Recombination in Small Pre-B Cells.

B cell development requires the ordered rearrangement of Ig genes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags. Igκ rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26 transformation-specific transcription factor Spi-C is activated in response to dsDNA double-stranded breaks in small pre-B cells to negatively regulate pre-BCR signaling and Igκ rearrangement. However, it is not clear if Spi-C regulates Igκ rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation of Igκ L chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulated Igκ rearrangement, Igκ transcript levels, and Rag1 transcript levels. We found that Igκ and Rag1 transcript levels were increased in small pre-B cells from Spic-/- mice. In contrast, Igκ and Rag1 transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in the Rag1 promoter region. These results suggest that Spi-C and PU.1 counterregulate Igκ transcription and Rag1 transcription to effect Igκ recombination in small pre-B cells.

Enhancer-instructed epigenetic landscape and chromatin compartmentalization dictate a primary antibody repertoire protective against specific bacterial pathogens.

Antigen receptor loci are organized into variable (V), diversity (D) and joining (J) gene segments that rearrange to generate antigen receptor repertoires. Here, we identified an enhancer (E34) in the murine immunoglobulin kappa (Igk) locus that instructed rearrangement of Vκ genes located in a sub-topologically associating domain, including a Vκ gene encoding for antibodies targeting bacterial phosphorylcholine. We show that E34 instructs the nuclear repositioning of the E34 sub-topologically associating domain from a recombination-repressive compartment to a recombination-permissive compartment that is marked by equivalent activating histone modifications. Finally, we found that E34-instructed Vκ-Jκ rearrangement was essential to combat Streptococcus pneumoniae but not methicillin-resistant Staphylococcus aureus or influenza infections. We propose that the merging of Vκ genes with Jκ elements is instructed by one-dimensional epigenetic information imposed by enhancers across Vκ and Jκ genomic regions. The data also reveal how enhancers generate distinct antibody repertoires that provide protection against lethal bacterial infection.

An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis.

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci.

Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

The order of immunoglobulin light chain κ and λ usage in primary and secondary lymphoid tissues of germ-free and conventional piglets.

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3' V and 5' J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5' IGLV and 3' IGLJ genes are used. A similar shift toward the use of more 5' IGKV and 3' IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

Comparative Aspects of Immunoglobulin Gene Rearrangement Arrays in Different Species.

Studies in humans and mice indicate the critical role of the surrogate light chain in the selection of the productive immunoglobulin repertoire during B cell development. However, subsequent studies using mutant mice have also demonstrated that alternative pathways are allowed. Our recent investigation has shown that some species, such as pig, physiologically use preferential rearrangement of authentic light chains, and become independent of surrogate light chains. Here we summarize the findings from swine and compare them with results in other species. In both groups, allelic and isotypic exclusions remain intact, so the different processes do not alter the paradigm of B-cell monospecificity. Both groups also retained some other essential processes, such as segregated and sequential rearrangement of heavy and light chain loci, preferential rearrangement of light chain kappa before lambda, and functional κ-deleting element recombination. On the other hand, the respective order of heavy and light chains rearrangement may vary, and rearrangement of the light chain kappa and lambda on different chromosomes may occur independently. Studies have also confirmed that the surrogate light chain is not required for the selection of the productive repertoire of heavy chains and can be substituted by authentic light chains. These findings are important for understanding evolutional approaches, redundancy and efficiency of B-cell generation, dependencies on other regulatory factors, and strategies for constructing therapeutic antibodies in unrelated species. The results may also be important for explaining interspecies differences in the proportional use of light chains and for the understanding of divergences in rearrangement processes. Therefore, the division into two groups may not be definitive and there may be more groups of intermediate species.

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2-4 and Cκ Domains Is an Alternative Reagent to Human IgE.

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2-4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A ß-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements.

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Generation of bispecific antibodies by structure-guided redesign of IgG constant regions.

Bispecific antibodies (BsAbs) form an exciting class of bio-therapeutics owing to their multispecificity. Although numerous formats have been developed, generation of hetero-tetrameric IgG1-like BsAbs having acceptable safety and pharmacokinetics profiles from a single cell culture system remains challenging due to the heterogeneous pairing between the four chains. Herein, we employed a structure-guided approach to engineer mutations in the constant domain interfaces (CH1-CL and CH3-CH3) of heavy and κ light chains to prevent heavy-light mispairing in the antigen binding fragment (Fab) region and heavy-heavy homodimerization in the Fc region. Transient co-transfection of mammalian cells with heavy and light chains of pre-existing antibodies carrying the engineered constant domains generates BsAbs with percentage purity ranging from 78% to 85%. The engineered BsAbs demonstrate simultaneous binding of both antigens, while retaining the thermal stability, Fc-mediated effector properties and FcRn binding properties of the parental antibodies. Importantly, since the variable domains were not modified, the mutations may enable BsAb formation from antibodies belonging to different germline origins and isotypes. The rationally designed mutations reported in this work could serve as a starting point for generating optimized solutions required for large scale production.

Consequences of the different order of immunoglobulin gene rearrangements in swine.

Swine use a reverse order of immunoglobulin chain rearrangement compared to humans and mice, and this altered and modified order should have measurable consequences. Here we perform new and defining experiments with developing and mature B cells, characterizing the B cell populations that do not exist in other species. First, we have finally confirmed that light chains κ and λ are rearranged and expressed on the surface before any heavy chain rearrangements using western-blot. And second, we have analyzed a pool of mature B cells on the single-cell level to demonstrate that many κ+ mature B cells carry λ transcripts. According to these findings, we believe that there may be more groups of mammals; one of which uses a pre-BCR-driven developmental pathway for B cell generation (like mice and humans), the second group uses a pre-BCR-independent one (like swine), and some may be even intermediate.

TREC/KREC levels in children with ataxia-telangiectasia.

The aim of the study was to determine the TREC/KREC levels in the patients diagnosed with ataxia-telangiectasia (AT) and to establish their informative value for early diagnosis of this pathology. TRECs and KREC assay was performed using real-time polymerase chain reaction on the DNA of 25 patients diagnosed with AT aged 3 to 14 years and of 173 healthy individuals of the control group aged 1 to 12 years. Clinical and laboratory characteristics of patients were ascertained using their medical records. In the patients with AT, the mean level of TRECs was 542.84 per 106 cells, ranging from 4 to 4720, while mean level of KRECs was 1317.64 per 106 cells, ranging from 146 to 9300. In 84% of the patients, TREC levels were less than 1000, which was significantly lower than in the control group, while KREC levels were reduced in 48% of the patients. A correlation was found between the levels of TREC and the absolute values of CD4 (r = 0.5455). Measurement of TREC/KREC levels opens new opportunities for early AT detection in children as a part of the newborn screening. Reduced time to diagnosis will allow to carry out timely in-depth immunological and genetic testing, prevent the development of severe infections, and improve quality of life.

Investigating the Variation of TREC/KREC in Combined Immunodeficiencies.

T-cell receptor excision circles (TREC)/Kappa-deleting recombination excision circles (KREC) assay has been recently recognized for detecting patients with primary (T- and/or B-cell) immunodeficiency (PID). We aimed to investigate the alterations of these biomarkers in some combined immunodeficiency patients compared to the healthy controls in different age groups. TREC and KREC were assessed in a total of 82 PID patients, most of them with exact genetic diagnosis (3 months to 42 years); using quantitative real-time-polymerase chain reaction (PCR). Patients had a final diagnosis of common variable immunodeficiency (n=23), ataxia-telangiectasia (AT) (n=17), hyper-IgE syndrome (HIES) (7 with DOCK8 deficiency, 4 with signal transducer and activator of transcription 3 (STAT3) deficiency, and 8 children with unknown genetic defects), Wiskott-Aldrich syndrome (WAS) (n=20), purine nucleoside phosphorylase (PNP)deficiency(n=1), dedicator of cytokinesis2 (DOCK2) deficiency (n=1), recombinase activating gene1 (RAG1) deficiency (n=1). Very low to zero amounts of TREC and/or KREC were detected in 14 out of 23 cases of common variable immunodeficiency (CVID), 14 out of 17 cases of AT, 8 out of 20 cases of WAS, 6 out of 7 cases of DOCK8-deficiency patients, 4 out of 8 cases of HIES with unknown genetic defects and all patients with defects in DOCK2, PNP, and RAG1. STAT3-deficient patients were normal for both biomarkers. All patients showed a significant difference in both markers compared to age-matched healthy controls. Our findings highlight that apart from severe types of T/B cell defects, this assay can also be used for early diagnosis the patients with late-onset of disease and even PIDs without a positive family history.

Immunoglobulin gene rearrangement in Koreans with multiple myeloma: Clonality assessment and repertoire analysis using next-generation sequencing.

INTRODUCTION: We assessed the applicability of next-generation sequencing (NGS)-based IGH/IGK clonality testing and analyzed the repertoire of immunoglobulin heavy chain (IGH) or immunoglobulin kappa light chain (IGK) gene usage in Korean patients with multiple myeloma (MM) for the first time. METHODS: Fifty-nine bone marrow samples from 57 Korean patients with MM were analyzed, and NGS-based clonality testing that targeted the IGH and IGK genes was performed using IGH FR1 and IGK primer sets. RESULTS: Clonal IGH and IGK rearrangements were observed in 74.2% and 67.7% of samples from Korean patients with kappa-restricted MM, respectively (90.3% had one or both), and in 60.7% and 95.5% of samples from those with lambda-restricted MM, respectively (85.7% had one or both). In total, 88.1% of samples from Koreans with MM had clonal IGH and/or IGK rearrangement. Clonal rearrangement was not significantly associated with the bone marrow plasma cells as a proportion of all BM lymphoid cells. IGHV3-9 (11.63%) and IGHV4-31 (9.30%) were the most frequently reported IGHV genes and were more common in Koreans with MM than in Western counterparts. IGHD3-10 and IGHD3-3 (13.95% each) were the most frequent IGHD genes; IGHD3-3 was more common in Koreans with MM. No IGK rearrangement was particularly prevalent, but single IGKV-J rearrangements were less common in Koreans with kappa-restricted MM than in Western counterparts. IGKV4-1 was less frequent in Koreans regardless of light chain type. Otherwise, the usages of the IGH V, D, and J genes and of the IGK gene were like those observed in previous Western studies. CONCLUSION: NGS-based IGH/IGK clonality testing ought to be applicable to most Koreans with MM. The overrepresentation of IGHV3-9, IGHV4-31, and IGHD3-3 along with the underrepresentation of IGKV4-1 and the differences in IGK gene rearrangement types suggest the existence of ethnicity-specific variations in this disease.

Next-Generation Sequencing-Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGS.

Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Amplification of a minimally biased antibody repertoire for in vitro display using a universal primer-based amplification method.

Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene-specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.