The genomic and molecular landscape of splenic marginal zone lymphoma, biological and clinical implications.

Splenic marginal zone lymphoma (SMZL) is a rare, predominantly indolent B-cell lymphoma constituting fewer than 2% of lymphoid neoplasms. However, around 30% of patients have a shorter survival despite currently available treatments and the prognosis is especially poor for the 5-15% of cases that transform to a large cell lymphoma. Mounting evidence suggests that the molecular pathogenesis of SMZL is critically shaped by microenvironmental triggering and cell-intrinsic aberrations. Immunogenetic investigations have revealed biases in the immunoglobulin gene repertoire, indicating a role of antigen selection. Furthermore, cytogenetic studies have identified recurrent chromosomal abnormalities such as deletion of the long arm of chromosome 7, though specific disease-associated genes remain elusive. Our knowledge of SMZL's mutational landscape, based on a limited number of cases, has identified recurring mutations in KLF2, NOTCH2, and TP53, as well as genes clustering within vital B-cell differentiation pathways. These mutations can be clustered within patient subgroups with different patterns of chromosomal lesions, immunogenetic features, transcriptional signatures, immune microenvironments, and clinical outcomes. Regarding SMZL epigenetics, initial DNA methylation profiling has unveiled epigenetically distinct patient subgroups, including one characterized by elevated expression of Polycomb repressor complex 2 (PRC2) components. Furthermore, it has also demonstrated that patients with evidence of high historical cell division, inferred from methylation data, exhibit inferior treatment-free survival. This review provides an overview of our current understanding of SMZL's molecular basis and its implications for patient outcomes. Additionally, it addresses existing knowledge gaps, proposes future research directions, and discusses how a comprehensive molecular understanding of the disease will lead to improved management and treatment choices for patients.

The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1-mutated chronic lymphocytic leukemia.

SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1MUT and 17 SF3B1WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1MUT CLL.

A Framework to Support the Standardized Management of Real-World Data.

Relying on our experience on the development of data registration and management systems for clinical and biological data coming from patients with hematological malignancies, as well as on the design of strategies for data collection and analysis to support multi-center, clinical association studies, we designed a framework for the standardized collection and transformation of clinically relevant real-world data into evidence, to meet the challenges of gathering biomedical data collected during daily clinical practice in order to promote basic and clinical research.

A Conformal Prediction Approach to Enhance Predictive Accuracy and Confidence in Machine Learning Application in Chronic Diseases.

Heterogeneity in chronic malignancies raises an increasing interest for the integration and study of predictive models. This study presents a machine learning model approach to predict outcomes and improve their trustworthiness in multi-factorial diseases with highly heterogeneous outcomes, like Chronic Lymphocytic Leukemia (CLL). We incorporated Conformal Prediction to quantify our models uncertainty, and generate confident personalized prediction outcomes that can be integrated into clinical practice.

Realizing precision medicine in chronic lymphocytic leukemia: Remaining challenges and potential opportunities.

Patients with chronic lymphocytic leukemia (CLL) exhibit diverse clinical outcomes. An expanding array of genetic tests is now employed to facilitate the identification of patients with high-risk disease and inform treatment decisions. These tests encompass molecular cytogenetic analysis, focusing on recurrent chromosomal alterations, particularly del(17p). Additionally, sequencing is utilized to identify TP53 mutations and to determine the somatic hypermutation status of the immunoglobulin heavy variable gene. Concurrently, a swift advancement of targeted treatment has led to the implementation of novel strategies for patients with CLL, including kinase and BCL2 inhibitors. This review explores both current and emerging diagnostic tests aimed at identifying high-risk patients who should benefit from targeted therapies. We outline existing treatment paradigms, emphasizing the importance of matching the right treatment to the right patient beyond genetic stratification, considering the crucial balance between safety and efficacy. We also take into consideration the practical and logistical issues when choosing a management strategy for each individual patient. Furthermore, we delve into the mechanisms underlying therapy resistance and stress the relevance of monitoring measurable residual disease to guide treatment decisions. Finally, we underscore the necessity of aggregating real-world data, adopting a global perspective, and ensuring patient engagement. Taken together, we argue that precision medicine is not the mere application of precision diagnostics and accessibility of precision therapies in CLL but encompasses various aspects of the patient journey (e.g., lifestyle exposures and comorbidities) and their preferences toward achieving true personalized medicine for patients with CLL.

Genetic counselling legislation and practice in cancer in EU Member States.

BACKGROUND: Somatic and germline genetic alterations are significant drivers of cancer. Increasing integration of new technologies which profile these alterations requires timely, equitable and high-quality genetic counselling to facilitate accurate diagnoses and informed decision-making by patients and their families in preventive and clinical settings. This article aims to provide an overview of genetic counselling legislation and practice across European Union (EU) Member States to serve as a foundation for future European recommendations and action. METHODS: National legislative databases of all 27 Member States were searched using terms relevant to genetic counselling, translated as appropriate. Interviews with relevant experts from each Member State were conducted to validate legislative search results and provide detailed insights into genetic counselling practice in each country. RESULTS: Genetic counselling is included in national legislative documents of 22 of 27 Member States, with substantial variation in legal mechanisms and prescribed details (i.e. the 'who, what, when and where' of counselling). Practice is similarly varied. Workforce capacity (25 of 27 Member States) and genetic literacy (all Member States) were common reported barriers. Recognition and/or better integration of genetic counsellors and updated legislation and were most commonly noted as the 'most important change' which would improve practice. CONCLUSIONS: This review highlights substantial variability in genetic counselling across EU Member States, as well as common barriers notwithstanding this variation. Future recommendations and action should focus on addressing literacy and capacity challenges through legislative, regulatory and/or strategic approaches at EU, national, regional and/or local levels.

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-2024 update.

In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

B-cell receptor immunoglobulin stereotypy in chronic lymphocytic leukemia: Key to understanding disease biology and stratifying patients.

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.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/µL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

Genetic Characterization of Primary Mediastinal B-Cell Lymphoma: Pathogenesis and Patient Outcomes.

PURPOSE: Primary mediastinal large B-cell lymphoma (PMBCL) is a rare aggressive lymphoma predominantly affecting young female patients. Large-scale genomic investigations and genetic markers for risk stratification are lacking. PATIENTS AND METHODS: To elucidate the full spectrum of genomic alterations, samples from 340 patients with previously untreated PMBCL were investigated by whole-genome (n = 20), whole-exome (n = 78), and targeted (n = 308) sequencing. Statistically significant prognostic variables were identified using a multivariable Cox regression model and confirmed by L1/L2 regularized regressions. RESULTS: Whole-genome sequencing revealed a commonly disrupted p53 pathway with nonredundant somatic structural variations (SVs) in TP53-related genes (TP63, TP73, and WWOX) and identified novel SVs facilitating immune evasion (DOCK8 and CD83). Integration of mutation and copy-number data expanded the repertoire of known PMBCL alterations (eg, ARID1A, P2RY8, and PLXNC1) with a previously unrecognized role for epigenetic/chromatin modifiers. Multivariable analysis identified six genetic lesions with significant prognostic impact. CD58 mutations (31%) showed the strongest association with worse PFS (hazard ratio [HR], 2.52 [95% CI, 1.50 to 4.21]; P < .001) and overall survival (HR, 2.33 [95% CI, 1.14 to 4.76]; P = .02). IPI high-risk patients with mutated CD58 demonstrated a particularly poor prognosis, with 5-year PFS and OS rates of 41% and 58%, respectively. The adverse prognostic significance of the CD58 mutation status was predominantly observed in patients treated with nonintensified regimens, indicating that dose intensification may, to some extent, mitigate the impact of this high-risk marker. By contrast, DUSP2-mutated patients (24%) displayed durable responses (PFS: HR, 0.2 [95% CI, 0.07 to 0.55]; P = .002) and prolonged OS (HR, 0.11 [95% CI, 0.01 to 0.78]; P = .028). Upon CHOP-like treatment, these patients had very favorable outcome, with 5-year PFS and OS rates of 93% and 98%, respectively. CONCLUSION: This large-scale genomic characterization of PMBCL identified novel treatment targets and genetic lesions for refined risk stratification. DUSP2 and CD58 mutation analyses may guide treatment decisions between rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone and dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab.

The effects of concurrent increases in supplementation of calcium and phytase on growth performance, balance of Ca and P, and bone mineralization in nursery pigs.

The objective of this study was to investigate the effects of concomitantly increasing supplementation of Ca and phytase on growth performance, balance of Ca and P, and bone mineralization in nursery pigs. There were eight experimental diets. The positive control (PC) one and two were formulated to contain 0.64% and 0.85% total Ca, respectively, whereas the dietary concentrations of other nutrients were identical and adequate. The negative control (NC) was deficient in total Ca (0.48%) and total P (0.41%). Five combinations of incremental levels of Ca and phytase (0.48% and 1,750 phytase units [FYT]/kg, 0.52% and 2,000 FYT/kg, 0.55% and 2,250 FYT/kg, 0.59% and 2,600 FYT/kg, and 0.63% and 3,000 FYT/kg) were added to the NC to establish the remaining five experimental diets. Each diet was fed to six pens of six pigs (three barrows and three gilts per pen). All diets contained 3 g/kg TiO2, and fecal samples were collected from each pen during the trial. In the end, one pig per pen was euthanized to collect the right tibia and urine in bladder. The results showed that the pigs of NC gained less weight, consumed less feed, and utilized feed less efficiently than their counterparts fed the PC and the treatments with phytase (P < 0.01). With increasing supplementation of Ca and phytase, there was a tendency for gain:feed to decrease (P < 0.10). There was a significant reduction in bone dry weight; and in percentages, as well as weights of bone ash, Ca, and P; in pigs of NC compared with pigs of PC1, PC2, or phytase treatments. In comparison to PC2, PC1 and phytase treatments resulted in a higher percentage of bone P and greater weights of bone ash, Ca, and P (P < 0.05). There was no significant effect of concurrent supplementation of Ca and phytase on bone mineralization. The NC had significantly lower apparent total tract digestibility (ATTD) of Ca and P, lower concentrations of digestible Ca and P, but a higher ATTD Ca/ATTD P ratio than PC1, PC2, or the phytase treatments. The averages of ATTD of Ca and P in treatments with phytase were significantly higher than PC1 or PC2 (P < 0.01). With increasing addition of Ca and phytase, the ATTD of P, digestible Ca and P, and the ATTD Ca/ATTD P ratio increased linearly (P < 0.05), which contrasted with a linear reduction in ATTD of Ca (P < 0.05). Meanwhile, there was a linear (P < 0.01) increase in the concentration of urinary Ca. In conclusion, increasing the dietary supplementation of phytase in conjunction with the increasing dietary Ca level increased the dietary ATTD Ca/ATTD P ratio without damaging the absorption of P in the current study. The higher ATTD Ca/ATTD P ratio did not improve the bone mineralization markedly and thus the extra Ca was voided through urine.

High risk HPV-positive women cervicovaginal microbial profiles in a Greek cohort: a retrospective analysis of the GRECOSELF study.

Increasing evidence supports a role for the vaginal microbiome (VM) in the severity of HPV infection and its potential link to cervical intraepithelial neoplasia. However, a lot remains unclear regarding the precise role of certain bacteria in the context of HPV positivity and persistence of infection. Here, using next generation sequencing (NGS), we comprehensively profiled the VM in a series of 877 women who tested positive for at least one high risk HPV (hrHPV) type with the COBAS® 4,800 assay, after self-collection of a cervico-vaginal sample. Starting from gDNA, we PCR amplified the V3-V4 region of the bacterial 16S rRNA gene and applied a paired-end NGS protocol (Illumina). We report significant differences in the abundance of certain bacteria compared among different HPV-types, more particularly concerning species assigned to Lacticaseibacillus, Megasphaera and Sneathia genera. Especially for Lacticaseibacillus, we observed significant depletion in the case of HPV16, HPV18 versus hrHPVother. Overall, our results suggest that the presence or absence of specific cervicovaginal microbial genera may be linked to the observed severity in hrHPV infection, particularly in the case of HPV16, 18 types.

N-Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.

EuroClonality-NGS Recommendations for Evaluation of B-Cell Clonality Analysis by Next-Generation Sequencing: A Structured Approach with the DEPART Algorithm.

Next-generation sequencing (NGS)-based clonality analysis allows in-depth assessment of the clonal composition of a sample with high sensitivity for detecting small clones. Within the EuroClonality-NGS Working Group, a protocol for NGS Ig clonality analysis was developed and validated previously. This NGS-based approach was designed to generate small amplicons, making it suitable for samples with suboptimal DNA quality, especially material derived from formalin-fixed, paraffin-embedded tissue. Using expert assessment of NGS Ig clonality results as a reference, a structured algorithmic approach to the assessment of NGS-amplicon-based B-cell clonality analysis was developed. A structured approach with the Detection of clonality through Evaluation of sample quality and assessment of Pattern, Abundance and RaTio (DEPART) algorithm was proposed, which consecutively evaluates sample quality, the pattern of the clonotypes present, the abundance of the most dominant clonotypes, and the ratio between the dominant clonotypes and the background to evaluate the different Ig gene targets. Specific issues with respect to evaluation of the various Ig targets as well as the integration of results of individual targets into a molecular clonality conclusion are discussed and illustrated with case examples. Finally, the importance of interpretation of NGS-based clonality results in clinical and histopathologic contexts is discussed. It is expected that these recommendations will have clinical utility to facilitate proper evaluation of clonality assessment.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

CLL stereotyped B-cell receptor immunoglobulin sequences are recurrent in the B-cell repertoire of healthy individuals: Apparent lack of central and early peripheral tolerance censoring.

Introduction: The leukemic cells of patients with chronic lymphocytic leukemia (CLL) are often unique, expressing remarkably similar IGHV-IGHD-IGHJ gene rearrangements, "stereotyped BCRs". The B-cell receptors (BCRs) on CLL cells are also distinctive in often deriving from autoreactive B lymphocytes, leading to the assumption of a defect in immune tolerance. Results: Using bulk and single-cell immunoglobulin heavy and light chain variable domain sequencing, we enumerated CLL stereotype-like IGHV-IGHD-IGHJ sequences (CLL-SLS) in B cells from cord blood (CB) and adult peripheral blood (PBMC) and bone marrow (BM of healthy donors. CLL-SLS were found at similar frequencies among CB, BM, and PBMC, suggesting that age does not influence CLL-SLS levels. Moreover, the frequencies of CLL-SLS did not differ among B lymphocytes in the BM at early stages of development, and only re-circulating marginal zone B cells contained significantly higher CLL-SLS frequencies than other mature B-cell subpopulations. Although we identified CLL-SLS corresponding to most of the CLL major stereotyped subsets, CLL-SLS frequencies did not correlate with those found in patients. Interestingly, in CB samples, half of the CLL-SLS identified were attributed to two IGHV-mutated subsets. We also found satellite CLL-SLS among the same normal samples, and they were also enriched in naïve B cells but unexpectedly, these were ~10-fold higher than standard CLL-SLS. In general, IGHV-mutated CLL-SLS subsets were enriched among antigen-experienced B-cell subpopulations, and IGHV-unmutated CLL-SLS were found mostly in antigen-inexperienced B cells. Nevertheless, CLL-SLS with an IGHV-mutation status matching that of CLL clones varied among the normal B-cell subpopulations, suggesting that specific CLL-SLS could originate from distinct subpopulations of normal B cells. Lastly, using single-cell DNA sequencing, we identified paired IGH and IGL rearrangements in normal B lymphocytes resembling those of stereotyped BCRs in CLL, although some differed from those in patients based on IG isotype or somatic mutation. Discussion: CLL-SLS are present in normal B-lymphocyte populations at all stages of development. Thus, despite their autoreactive profile they are not deleted by central tolerance mechanisms, possibly because the level of autoreactivity is not registered as dangerous by deletion mechanisms or because editing of L-chain variable genes occurred which our experimental approach could not identify.