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Introduction: Analysis of an individual's immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated. Methods: The Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3' or 5' truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanyingmetadata. Results and discussion: The Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The versiontracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data. [ABSTRACT FROM AUTHOR]

IntroductionAnalysis of an individual’s immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated.MethodsThe Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3’ or 5’ truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata.Results and discussionThe Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The version-tracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data.

Two main variants of Richter syndrome (RS) are recognized, namely, the diffuse large B-cell lymphoma (DLBCL) and the Hodgkin's lymphoma (HL) variant. Clonal relationship, defined as an identity of the immunoglobulin heavy chain variable (IGHV) region sequence between chronic lymphocytic leukemia (CLL) and RS clones, characterizes patients with a poor prognosis. Due to method sensitivity, this categorization is performed without considering the possibility of small-size ancillary clones, sharing the same phenotype with the preexisting predominant CLL clone, but with different IGHV rearrangements. Here we describe and molecularly profile the peculiar case of a patient with a CLL-like monoclonal B-cell lymphocytosis (MBL), who sequentially developed a DLBCL, which occurred concomitantly to progression of MBL to CLL, and a subsequent HL. Based on standard IGHV clonality analysis, DLBCL was considered clonally unrelated to the concomitantly expanded CLL clone and treated as a de novo lymphoma, achieving a persistent response. Three years later, the patient further developed a clonally unrelated HL, refractory to bendamustine, which was successfully treated with brentuximab vedotin and radiotherapy, and later with pembrolizumab. We retrospectively performed additional molecular testing, by applying next-generation sequencing (NGS) of immunoglobulin repertoire (Ig-rep) techniques and a more sensitive allele-specific oligonucleotide-droplet digital PCR (ASO-ddPCR) strategy, in order to quantitatively investigate the presence of the rearranged IGHV genes in tumor specimens collected during the disease course. In this highly complex case, the application of modern and sensitive molecular technologies uncovered that DLBCL, initially considered as a de novo lymphoma, was instead the result of the transformation of a preexisting ancillary B-cell clone, which was already present at the time of first MBL diagnosis. A similar approach was also applied on the HL sample, showing its clonal unrelatedness to the previous MBL and DLBCL. [ABSTRACT FROM AUTHOR]

Thorough examination of clonotypic B-cell receptor immunoglobulin (BcR IG) gene rearrangement sequences in patients with mature B-cell malignancies has revealed significant repertoire restrictions, leading to the identification of subsets of patients expressing highly similar, stereotyped BcR IG. This discovery strongly suggests selection by common epitopes or classes of structurally similar epitopes in the development of these tumors. Initially observed in chronic lymphocytic leukemia (CLL), where the stereotyped fraction accounts for a substantial fraction of patients, stereotyped BcR IGs have also been identified in other mature B-cell malignancies, including mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL).Further comparisons across different entities have indicated that stereotyped IGs are predominantly "disease-biased," indicating distinct immune pathogenetic trajectories. Notably, accumulating evidence suggests that molecular subclassification of mature B-cell malignancies based on BcR IG stereotypy holds biological and clinical relevance. Particularly in CLL, patients belonging to the same subset due to the expression of a specific stereotyped BcR IG exhibit consistent biological backgrounds and clinical courses, especially for major and extensively studied subsets. Therefore, robust assignment to stereotyped subsets may aid in uncovering mechanisms underlying disease initiation and progression, as well as refining patient risk stratification. In this chapter, we offer an overview of recent studies on BcR IG stereotypy in mature B-cell malignancies and delineate past and present methodological approaches utilized for the identification of stereotyped BcR IG., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Two main variants of Richter syndrome (RS) are recognized, namely, the diffuse large B-cell lymphoma (DLBCL) and the Hodgkin’s lymphoma (HL) variant. Clonal relationship, defined as an identity of the immunoglobulin heavy chain variable (IGHV) region sequence between chronic lymphocytic leukemia (CLL) and RS clones, characterizes patients with a poor prognosis. Due to method sensitivity, this categorization is performed without considering the possibility of small-size ancillary clones, sharing the same phenotype with the preexisting predominant CLL clone, but with different IGHV rearrangements. Here we describe and molecularly profile the peculiar case of a patient with a CLL-like monoclonal B-cell lymphocytosis (MBL), who sequentially developed a DLBCL, which occurred concomitantly to progression of MBL to CLL, and a subsequent HL. Based on standard IGHV clonality analysis, DLBCL was considered clonally unrelated to the concomitantly expanded CLL clone and treated as a de novo lymphoma, achieving a persistent response. Three years later, the patient further developed a clonally unrelated HL, refractory to bendamustine, which was successfully treated with brentuximab vedotin and radiotherapy, and later with pembrolizumab. We retrospectively performed additional molecular testing, by applying next-generation sequencing (NGS) of immunoglobulin repertoire (Ig-rep) techniques and a more sensitive allele-specific oligonucleotide-droplet digital PCR (ASO-ddPCR) strategy, in order to quantitatively investigate the presence of the rearranged IGHV genes in tumor specimens collected during the disease course. In this highly complex case, the application of modern and sensitive molecular technologies uncovered that DLBCL, initially considered as a de novo lymphoma, was instead the result of the transformation of a preexisting ancillary B-cell clone, which was already present at the time of first MBL diagnosis. A similar approach was also applied on the HL sample, showing its clonal unrelatedness to the previous MBL and DLBCL.

Objective: to analyze the stereotyped subsets in cohort of Ukrainian chronic lymphocytic leukemia (CLL) patients in general and depending on the ionizing radiation (IR) exposure. Methods. Analysis was performed in the groups of 118 CLL patients irradiated due to the Chornobyl NPP accident (95 clean-up workers, 17 inhabitants of radionuclide contaminated areas, and 6 evacuees) and 294 IR non-exposed patients. The IGHV (immunoglobulin heavy chain variable region) gene mutational status, mutations of NOTCH1, TP53 and SF3B1 genes were studied by polymerase chain reaction followed by direct sequencing. Associations between clinical and molecular data of patients were analyzed with the SPSS software package, version 20.0. Results. The incidence of stereotyped CLL cases in Ukrainian cohort was high (50.5 %) and comparable in IR-exposed and non-exposed patients. The ratio of major and minor clusters as well as the frequency of individual clusters was comparable with reported data with some exceptions: a low incidence of subset #2; absence of subset #8; high frequency of minor subset #V4|J4.5.6|18|5. The distinctive features of IR-exposed CLL patients found were: 1) comparable frequency of stereotyped cases among mutated and unmutated (UM) IGHV genes cases (p = 0.557); 2) lack of differences IGHV gene repertoires among stereotyped and heterogeneous cases (p = 0.508); 3) «hetero-geneity» of stereotyped cases: all identified stereotyped clusters, with the exception of cluster #1, consisted of one case. Stereotyped cases with expression of UM IGHV clan I genes (except IGHV1-69 gene) were more susceptible to the appearance of NOTCH1 mutations. Patients of cluster #4 were younger, tended to have a longer time-to-treatment period and overall survival (OS) compared to subset #2. Patients of cluster #2 are more likely to have autoimmune hemolytic anemia (AIHA) and SF3F1 mutations. IGHV3-21 expression was associated with worse OS in univariate and multivariate analysis. AIHA was more common in patients with UM IGHV4-59 and IGHV3-11 genes. Conclusions. The revealed differences in distribution of stereotyped CLL cases in Ukrainian cohort are most likely to reflect variations in the genetic background, environmental factors (including IR exposure), and their interactions in different geographic areas. [ABSTRACT FROM AUTHOR]

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region. [ABSTRACT FROM AUTHOR]

Sequence convergence, otherwise stereotypy, of B-cell receptor immunoglobulin (BcR IG) from unrelated patients is a distinctive feature of the IG gene repertoire in chronic lymphocytic leukemia (CLL) whereby patients expressing a particular BcR IG archetype are classified into groups termed stereotyped subsets. From a biological perspective, the fact that a considerable fraction (∼41%) of patients with CLL express (quasi)identical or stereotyped BcR IG underscores the key role of antigen selection in the natural history of CLL. From a clinical perspective, at odds with the pronounced heterogeneity of CLL at large, patients belonging to the same stereotyped subset display consistent clinical presentation and outcome, including response to treatment, likely as a reflection of consistent biological background. Many major stereotyped subsets were recently shown to have satellites, that is, smaller subsets that are immunogenetically similar. Preliminary evidence supports that this similarity extends to shared biological and even clinical features, with important implications for patient stratification. Consequently, BcR IG stereotypy emerges as a powerful tool for dissecting the heterogeneity of CLL toward refined risk stratification and, eventually, more precise therapeutic interventions., Competing Interests: Declaration of competing interest Andreas Agathangelidis and Thomas Chatzikonstantinou have no conflict of interest to disclose. Kostas Stamatopoulos received honoraria and research support from Janssen, Abbvie, AstraZeneca, Gilead., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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Objective: to analyze the stereotyped subsets in cohort of Ukrainian chronic lymphocytic leukemia (CLL) patients in general and depending on the ionizing radiation (IR) exposure., Methods: Analysis was performed in the groups of 118 CLL patients irradiated due to the Chornobyl NPP accident (95 clean-up workers, 17 inhabitants of radionuclide contaminated areas, and 6 evacuees) and 294 IR non-exposed patients. The IGHV (immunoglobulin heavy chain variable region) gene mutational status, mutations of NOTCH1, TP53 and SF3B1 genes were studied by polymerase chain reaction followed by direct sequencing. Associations between clinical and molecular data of patients were analyzed with the SPSS software package, version 20.0., Results: The incidence of stereotyped CLL cases in Ukrainian cohort was high (50.5 %) and comparable in IR-exposed and non-exposed patients. The ratio of major and minor clusters as well as the frequency of individual clusters was comparable with reported data with some exceptions: a low incidence of subset #2; absence of subset #8; high frequency of minor subset #V4|J4.5.6|18|5. The distinctive features of IR-exposed CLL patients found were:1) comparable frequency of stereotyped cases among mutated and unmutated (UM) IGHV genes cases (p = 0.557);2) lack of differences IGHV gene repertoires among stereotyped and heterogeneous cases (p = 0.508); 3) «heterogeneity» of stereotyped cases: all identified stereotyped clusters, with the exception of cluster #1, consisted of one case. Stereotyped cases with expression of UM IGHV clan I genes (except IGHV1-69 gene) were more susceptible to the appearance of NOTCH1 mutations. Patients of cluster #4 were younger, tended to have a longer time-to-treatment period and overall survival (OS) compared to subset #2. Patients of cluster #2 are more likely to have autoimmune hemolytic anemia (AIHA) and SF3F1 mutations. IGHV3-21 expression was associated with worse OS in univariate and multivariate analysis. AIHA was more common in patients with UM IGHV4-59 and IGHV3-11 genes., Conclusions: The revealed differences in distribution of stereotyped CLL cases in Ukrainian cohort are most likely to reflect variations in the genetic background, environmental factors (including IR exposure), and their interactions in different geographic areas., (N. I. Bilous, I. V. Abramenko, A. A. Chumak, I. S. Dyagil, Z. V. Martina.)

According to the WHO classification, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are considered the same entity. However, the clinical presentations are different. As yet, detailed molecular-genetic comparisons between CLL and SLL are scarce. In this study, we evaluated the presence of genetic abnormalities and (immunoglobulin heavy chain variable genes) IGHV use and mutation status in lymph node samples of patients presenting with lymphadenopathy alone and, therefore, classified as SLL ( n = 21) or with persisting lymphadenopathy and absolute lymphocyte counts of ≥5 × 10/L and, therefore, classified as CLL/SLL ( n = 17). In addition, blood samples of CLL patients were evaluated ( n = 82). The majority of lymph node samples from SLL patients (66%) showed unmutated IGHV genes. This occurrence was significantly higher than in CLL (29%). Poor risk genetic aberrations (11q-, 17p-, and +12) were more prevalent in the SLL (45%) as compared to CLL (22%). Samples from CLL/SLL patients exhibited almost equal frequencies of poor risk genetic aberrations (53%), but lower frequencies of unmutated IGHV genes (35%), as compared to SLL. In summary, SLL not only differs clinically from CLL, but also genetically, with a higher propensity of adverse molecular parameters in SLL. CLL and SLL likely encompass a phenotypic spectrum within one disease entity. [ABSTRACT FROM AUTHOR]

A fraction of chronic lymphocytic leukemia (CLL) carries highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable ( IGHV) genes and heavy-chain complementarity-determining region-3 (HCDR3), often associated with a restricted selection of IG(K/L)V light chains. We analyzed the features of CLL expressing homologous HCDR3 in a cohort of 264 Ukrainian patients by merging them with a recently published reference series of 1426 cases. This approach allowed us to identify 96/264 (36%) cases as expressing homologous HCDR3, subdivided into 47 subsets. Among these, 27 apparently novel subsets were identified, although most of them were composed of two sequences per subset (‘potential subsets’). CLL cases belonging to several stereotyped subsets showed HCDR3 homologies with various autoreactive clones. Our analysis identified molecular and clinical features of a Ukrainian cohort of patients with CLL. [ABSTRACT FROM AUTHOR]

The choice of 98% sequence homology for immunoglobulin heavy chains to distinguish between mutated and unmutated versions of chronic lymphocytic leukaemia (CLL) was arbitrary and was chosen to account for supposed polymorphisms. Some authors chose 97% or even 95%. This study examined survival curves for cohorts of patients with varying degrees of sequence homology. All patients with <97% homology behaved as if mutated. Those with 97–98% homology were more aggressive than the mutated cases, but less aggressive than those with >98% homology. [ABSTRACT FROM AUTHOR]

The finding that (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins IGs) are expressed in a significant fraction of chronic lymphocytic leukemia (CLL) highlighted the importance of antigen selection in disease pathogenesis. Subsets of patients sharing the same stereotyped BcR IG display consistent biological features and, at least for certain subsets, clinical presentation and outcome, including the response to particular treatment. On these grounds, BcR IG stereotypy emerges as a useful tool for dissecting the pronounced heterogeneity of CLL toward refining risk stratification and therapeutic management aligned with the principles of precision medicine., Competing Interests: Disclosure K. Stamatopoulos has received honoraria and research support from AbbVie, Janssen, AstraZeneca, and Gilead. Conflict of Interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: to determine the association between the expression of lipoprotein lipase (LPL) and c-MYC genes inperipheral blood cells of chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophedepending on the mutational status of IGHV genes., Methods: Analysis was performed in the group of 69 CLL patients irradiated due to the Chornobyl NPP accident (58clean-up workers of 1986 year, 6 inhabitants of radionuclide contaminated areas, and 5 evacuees). The IGHV genemutational status was studied by polymerase chain reaction (PCR) followed by direct sequencing. LPL and c-MYCexpression was evaluated by Quantitative Real-time PCR. Data were analyzed with the SPSS software package, version 20.0., Results: Relative LPL expression levels in CLL samples ranged from 0 to 1663.5 (mean 138.47 ± 30.69, median 26.1).A strong correlation between individual LPL expression levels and IGHV mutational status was found (r = 0.684;p < 0.0001). The average relative c-MYC expression level was 5.7 ± 0.87 (median 2.86; range 0-48.5). No association between c-MYC expression and IGHV mutational status was found. Among unmutated IGHV cases, a correlationbetween LPL and c-MYC gene expression levels was identified: r = 0.351; p = 0.013., Conclusions: Our data confirm the dominant concept that unmutated IGHV CLL cases are more sensitive to the actionof proliferative stimuli compared to mutated IGHV CLL cases. This is manifested by an increase in the expression ofa functionally significant LPL gene, is one for the strongest negative prognostic markers in CLL., (N. I. Bilous, I. V. Abramenko, A. A. Chumak, I. S. Diagil, Z. V. Martina.)

Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway.