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Aims: Cytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities., Methods: Aspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH . Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed., Results: Up to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV , indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells., Conclusions: This automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma., Competing Interests: Competing interests: HH, KF and WNE are inventors of a patent application covering the immuno-flowFISH technique. The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (V H ), diversity (D H ), and joining (J H ) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the V H genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development., Competing Interests: Declaration of interests P.F. and S.S. are co-founders and shareholders of Enhanc3D Genomics Ltd., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: Chronic lymphocytic leukemia (CLL) is a highly heterogenous hemopathy. Genetic stratification of CLL patients has important prognostic and therapeutic values - mainly immunoglobulin heavy chain variable region gene ( IGHV ) mutational status and the presence of cytogenetic abnormalities. The genetics of CLL in Lebanon is scarcely described in the literature. Patients & methods: In this work, we studied the genetic biomarkers of 312 Lebanese CLL patients. Results: Prominent IGHV genes were IGHV4-34 , IGHV1-69 and IGHV3-30 ; and CLL #1 and #5 presented major subsets. Some similarities as well as major differences were highlighted when comparing our data with previously published data. Conclusion: The distribution of IGHV alleles in our series differed from previously described distributions, suggesting involvement of antigenic selection and regional variables in CLL pathogenesis.

Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease., (© 2023. The Author(s).)

Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) is a heterogeneous disease in which the role of immunoglobulin heavy-chain genes (IGHs) remains unknown. To determine the clinical relevance of the IGH repertoire in patients with LPL/WM, we performed immunoglobulin gene rearrangement and complementarity determining region 3 (CDR3) analysis. The IGH variable gene (IGHV) repertoire was remarkably biased in LPL/WM. IGHV3-23, IGHV4-34, IGHV3-30, IGHV3-7, and IGHV3-74 accounted for one-half of the cohort's repertoire. Most cases (97.1%) were found to carry mutated IGHV genes, based on a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating there was specific antigen selection in LPL/WM. Patients with IGHV3-7 were significantly more likely to harbor the 6q deletion (P < .001) and an abnormal karyotype (P = .004). The IGHV hypermutation rate in patients with the MYD88 L265P mutation was significantly higher than that of wild-type patients (P = .050). IGHV3-23 and IGHV3-74 segments were more frequently detected in patients with MYD88-mutated LPL/WM (P = .050), whereas IGHV3-7 presented more frequently in MYD88 wild-type patients (P = .042). Patients with IGHV4, especially IGHV4-34, had higher levels of lactate dehydrogenase, and IGHV4 was a predictive marker of shorter progression-free survival. These results showed for the first time that the IGHV repertoire has clinical relevance in LPL/WM., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA "scars" (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5'-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5'-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Thörnqvist and Ohlin.)

High-throughput sequencing technologies brought a renewed interest for immune repertoires. Fish Ab and B cell repertoires are no exception, and their comprehensive analysis can both provide new insights into poorly understood immune mechanisms, and identify markers of protection after vaccination. However, the lack of genomic description and standardized nomenclature of IG genes hampers accurate annotation of Ig mRNA deep sequencing data. Complete genome sequences of Atlantic salmon and rainbow trout (Swanson line) recently allowed us to establish a comprehensive and coherent annotation of Salmonid IGH genes following IMGT standards. Here we analyzed the IGHV, D, and J genes from the newly released genome of a second rainbow trout line (Arlee). We confirmed the validity of salmonid IGHV subgroups, and extended the description of the rainbow trout IGH gene repertoire with novel sequences, while keeping nomenclature continuity. This work provides an important resource for annotation of high-throughput Ab repertoire sequencing data., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection., (© 2021 by The American Society of Hematology.)

Most patients with chronic lymphocytic leukaemia (CLL) are nowadays diagnosed without any symptoms and do not require therapy. A prognostic score identifying patients within this large group who are at high risk of disease progression would be highly beneficial. The recently published International Prognostic Score for Early asymptomatic patients (IPS-E) uses combination of absolute lymphocyte count (ALC) >15 × 10 9 /l, palpable lymphadenopathy, and unmutated immunoglobulin heavy-chain variable-region (IGHV) gene to predict the time to first-line therapy (TTFT). Patients at low, intermediate, and high risk had estimated 5-year TTFT of 8%, 28%, and 61%. We performed an external validation of the IPS-E score using an unselected, consecutive group of 130 Binet A patients. The 5-year TTFT was 11%, 36%, and 78% (C-statistic 0·74). Furthermore, we propose an alternative system (AIPS-E) using cytogenetic aberrations instead of palpable lymphadenopathy. This system yielded 5-year TTFT of 14%, 40%, and 72%. These results were externally validated in 388 Binet A patients from five Czech centres; the 5-year TTFT was 16%, 37%, and 80% (C-statistic 0·74). In conclusion, we have successfully validated the IPS-E score for patients with early stage CLL. In addition, we propose a modified scoring system, the AIPS-E, combining IGHV, fluorescence in situ hybridisation, and ALC., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

The somatic hypermutation (SHM) of Immunoglobulin (Ig) genes is a key process during antibody affinity maturation in B cells. The mutagenic enzyme activation induced deaminase (AID) is required for SHM and has a preference for WRC hotspots in DNA. Error-prone repair mechanisms acting downstream of AID introduce further mutations, including DNA polymerase eta (Polη), part of the non-canonical mismatch repair pathway (ncMMR), which preferentially generates mutations at WA hotspots. Previously proposed mechanistic models lead to a variety of predictions concerning interactions between hotspots, for example, how mutations in one hotspot will affect another hotspot. Using a large, high-quality, Ig repertoire sequencing dataset, we evaluated pairwise correlations between mutations site-by-site using an unbiased measure similar to mutual information which we termed "mutational association" (MA). Interactions are dominated by relatively strong correlations between nearby sites (short-range MAs), which can be almost entirely explained by interactions between overlapping hotspots for AID and/or Polη. We also found relatively weak dependencies between almost all sites throughout each gene (longer-range MAs), although these arise mostly as a statistical consequence of high pairwise mutation frequencies. The dominant short-range interactions are also highest within the most highly mutating IGHV sub-regions, such as the complementarity determining regions (CDRs), where there is a high hotspot density. Our results suggest that the hotspot preferences for AID and Polη have themselves evolved to allow for greater interactions between AID and/or Polη induced mutations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krantsevich, Tang and MacCarthy.)

The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells 1 . Upon binding a recombination centre-based J H , RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ H -based recombination centre 2 . CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning 3-5 ; however, their inactivation allows scanning to proximal V H s, where additional CBEs activate rearrangement and impede scanning any further upstream 5 . Distal V H utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction 6-8 . Here we test the potential of linear RAG scanning to mediate distal V H usage in G1-arrested v-Abl pro-B cell lines 9 , which undergo robust D-to-J H but little V H -to-DJ H rearrangements, presumably owing to lack of locus contraction 2,5 . Through an auxin-inducible approach 10 , we degraded the cohesin component RAD21 10-12 or CTCF 12,13 in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-J H joining, in which synapsis occurs by diffusion 2 . Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust V H -to-DJ H joining of, distal V H s, with patterns similar to those of 'locus-contracted' primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus.

Composite lymphoma is defined as two or more morphologically and immunophenotypically distinct lymphoma clones that occur in the same tissue site. The occurrence of cutaneous composite lymphoma (CCL) is extremely rare. Here we report a unique case of CCL consisting of Hodgkin lymphoma (HL) and mycosis fungoides (MF). Our patient presented with longstanding erythematous plaques on the skin and later developed axillary lymph node enlargement. Histopathologically, the skin lesions were characterized by a dense dermal lymphocytic infiltrate with prominent epidermotropism of pleomorphic T-cells, consistent with typical MF. Nonetheless, scattered large atypical cells resembling Reed-Sternberg (R-S) cells were interspersed among these atypical T-cells in the deep dermis. Immunophenotyping suggested a HL origin of these R-S cells. Monoclonality of T-cell receptor beta gene was detected in the skin, monoclonal immunoglobulin heavy chain gene rearrangement was identified in these R-S cells microdissected from the deep dermis, confirming the origin from HL. The lymph node biopsy showed nodular sclerosis classic Hodgkin lymphoma. Therefore, CCL of HL and MF, with lymph node HL was diagnosed. The lesions of this patient responded to a sequential treatment to HL and MF. Being aware of this rare CCL facilitates correct diagnosis and proper clinical management., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: We aimed to determine the biological processes and pathways involved in cervical carcinogenesis associated with high-risk human papillomavirus (HPV) infection., Materials and Methods: Total RNA was extracted from three formalin-fixed paraffin-embedded (FFPE) samples each of normal cervix, HPV-infected low-grade squamous intraepithelial lesion (LSIL), high-grade SIL (HSIL) and squamous cell carcinoma (SCC). Transcriptomic profiling by microarrays was conducted followed by downstream Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses., Results: We examined the difference in GOs enriched for each transition stage from normal cervix to LSIL, HSIL, and SCC, and found 307 genes to be differentially expressed. In the transition from normal cervix to LSIL, the extracellular matrix (ECM) genes were significantly downregulated. The MHC class II genes were significantly upregulated in the LSIL to HSIL transition. In the final transition from HSIL to SCC, the immunoglobulin heavy locus genes were significantly upregulated and the ECM pathway was implicated., Conclusion: Deregulation of the immune-related genes including MHC II and immunoglobulin heavy chain genes were involved in the transitions from LSIL to HSIL and SCC, suggesting immune escape from host anti-tumour response. The extracellular matrix plays an important role during the early and late stages of cervical carcinogenesis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments 1 and topologically associated domains (TADs) 2,3 consisting of chromatin loops 4 . TADs are formed by chromatin-loop extrusion 5-7 , which depends on the loop-extrusion function of the ring-shaped cohesin complex 8-12 . Conversely, the cohesin-release factor Wapl 13,14 restricts loop extension 10,15 . The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination 16 , which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax5 17,18 . However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.

Objectives: MALT lymphoma occurs in various organs and has several characteristic genetic aberrations. Thyroid MALT lymphoma has been reported to include t(3;14)(p14.1;q32)/FOXP1-IGH as a specific genetic aberration, but the number of studies is limited., Method and Results: We examined 86 thyroid lymphoma cases using fluorescence in situ hybridization (FISH) for the detection of t(3;14)/FOXP1-IGH in formalin fixed paraffin-embedded tissue (FFPE). Histopathological diagnoses of the analyzed specimen were as follows: thyroid MALT lymphoma (n = 59), DLBCL (n = 23), follicular lymphoma (n = 4), and benign lesions (n = 14) included Hashimoto's thyroiditis (n = 13) and other (n = 1). Of the 100 analyzed cases, thirty-six (36 %) thyroid lymphoma cases were positive for t(3;14)/FOXP1-IGH. Thirty-three (55.9 %) of the 59 MALT lymphoma cases were positive for t(3;14)/FOXP1-IGH. Three (13.0 %) of the 23 DLBCL cases were positive for t(3;14)/FOXP1-IGH. All 4 follicular lymphomas examined were negative for t(3;14)/FOXP1-IGH. None of the benign cases was positive for t(3;14)/FOXP1-IGH, including Hashimoto's thyroiditis (0/13) and benign tissue (0/1)., Conclusions: Our study found that t(3;14)/FOXP1-IGH was frequently found in thyroid MALT lymphoma. A detection of t(3;14)/FOXP1-IGH is extremely useful for the differential diagnosis between primary MALT lymphoma of the thyroid and other thyroid disorders., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination 1 . The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen 1 . Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs) 2,3 . Although most GCs are transient 3 , those in intestinal Peyer's patches (PPs)-which depend on the gut microbiota-are chronic 4 , and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses.

Dengue is one of the most serious mosquito-borne viral diseases occurring in humans. To combat the complexity of 4 antigenically distinct serotypes, the ideal vaccine for dengue should be able to stimulate cross-neutralizing antibodies. Recently, genetics-based immune responses have been studied to guide vaccine design against several viral pathogens. Despite a recent approval of dengue vaccine, information on genetics-based immune responses against dengue virus (DENV) is still limited. Consequently, we aimed to determine the profiles of immunoglobulin heavy chain genes from DENV2 infected patients. The immunoglobulin heavy chain variable region genes (IGHV) were amplified from peripheral blood mononuclear cells of DENV2 secondary infected patients in the acute, convalescence, and recovery phases. Antibody heavy chain genes were sequenced using next-generation sequencing, and analyzed to identify correlations with neutralizing and enhancing activities of the serum samples. IGHV1-69, 3-23, and 3-30 were frequently discovered in our Thai DENV2 infected patients. Our findings provide new data on the human B cell response during secondary DENV2 infections in Thai dengue patients that offer supportive information for dengue vaccine design and therapeutics development.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries but very rare in Asia. Peripheral blood or bone marrow mononuclear cells obtained at initial diagnosis from 194 patients with CLL were analysed to determine the ethnic difference in genetic abnormalities. Mutated IGHV was detected in 71·2% of Taiwanese CLL and IGHV3-23 was the most frequently used gene. Stereotyped BCR was present in 18·3% with subset 8 being the most frequent. All cases with subset 8 belonged to IGHV 4-39 and were exclusively associated with un-mutated IGHV and poor outcome. Mutation frequencies of SF3B1 (9·7%), NOTCH1 (8·6%), BIRC3 (1·1%), ATM (16·9%) or TP53 (8·1%), and frequencies of cytogenetic abnormalities including trisomy 12 (18·6%), del(17p) (10·4%), del(13q) (43·7%) and IGH translocation (10·1%) were comparable to those reported from Western countries, except del(11q) (6·9%) which was lower in our patients. Patients with un-mutated IGHV, subset 8, disrupted TP53, trisomy 12, and SF3B1 mutations had a worse outcome compared to patients without these mutations. In conclusion, IGHV3-23 usage, stereotyped subset 8 and lower frequency of del(11q) show an ethnicity-dependent association in Taiwanese CLL patients., (© 2019 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Porcine thymus contains three independent populations of cells that have rearranged immunoglobulin heavy chain VDJ H genes. The first population can be found exclusively in medulla and it consists of existing mature B cells and plasma cells. The second consists of developing B cells characterized by the presence of selected VDJ H rearrangement, similar to B cell lymphogenesis in the bone marrow. The third population is entirely unaffected by selection mechanism for productive VDJ H rearrangement and represents T lineage cells that rearrange immunoglobulin genes. Transcription of unselected VDJ H repertoire is not allowed in T cells. Sequence analysis of unselected VDJ H repertoire from T cells also revealed important consequences for B cell lymphogenesis and selection of B cell repertoire. As far as we know, this is the first evidence that some species completely rearrange VDJ H genes in T cells. Our results also support the finding that B cells actively develop in the thymus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes., Competing Interests: The authors have declared that no competing interests exist.

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

B cell acute lymphoblastic leukemia (B ALL) is a genetically heterogeneous neoplasm often demonstrating extensive subclone diversity within each patient's disease. The immunoglobulin heavy chain (IGH) locus is a marker of clonal variation in B ALL due to its intrinsic role in B lymphocyte development and its diverse Vh(D)Jh rearrangement patterns. B ALL IGH evolution may contribute to limitations in minimal residual disease (MRD) monitoring methods. Evolving technologies for IGH high-throughput sequencing (HTS) have demonstrated MRD detection as sensitive as 1 cell in 1,000,000. These methods may enhance the surveillance of B ALL in the setting of extensive subclone evolution and provide opportunities for detection and intervention before the onset of relapse. However, HTS MRD methods will need to be evaluated in the context of clinical trials in order to gain further insights about the clinical relevance of such sensitive B ALL MRD detection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Polysaccharide vaccines such as the Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi induce efficient Ab responses in adults but not in young children. The reasons for this difference are not understood. IL-7 dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many V H gene segments that are distal to D H -J H in the IgH locus and for the complete diversification of the BCR repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of Ags because of a restricted BCR repertoire. Compared with adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced Ab response to ViPS. Additionally, ViPS-binding B cells in adult wildtype mice predominantly used distal V H gene segments. Transgenic expression of either IL-7 or a BCR encoded by a distal V H gene segment permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted V H gene usage early in life results in a paucity of Ag-specific B cell precursors, thus limiting antipolysaccharide responses., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Objective: To evaluate the application of FISH testing of bcl-2/IgH gene translocation and IgH/L gene rearrangement in different stages of follicular lymphoma. Methods: In 32 follicular lymphoma cases, which were collected at Guangdong General Hospital from September 2014 to December 2016, the bcl-2/IgH gene ectopic state was detected by FISH while the IgH/L gene rearrangement was tested using PCR-GeneScan to analyze the relationship between bcl-2/IgH gene translocation, different stages of follicular lymphoma and clonal immunoglobulin (IgH/L) gene rearrangements. Results: From the paraffin sections of all 32 follicular lymphomas, 17 cases showed bcl-2/IgH gene translocation, and the percentages of FL1, FL2 and FL3 translocation were 12/13, 3/5 and 2/14, respectively. Among the 24 cases of IgH/L gene arrangements identified from the total sample, the occurrence rates of FL1, FL2 and FL3 gene arrangement were 7/13, 4/5 and 13/14, respectively. Spearman's rank correlation analysis and χ(2) analysis showed that bcl-2/IgH gene translocation was negatively correlated with follicular lymphoma stage and the association was statistically significant. In more advanced stages of follicular lymphoma, the occurrence of bcl-2/IgH gene translocation tended to decrease with distinct FL1, FL2 and Fl3 gene expression ( P <0.05). As IgH/L gene rearrangement in FL3 was higher than that in FL1 and FL2, its detection may be complimentary to FISH test for bcl-2/IgH gene translocation in diagnosing follicular lymphoma. Conclusions: The combined use of FISH and PCR-GeneScan increases the positive rate of follicular lymphoma diagnosis, and this combination is more sensitive than FISH or clonal analysis only to detect the chromosomal abnormality or the gene rearrangement.

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., (© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Inference of antibody gene repertoires using transcriptome data has emerged as an alternative approach to the complex process of sequencing of adaptive immune receptor germline gene loci. The diversity introduced during rearrangement of immunoglobulin heavy chain variable (IGHV), diversity, and joining genes has however been identified as potentially affecting inference specificity. In this study, we have addressed this issue by analysing the nucleotide composition of unmutated human immunoglobulin heavy chains-encoding transcripts, focusing on the 3ö most bases of 47 IGHV germline genes. Although transcripts derived from some of the germline genes predominately incorporated the germline encoded base even at position 320, the last base of most IGHV genes, transcripts originating in other genes presented other nucleotides to the same extent at this position. In transcripts derived from two of the germline genes, IGHV3-13*01 and IGHV4-30-2*01, the predominating nucleotide (G) was in fact not that of the gene (A). Hence, we suggest that inference of IGHV genes should be limited to bases preceding nucleotide 320, as inference beyond this would jeopardize the specificity of the inference process. The different degree of incorporation of the final base of the IGHV gene directly influences the distribution of amino acids of the ascending strand of the third complementarity determining region of the heavy chain. Thereby it influences the nature of this specificity-determining part of the antibody population. In addition, we also present data that indicate the existence of a common so far un-recognized allelic variant of IGHV3-7 that carries an A318G difference in relation to IGHV3-7*02., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Fuchs' uveitis (FU) is occasionarlly complicated with heavy vitreous opacity. We have performed vitrectomy procedures to remove vitreous opacity in affected patients as part of differential diagnosis for primary vitreoretinal lymphoma (PVRL)., Case Presentation: We retrospectively reviewed the clinical records of five patients who first visited the Uveitis Clinic of the University of Tokyo Hospital between 2009 and 2013, were diagnosed with FU and underwent a vitrectomy for removal of dense vitreous opacity. All were diagnosed as FU by ocular findings and elevation of Goldmann-Witmer coefficient (GWC) value for the rubella virus (RV) antibody. In examinations of the vitreous body, cytological diagnosis, elevation of IL-10/IL-6 ratio, and the kappa/lambda ratio in flow cytometry findings were negative in all cases, whereas monoclonal immunoglobulin heavy chain (IgH) gene rearrangement was positive in 4 cases and negative in 1 case., Conclusions: Although monoclonal IgH gene rearrangement is thought to be a reliable biomarker for PVRL, a high percentage of vitreous specimens from our FU patients showed pseudo-positive results. Ophthalmologists must take care regarding possible pseudo-positive findings when performing differential diagnosis between FU and PVRL. Combinations of results of cytological diagnosis, IL-10/IL-6 ratio, kappa/lambda ratio, and IgH gene rearrangement may be necessary for a definitive diagnosis of PVRL and differentiation from FU.

The immunoglobulin heavy chain (IgH) 3' regulatory region (3'RR) superenhancer controls B2 B-cell IgH transcription and cell fate at the mature stage but not early repertoire diversity. B1 B cells represent a small percentage of total B cells differing from B2 B cells by several points such as precursors, development, functions, and regulation. B1 B cells act at the steady state to maintain homeostasis in the organism and during the earliest phases of an immune response, setting them at the interface between innate and acquired immunity. We investigated the role of the 3'RR superenhancer on B1 B-cell fate. Similar to B2 B cells, the 3'RR controls μ transcription and cell fate in B1 B cells. In contrast to B2 B cells, 3'RR deletion affects B1 B-cell late repertoire diversity. Thus, differences exist for B1 and B2 B-cell 3'RR control during B-cell maturation. For the first time, these results highlight the contribution of the 3'RR superenhancer at this interface between innate and acquired immunity., (© 2018 by The American Society of Hematology.)

Chronic lymphocytic leukaemia (CLL) consists of two biologically and clinically distinct subtypes defined by the abundance of somatic hypermutation (SHM) affecting the Ig variable heavy-chain locus (IgHV). The molecular mechanisms underlying these subtypes are incompletely understood. Here, we present a comprehensive whole-genome sequencing analysis of somatically acquired genetic events from 46 CLL patients, including a systematic comparison of coding and non-coding single-nucleotide variants, copy number variants and structural variants, regions of kataegis and mutation signatures between IgHV mut and IgHV unmut subtypes. We demonstrate that one-quarter of non-coding mutations in regions of kataegis outside the Ig loci are located in genes relevant to CLL. We show that non-coding mutations in ATM may negatively impact on ATM expression and find non-coding and regulatory region mutations in TCL1A, and in IgHV unmut CLL in IKZF3, SAMHD1,PAX5 and BIRC3. Finally, we show that IgHV unmut CLL is dominated by coding mutations in driver genes and an aging signature, whereas IgHV mut CLL has a high incidence of promoter and enhancer mutations caused by aberrant activation-induced cytidine deaminase activity. Taken together, our data support the hypothesis that differences in clinical outcome and biological characteristics between the two subgroups might reflect differences in mutation distribution, incidence and distinct underlying mutagenic mechanisms.

After therapy or stem cell transplantation, multiple myeloma patients achieving complete response or stringent complete response can still have a significant risk of disease relapse. This highlights the importance of using highly sensitive laboratory methods for minimal residual disease detection and prognostication. Older methods such as allele-specific oligonucleotide real time quantitative polymerase chain reaction and fluorescent polymerase chain reaction have their drawbacks. Meanwhile, the recent generation of multiparametric flow cytometry and next-generation sequencing (NGS)-based detection methods currently offer the highest technical sensitivities, and are likely to gain more widespread use and be recognized as the standard of care for disease monitoring in myeloma patients., (Copyright © 2018. Published by Elsevier Inc.)

The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin V H gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in V H usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific V H usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent V H usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As the therapeutic landscape for chronic lymphocytic leukemia (CLL) continues to expand, biological predictors of response to therapy are becoming increasingly important. One such predictive biomarker is the mutational status of the variable region of the immunoglobulin heavy chain (IGHV) gene, which is a powerful predictor of duration of response and overall survival with chemoimmunotherapy (CIT). As this test may influence choice of therapy between CIT and novel agents, it is critical that providers understand how mutational status is determined and the limitations of testing. Here, we describe the details of IGHV mutational status testing, highlighting the appropriate way to interpret this information and best apply it to the care of patients with CLL., (© 2017 Wiley Periodicals, Inc.)

Objectives: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with white patients. In this study, we aimed to identify genetic factors that may account for this difference., Methods: We analyzed immunoglobulin heavy chain (IGH) genes' mutational status by performing next-generation sequencing in 25 Senegalese and 50 Italian patients with CLL., Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series., Conclusions: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Analysis of antibody repertoire development and specific antibody responses important for e.g. autoimmune conditions, allergy, and protection against disease is supported by high throughput sequencing and associated bioinformatics pipelines that describe the diversity of the encoded antibody variable domains. Proper assignment of sequences to germline genes are important for many such processes, for instance in the analysis of somatic hypermutation. Germline gene inference from antibody-encoding transcriptomes, by using tools such as TIgGER or IgDiscover, has a potential to enhance the quality of such analyses. These tools may also be used to identify germline genes not previously known. In this study, we exploited such software for germline gene inference and define aspects of analysis settings and pre-existing knowledge of germline genes that affect the outcome of gene inference. Furthermore, we demonstrate the capacity of IGHJ and IGHD haplotype inference, whenever subjects are heterozygous with respect to such genes, to lend support to IGHV gene inference in general, and to the identification of novel alleles presently not recognized by germline gene reference directories. We propose that such haplotype analysis shall, whenever possible, be used in future best practice to support the outcome of germline gene inference. IGHJ-directed haplotype inference was also used to identify haplotypes not expressing some IGHV germline genes. In particular, we identified a haplotype that did not express several major germline genes such as IGHV1-8, IGHV3-9, IGHV3-15, IGHV1-18, IGHV3-21, and IGHV3-23. We envisage that haplotype analysis will provide an efficient approach to identify subjects for further studies of the link between the available immunoglobulin repertoire and outcomes of immune responses., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

CD49d is a surface integrin that is expressed on chronic lymphocytic leukaemia (CLL) cells, and strongly correlates with more aggressive disease. Given its association with cell-cell adhesion and leucocyte trafficking, we hypothesized that patients with high CD49d expression would experience a clinical course dominated by lymphadenopathy. CD49d expression was measured by flow cytometry and considered positive if expressed by ≥30% of CLL cells. The study included 797 newly diagnosed CLL/small lymphocytic leukaemia patients; 279 (35%) were CD49d positive. CD49d-positive patients were more likely to present with lymphadenopathy (P < 0·001); a finding that persisted after adjusting for fluorescence in situ hybridisation (FISH) and IGHV mutation status [odds ratio (OR) 2·51; 95% confidence interval (CI) 1·64-3·83; P < 0·001]. Among CLL Rai 0 patients, CD49d positivity was associated with shorter time to development of lymphadenopathy (3·2 years vs not reached, P < 0·01). This association was maintained after adjusting for either FISH [hazard ratio (HR) 2·18; 95% CI 1·25-3·81; P = 0·006) or IGHV status (HR 2·02; 95% CI 1·11-3·69; P = 0·02) individually, but was attenuated when adjusting by both (HR 1·72; 95% CI 0·88-3·38; P = 0·11).These data demonstrate that CD49d-positive CLL patients experience a disease course dominated by lymphadenopathy. These findings could have implications for therapy selection and disease monitoring., (© 2017 John Wiley & Sons Ltd.)

Differences in chronic lymphocytic leukemia between the Asian and the Western population are widely known. To further clarify these ethnic differences, we profiled the molecular genetics in a cohort of 83 newly diagnosed patients from Taiwan. In detail, we assessed: (i) the usage and the mutational status of the clonotypic immunoglobulin heavy-chain variable region ( IgHV ) genes, (ii) the presence of VH CDR3 stereotypes, and (iii) TP53, NOTCH1, SF3B1, BIRC3 , and MYD88 mutations. The IgHV gene repertoire was biased and distinct from that observed in the West with the most common IgHV genes being IgHV3-23, IgHV3-7 , and IgHV3-48 In terms of IgHV gene mutational status, 63.8% of patients carried mutated rearrangements, whereas 22.4% of patients were assigned to stereotyped subsets (6.9% to major subsets and 15.5% to minor ones). The frequencies of NOTCH1 , SF3B1, BIRC3 and MYD88 mutations were 9.6%, 7.2%, 1.2%, and 2.4%, respectively; however, the frequency of TP53 mutations was significantly higher (20.5%). Patients with TP53 mutations or del(17p), SF3B1 mutations and unmutated IgHV had a worse outcome compared to the other patients. In conclusion, the differences observed in IgHV properties suggest different pathogenetic factors implicated in the development of chronic lymphocytic leukemia, while the high frequency of TP53 mutations could in part explain the dismal outcome of these patients in Taiwan., (Copyright© Ferrata Storti Foundation.)

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ-PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ-PCR≥10 -5 ). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR-negative/minor cluster region-negative/minor BCL2-negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression-free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM-; 6 PB-/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ-PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized., (© 2017 John Wiley & Sons Ltd.)

Composite lymphomas (CLs) are defined as two unrelated lymphomas occurring at the same time within the same tissue. The incidence of these tumors is low. Of all possible combinations between lymphomas, the least frequent are the ones combining peripheral T-cell lymphoma (PTCL) and Hodgkin lymphoma (HL). We recently identified five cases of CL composed of PTCL and classical HL, mixed cellularity type. We investigated histological and clinical features of these cases. Immunostaining was performed on paraffin sections. PTCL cells were positive for CD8 and TIA-1 in four of the five cases. Hodgkin and Reed-Sternberg (HRS) cells were positive for CD30 and weakly positive for PAX5 in all cases, positive for CD15 in three of five cases, positive for CD20 in one of five cases, and negative for EBER. Monoclonal rearrangement of the T-cell receptor (TCR) and immunoglobulin heavy chain (IGH) genes was confirmed by polymerase chain reaction (PCR) using whole paraffin sections. We concluded more precisely the monoclonality of the IGH rearrangement of HRS cells based on single-cell PCR for IGH and DNA sequencing analysis after laser microdissection of single cells in one case. HL can occur in CD8-positive and TIA-1-positive PTCL. Clinicians should recognize the possibility of these CL., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)