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.

• MeSH
• alternativní sestřih MeSH
• chronická lymfatická leukemie * genetika   patologie   metabolismus   MeSH
• fosfoproteiny * genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• proteiny obsahující bromodoménu MeSH
• restrukturace chromatinu * MeSH
• sestřihové faktory * genetika   metabolismus   MeSH
• spliceozomy * metabolismus   genetika   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BRD9 protein, human MeSH Prohlížeč
• fosfoproteiny * MeSH
• proteiny obsahující bromodoménu MeSH
• sestřihové faktory * MeSH
• SF3B1 protein, human MeSH Prohlížeč
• transkripční faktory MeSH

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have a higher risk of developing other malignancies (OMs) compared to the general population. However, the impact of CLL-related risk factors and CLL-directed treatment is still unclear and represents the focus of this work. METHODS: We conducted a retrospective international multicenter study to assess the incidence of OMs and detect potential risk factors in 19,705 patients with CLL, small lymphocytic lymphoma, or high-count CLL-like monoclonal B-cell lymphocytosis, diagnosed between 2000 and 2016. Data collection took place between October 2020 and March 2022. FINDINGS: In 129,254 years of follow-up after CLL diagnosis, 3513 OMs were diagnosed (27.2 OMs/1000 person-years). The most common hematological OMs were Richter transformation, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Non-melanoma skin (NMSC) and prostate cancers were the most common solid tumors (STs).The only predictor for MDS and AML development was treatment with fludarabine and cyclophosphamide with/without rituximab (FC ± R) (OR = 3.7; 95% CI = 2.79-4.91; p < 0.001). STs were more frequent in males and patients with unmutated immunoglobulin heavy variable genes (OR = 1.77; 95% CI = 1.49-2.11; p < 0.001/OR = 1.89; 95% CI = 1.6-2.24; p < 0.001).CLL-directed treatment was associated with non-melanoma skin and prostate cancers (OR = 1.8; 95% CI = 1.36-2.41; p < 0.001/OR = 2.11; 95% CI = 1.12-3.97; p = 0.021). In contrast, breast cancers were more frequent in untreated patients (OR = 0.17; 95% CI = 0.08-0.33; p < 0.001).Patients with CLL and an OM had inferior overall survival (OS) than those without. AML and MDS conferred the worst OS (p < 0.001). INTERPRETATION: OMs in CLL impact on OS. Treatment for CLL increased the risk for AML/MDS, prostate cancer, and NMSC. FCR was associated with increased risk for AML/MDS. FUNDING: AbbVie, and EU/EFPIAInnovative Medicines Initiative Joint Undertaking HARMONY grant n° 116026.

• Klíčová slova
• Chronic lymphocytic leukemia, Other cancers, Other malignancies, Second primary malignancies,
• Publikační typ
• časopisecké články MeSH

Mounting evidence underscores the clinical value of cytogenetic analysis in chronic lymphocytic leukemia (CLL), particularly as it allows the identification of complex karyotype, that has recently emerged as a prognostic and potentially predictive biomarker. That said, explicit recommendations regarding the methodology and clinical interpretation of either chromosome banding analysis (CBA) or chromosome microarray analysis (CMA) are still lacking. We herein present the consensus of the Cytogenetic Steering Scientific Committee of ERIC, the European Research Initiative on CLL, regarding methodological issues as well as clinical interpretation of CBA/CMA and discuss their relevance in CLL. ERIC considers CBA standardized and feasible for CLL on the condition that standards are met, extending from the use of novel mitogens to the accurate interpretation of the findings. On the other hand, CMA, is also standardized, however, robust data on its clinical utility are still scarce. In conclusion, cytogenetic analysis is not yet mature enough to guide treatment choices in CLL. That notwithstanding, ERIC encourages the wide application of CBA, and potentially also CMA, in clinical trials in order to obtain robust evidence regarding the predictive value of specific cytogenetic profiles towards refining risk stratification and improving the management of patients with CLL.

• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• chronická lymfatická leukemie genetika   MeSH
• frekvence genu MeSH
• genová přestavba MeSH
• lidé MeSH
• somatická hypermutace imunoglobulinových genů MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• variabilní oblast imunoglobulinu genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• těžké řetězce imunoglobulinů MeSH
• variabilní oblast imunoglobulinu MeSH

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

• MeSH
• chronická lymfatická leukemie * diagnóza   genetika   MeSH
• lidé MeSH
• mutace MeSH
• reprodukovatelnost výsledků MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.

• MeSH
• čas zasáhnout při rozvinutí nemoci MeSH
• chromozomální aberace MeSH
• chronická lymfatická leukemie etiologie   mortalita   patologie   terapie   MeSH
• imunogenetika MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• mutace MeSH
• náchylnost k nemoci * MeSH
• nádorové biomarkery * MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery * MeSH

• MeSH
• chronická lymfatická leukemie farmakoterapie   mortalita   MeSH
• dospělí MeSH
• farmakoterapie mortalita   MeSH
• imunoterapie mortalita   MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• mladý dospělý MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

The rapid progress in next-generation sequencing technologies has significantly contributed to our knowledge of the genetic events associated with the development, progression and treatment resistance of chronic lymphocytic leukemia patients. Together with the discovery of new driver mutations, next-generation sequencing has revealed an immense degree of both intra- and inter-tumor heterogeneity and enabled us to describe marked clonal evolution. Advances in immunogenetics may be implemented to detect minimal residual disease more sensitively and to track clonal B cell populations, their dynamics and molecular characteristics. The interpretation of these aspects is indispensable to thoroughly examine the genetic background of chronic lymphocytic leukemia. We review and discuss the recent results provided by the different next-generation sequencing techniques used in studying the chronic lymphocytic leukemia genome, as well as future perspectives in the methodologies and applications.

• Klíčová slova
• CLL prognosis, chronic lymphocytic leukemia, clonal evolution, immunogenetics, next-generation sequencing,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• chronická lymfatická leukemie genetika   MeSH
• geny pro imunoglobuliny * MeSH
• lidé MeSH
• sekvenční analýza DNA normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Evropa MeSH

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

• MeSH
• chronická lymfatická leukemie klasifikace   farmakoterapie   genetika   mortalita   MeSH
• dospělí MeSH
• geny p53 MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• proporcionální rizikové modely MeSH
• protein 2 časné růstové odpovědi genetika   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EGR2 protein, human MeSH Prohlížeč
• protein 2 časné růstové odpovědi MeSH