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.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• chronická lymfatická leukemie * genetika   patologie   metabolismus   farmakoterapie   MeSH
• lidé MeSH
• myši MeSH
• NF-kappa B * metabolismus   MeSH
• piperidiny farmakologie   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteiny I-kappa B * genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenin MeSH
• ibrutinib MeSH Prohlížeč
• NF-kappa B * MeSH
• NFKBIE protein, human MeSH Prohlížeč
• piperidiny MeSH
• proteiny I-kappa B * MeSH
• protoonkogenní proteiny MeSH

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

• MeSH
• chronická lymfatická leukemie * genetika   MeSH
• fenotyp MeSH
• lidé MeSH
• mutace MeSH
• myeloidní diferenciační faktor 88 genetika   MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• myeloidní diferenciační faktor 88 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

• MeSH
• chromozomální aberace * MeSH
• chronická lymfatická leukemie genetika   patologie   terapie   MeSH
• kombinovaná terapie MeSH
• lidé MeSH
• longitudinální studie MeSH
• mutace * MeSH
• nádorové biomarkery genetika   MeSH
• následné studie MeSH
• prognóza MeSH
• sekvenování exomu MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• nádorové biomarkery 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

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

• 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

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s).

• MeSH
• chronická lymfatická leukemie genetika   metabolismus   mortalita   MeSH
• cytogenetické vyšetření MeSH
• frekvence genu MeSH
• genová přestavba B-lymfocytů MeSH
• geny pro imunoglobuliny MeSH
• hypervariabilní oblasti genetika   MeSH
• imunoglobuliny - vazebný region genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• mutace * MeSH
• nádorové biomarkery * MeSH
• prognóza MeSH
• receptory antigenů B-buněk genetika   metabolismus   MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• variabilní oblast imunoglobulinu genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hypervariabilní oblasti MeSH
• imunoglobuliny - vazebný region MeSH
• nádorové biomarkery * MeSH
• receptory antigenů B-buněk MeSH
• těžké řetězce imunoglobulinů MeSH
• variabilní oblast imunoglobulinu MeSH

Fludarabine, cyclophosphamide, and rituximab (FCR) is first-line treatment of medically fit chronic lymphocytic leukemia (CLL) patients; however, despite good response rates, many patients eventually relapse. Although recent high-throughput studies have identified novel recurrent genetic lesions in adverse prognostic CLL, the mechanisms leading to relapse after FCR therapy are not completely understood. To gain insight into this issue, we performed whole-exome sequencing of sequential samples from 41 CLL patients who were uniformly treated with FCR but relapsed after a median of 2 years. In addition to mutations with known adverse-prognostic impact (TP53, NOTCH1, ATM, SF3B1, NFKBIE, and BIRC3), a large proportion of cases (19.5%) harbored mutations in RPS15, a gene encoding a component of the 40S ribosomal subunit. Extended screening, totaling 1119 patients, supported a role for RPS15 mutations in aggressive CLL, with one-third of RPS15-mutant cases also carrying TP53 aberrations. In most cases, selection of dominant, relapse-specific subclones was observed over time. However, RPS15 mutations were clonal before treatment and remained stable at relapse. Notably, all RPS15 mutations represented somatic missense variants and resided within a 7 amino-acid, evolutionarily conserved region. We confirmed the recently postulated direct interaction between RPS15 and MDM2/MDMX and transient expression of mutant RPS15 revealed defective regulation of endogenous p53 compared with wild-type RPS15. In summary, we provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment and highlight a novel mechanism underlying clinical aggressiveness involving a mutated ribosomal protein, potentially representing an early genetic lesion in CLL pathobiology.

• MeSH
• chemorezistence genetika   MeSH
• chronická lymfatická leukemie farmakoterapie   genetika   mortalita   patologie   MeSH
• cyklofosfamid aplikace a dávkování   MeSH
• exom MeSH
• imunoprecipitace MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• lokální recidiva nádoru genetika   patologie   MeSH
• missense mutace * MeSH
• mutační analýza DNA MeSH
• nádorový supresorový protein p53 genetika   MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• ribozomální proteiny genetika   MeSH
• rituximab aplikace a dávkování   MeSH
• separace buněk MeSH
• transfekce MeSH
• vidarabin aplikace a dávkování   analogy a deriváty   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklofosfamid MeSH
• fludarabine MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• ribosomal protein S15 MeSH Prohlížeč
• ribozomální proteiny MeSH
• rituximab MeSH
• vidarabin MeSH