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Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified. © 2021 American Society of Hematology

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.

Background About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptorimmunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggeststhat B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never beenexplored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn.Methods For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogeneticdata were available. These patients were followed up in 15 academic institutions throughout Europe (in CzechRepublic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collectedbetween June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptorimmunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressingunmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome ofour analysis was time to fi rst treatment, defi ned as the time between diagnosis and date of fi rst treatment.Findings 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 majorsubsets. Stereotyped subsets showed signifi cant diff erences in terms of age, sex, disease burden at diagnosis,CD38 expression, and cytogenetic aberrations of prognostic signifi cance. Patients within a specifi c subset generallyfollowed the same clinical course, whereas patients in diff erent stereotyped subsets—despite having the sameimmunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status—showedsubstantially diff erent times to fi rst treatment. By integrating B-cell receptor immunoglobulin stereotypy (forsubsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, whichcollectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separateclinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2).Interpretation The molecular classifi cation of chronic lymphocytic leukaemia based on B-cell receptorimmunoglobulin stereotypy improves the Döhner hierarchical model and refi nes prognostication beyondimmunoglobulin mutational status, with potential implications for clinical decision making, especially withinprospective clinical trials.Funding European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry ofHealth; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on ChronicLymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and NationalCancer Institute (NIH).

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2.Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL. © 2015 by The American Society of Hematology 10.1182/blood-2014-09-600874.

Νext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted. © 2014, Springer-Verlag Berlin Heidelberg.

IMGT(®), the international ImMunoGeneTics information system(®) (http://www.imgt.org), created in 1989 (Centre National de la Recherche Scientifique, Montpellier University), is acknowledged as the global reference in immunogenetics and immunoinformatics. The accuracy and the consistency of the IMGT(®) data are based on IMGT-ONTOLOGY which bridges the gap between genes, sequences, and three-dimensional (3D) structures. Thus, receptors, chains, and domains are characterized with the same IMGT(®) rules and standards (IMGT standardized labels, IMGT gene and allele nomenclature, IMGT unique numbering, IMGT Collier de Perles), independently from the molecule type (genomic DNA, complementary DNA, transcript, or protein) or from the species. More particularly, IMGT(®) tools and databases provide a highly standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) V and C domains. IMGT/V-QUEST analyzes the V domains of IG or TR rearranged nucleotide sequences, integrates the IMGT/JunctionAnalysis and IMGT/Automat tools, and provides IMGT Collier de Perles. IMGT/HighV-QUEST analyzes sequences from high-throughput sequencing (HTS) (up to 150,000 sequences per batch) and performs statistical analysis on up to 450,000 results, with the same resolution and high quality as IMGT/V-QUEST online. IMGT/DomainGapAlign analyzes amino acid sequences of V and C domains and IMGT/3Dstructure-DB and associated tools provide information on 3D structures, contact analysis, and paratope/epitope interactions. These IMGT(®) tools and databases, and the IMGT/mAb-DB interface with access to therapeutical antibody data, provide an invaluable help for antibody engineering and antibody humanization.

Background: About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptorimmunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggeststhat B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never beenexplored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn.Methods: For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogeneticdata were available. These patients were followed up in 15 academic institutions throughout Europe (in CzechRepublic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collectedbetween June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptorimmunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressingunmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome ofour analysis was time to fi rst treatment, defi ned as the time between diagnosis and date of fi rst treatment.Findings: 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 majorsubsets. Stereotyped subsets showed signifi cant diff erences in terms of age, sex, disease burden at diagnosis,CD38 expression, and cytogenetic aberrations of prognostic signifi cance. Patients within a specifi c subset generallyfollowed the same clinical course, whereas patients in diff erent stereotyped subsets-despite having the sameimmunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showedsubstantially diff erent times to fi rst treatment. By integrating B-cell receptor immunoglobulin stereotypy (forsubsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, whichcollectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separateclinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2).Interpretation: The molecular classifi cation of chronic lymphocytic leukaemia based on B-cell receptorimmunoglobulin stereotypy improves the Döhner hierarchical model and refi nes prognostication beyondimmunoglobulin mutational status, with potential implications for clinical decision making, especially withinprospective clinical trials.

IMGT(®), the international ImMunoGeneTics information system(®) (http://www.imgt.org), was created in 1989 to manage the huge diversity of the antigen receptors, immunoglobulins (IG) or antibodies, and T cell receptors (TR). Standardized sequence and structure analysis of antibody using IMGT(®) databases and tools allows one to bridge, for the first time, the gap between antibody sequences and three-dimensional (3D) structures. This is achieved through the IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts of classification (IMGT gene and allele nomenclature), description (IMGT standardized labels), and numerotation (IMGT unique numbering and IMGT Colliers de Perles). IMGT(®) is the international reference for immunogenetics and immunoinformatics and its standards are particularly useful for antibody humanization and evaluation of immunogenicity. IMGT(®) databases for antibody nucleotide sequences and genes include IMGT/LIGM-DB and IMGT/GENE-DB, respectively, whereas nucleotide sequence analysis is performed by the IMGT/V-QUEST, IMGT/HighV-QUEST, and IMGT/JunctionAnalysis tools. In this chapter, we focus on IMGT(®) databases and tools for amino acid sequences, two-dimensional (2D) and three-dimensional (3D) structures: the IMGT/DomainGapAlign and IMGT/Collier-de-Perles tools, the IMGT/2Dstructure-DB database for amino acid sequences of monoclonal antibodies (mAb, suffix -mab) and fusion proteins for immune applications (FPIA, suffix -cept) of the World Health Organization/International Nonproprietary Name (WHO/INN) programme and other proteins of interest, and the IMGT/3Dstructure-DB database for crystallized antibodies and its associated tools (IMGT/StructuralQuery, IMGT/DomainSuperimpose).

IMGT/V-QUEST is the highly customized and integrated online IMGT(®) tool for the standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) rearranged nucleotide sequences. The analysis of these antigen receptors represents a crucial challenge for the study of the adaptive immune response in normal and disease-related situations. The expressed IG and TR repertoires represent a potential of 10(12) IG and 10(12) TR per individual. This huge diversity results from mechanisms that occur at the DNA level during the IG and TR molecular synthesis. These mechanisms include the combinatorial rearrangements of the variable (V), diversity (D) and joining (J) genes, the N-diversity (deletion and addition at random of nucleotides during the V-(D)-J rearrangement) and, for IG, somatic hypermutations. IMGT/V-QUEST identifies the V, D, J genes and alleles by alignment with the germline IG and TR gene and allele sequences of the IMGT reference directory. The tool describes the V-REGION mutations and identifies the hot spot positions in the closest germline V gene. IMGT/V-QUEST integrates IMGT/JunctionAnalysis for a detailed analysis of the V-J and V-D-J junctions and IMGT/Automat for a complete annotation of the sequences and also provides IMGT Collier de Perles. IMGT/HighV-QUEST, the high-throughput version of IMGT/V-QUEST, implemented to answer the needs of deep sequencing data analysis from Next Generation Sequencing (NGS), allows the analysis of thousands of IG and TR sequences in a single run. IMGT/V-QUEST and IMGT/HighV-QUEST are available at the IMGT(®) Home page, http://www.imgt.org.

The current study investigated the presence, frequency, and status of somatic hypermutations as well as their role in children with B lineage ALL. The obtained sequences were analyzed using IMGT/V-QUEST. Totally, 150 IGH sequences were evaluated; 139 from the 111 patients at the time of diagnosis and 11 from 8/111 patients at the time of relapse. The findings of the current report revealed the presence of somatically mutated V genes in childhood B lineage ALL. A higher frequency of somatic hypermutations was noted in unproductive rearrangements and was generally attributed to nucleotide mutation type, region, and IGHV gene subgroup biases. © 2011 Informa Healthcare USA, Inc.

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IMGT, the international ImMunoGeneTics database, is an integrated database specializing in immunoglobulins, T-cell receptors (TcR) and major histocompatibility complex (MHC) of all vertebrate species, initiated and co-ordinated by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes two databases: LIGM-DB (for immunoglobulins and TcR) and MHC/HLA-DB. IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 19 000 immunoglobulin and TcR sequences from 78 species. MHC/HLA-DB contains class I and class II human leukocyte antigen alignment tables. An IMGT tool, DNAPLOT, developed for immunoglobulins, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including sequences, oligonucleotide primers, gene maps and other genetic data of immunoglobulins, TcR and MHC molecules, and to provide a graphical user-friendly data access. IMGT will have important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT can be accessed at http://imgt.cnusc.fr:8104 and http://www.ebi.ac.uk/IMGT