Nejvíce citovaný článek - PubMed ID 25480502
Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting
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.
Chronic lymphocytic leukemia is associated with a highly heterogeneous disease course in terms of clinical outcomes and responses to chemoimmunotherapy. This heterogeneity is partly due to genetic aberrations identified in chronic lymphocytic leukemia cells such as mutations of TP53 and/or deletions in chromosome 17p [del(17p)], resulting in loss of one TP53 allele. These aberrations are associated with markedly decreased survival and predict impaired response to chemoimmunotherapy thus being among the strongest predictive markers guiding treatment decisions in chronic lymphocytic leukemia. Clinical trials demonstrate the importance of accurately testing for TP53 aberrations [both del(17p) and TP53 mutations] before each line of treatment to allow for appropriate treatment decisions that can optimize patients' outcomes. The current report reviews the diagnostic methods to detect TP53 disruption better, the role of TP53 aberrations in treatment decisions and current therapies available for patients with chronic lymphocytic leukemia carrying these abnormalities. The standardization in sequencing technologies for accurate identification of TP53 mutations and the importance of continued evaluation of TP53 aberrations throughout initial and subsequent lines of therapy remain unmet clinical needs as new therapeutic alternatives become available.
- MeSH
- chromozomální delece * MeSH
- chronická lymfatická leukemie diagnóza genetika terapie MeSH
- lidé MeSH
- lidské chromozomy, pár 17 genetika MeSH
- mutace * MeSH
- nádorový supresorový protein p53 genetika MeSH
- přežití bez známek nemoci MeSH
- prognóza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- nádorový supresorový protein p53 MeSH
In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.
- MeSH
- chronická lymfatická leukemie genetika MeSH
- geny p53 genetika MeSH
- lidé MeSH
- mutační analýza DNA metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- směrnice pro lékařskou praxi MeSH
- Geografické názvy
- Evropa 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
- adenin analogy a deriváty MeSH
- alely MeSH
- bicyklické sloučeniny heterocyklické terapeutické užití MeSH
- chinazolinony terapeutické užití MeSH
- chronická lymfatická leukemie diagnóza farmakoterapie genetika patologie MeSH
- cílená molekulární terapie MeSH
- exprese genu MeSH
- fosfatidylinositol-3-kinasy třídy I antagonisté a inhibitory genetika metabolismus MeSH
- frekvence genu MeSH
- lidé MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádorový supresorový protein p53 nedostatek genetika MeSH
- piperidiny MeSH
- prognóza MeSH
- proteinkinasa BTK MeSH
- protinádorové látky terapeutické užití MeSH
- puriny terapeutické užití MeSH
- pyrazoly terapeutické užití MeSH
- pyrimidiny terapeutické užití MeSH
- sulfonamidy terapeutické užití MeSH
- tyrosinkinasy antagonisté a inhibitory genetika metabolismus MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- úvodníky MeSH
- Názvy látek
- adenin MeSH
- bicyklické sloučeniny heterocyklické MeSH
- chinazolinony MeSH
- fosfatidylinositol-3-kinasy třídy I MeSH
- ibrutinib MeSH Prohlížeč
- idelalisib MeSH Prohlížeč
- nádorové biomarkery MeSH
- nádorový supresorový protein p53 MeSH
- PIK3CD protein, human MeSH Prohlížeč
- piperidiny MeSH
- proteinkinasa BTK MeSH
- protinádorové látky MeSH
- puriny MeSH
- pyrazoly MeSH
- pyrimidiny MeSH
- sulfonamidy MeSH
- TP53 protein, human MeSH Prohlížeč
- tyrosinkinasy MeSH
- venetoclax MeSH Prohlížeč
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