RESUMO
BACKGROUND: Chronic lymphocytic leukemia (CLL) is clinically and biologically highly variable disease which is closely related with multiple cellular and molecular markers, including sequence motifs of B-cell receptors. These motifs are highly similar (stereotyped) within one third of CLL patients and create homogeneous groups called stereotyped CLL subsets. The homogeneity is reflected also in clinical and biological characteristics of the disease. To facilitate access to the information about individual subsets, we have created a publicly available web-based tool Encyclopedia of CLL Subsets. MATERIALS AND METHODS: The Encyclopedia of CLL subsets belongs to our bioinformatics platform Antigen Receptor Research Tool (ARResT) developed for analysis, clustering, and annotation of immunoglobulin sequences. To gather primary knowledge about the subsets, we have analyzed a dataset of 7,500 CLL patients published by Agathangelidis et al in 2012 [1]. We have created an overview of major stereotyped subsets and their characteristics. Additional clinical and cytogenomic information about individual subsets has been obtained by machine text processing of available literature from server PubMed and is regularly updated. RESULTS: We have created a unique web-based application Encyclopedia of CLL Subsets available from http: //arrest.tools/subsets for an interactive access to the information about stereotyped CLL subsets. A user can obtain and compare basic information about the major subsets including their clinical and cytogenomic characteristics. These have been manually curated from machine processed results from PubMed database by experts in CLL research. Through the Encyclopedias user interface, user can also directly use our published tool ARResT/AssignSubsets to assign new immunoglobulin sequences to the major subsets. CONCLUSION: The Encyclopedia of CLL Subsets is a publicly available online tool facilitating access to the most recent research knowledge about stereotyped CLL subsets and enabling analysis of own data and interpretation of the results. This gives the Encyclopedia a great potential for its use in clinical routine. This work was supported by Czech Ministry of Health grant No. 34272A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.
Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Bases de Dados Factuais , Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/classificação , Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Antígenos de Linfócitos B/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are defined as RNA molecules longer than 200 nucleotides with poor protein-coding capacity and key functions in regulation of gene expression. Dysregulations of lncRNAs (e.g. HOTAIR and MALAT I) were detected in plasma of breast cancer (BC) patients. Plasma samples are examined as liquid biopsies for purposes of non-invasive diagnostics therefore the research of plasma lncRNAs as potential plasma biomarkers became highly topical. 84 lncRNAs were profiled in 18 plasma samples - 9 BC patients and 9 age-matched healthy - using Human Inflammatory Response & Autoimmunity RT2 lncRNA PCR Array. Total RNA from plasma samples was isolated using miRNeasy Serum/Plasma Kit. Although a pre-amplification recommended for quantification from small starting RNA amounts was used, only 3 lncRNAs (A2ML1-AS1, GAS5 and SNHG5) were detected in all plasma samples. A total of 72 lncRNAs (e.g. HOTAIR or MALAT I) were detected only in some samples and 9 lncRNAs were not detected in any samples. No significant differences were observed in levels of plasma lncRNAs between the BC patients and healthy controls despite the fact that our panel contained also the lncRNAs whose levels were previously reported as significantly different in plasma or cancer tissues (e.g. GAS5, HOTAIR, MALAT I) in BC patients. Detection of lncRNAs in plasma is due to their low concentrations quite difficult as compared with tissues. Our findings suggest that analysis of plasma lncRNAs using this technology is not suitable for use as non-invasive diagnostic tool in BC patients.
Assuntos
Neoplasias da Mama/diagnóstico , Reação em Cadeia da Polimerase , RNA Longo não Codificante/sangue , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Chromosome rearrangements play an important role in cancer pathophysiology. Recently, chromothripsis has been proposed among the mechanisms leading to their formation. Chromothripsis leads to fragmentation of chromosomes and their reconstitution with tens to hundreds of rearrangements clustered in small genome regions. In contrast to the traditional concept of malignant transformation, abnormalities caused by chromothripsis are not accumulated gradually but arise during a single event. The resulting structural variants are extensive and often cause oncogene activation or tumor suppressor inactivation. Chromothripsis is associated with many tumor types, especially with brain and bone tumors. Besides that, it has been described also in congenital disorders. The exact mechanism of chromothripsis origin has not been clarified yet; however, several hypotheses have been prosed, among which DNA damage in micronucleus seems to be most likely. Similarly, an impact of chromothripsis on cellular processes has not been fully understood, yet its association with unfavorable prognosis has been observed. PURPOSE: The purpose of this article is to summarize the current knowledge about chromothripsis and to present gathered pieces of information in a structured way. We focused on describing the basic features of chromothripsis, potential mechanisms of its origin, its impact on cellular processes and providing an overview of diseases where chromothripsis has been noted, with particular attention to cancer. Finally, we suggest a potential use of current knowledge about chromothripsis in the optimization of personalized treatment. Supported by Ministry of Health of the Czech Republic, grant no. 15-31834A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 31. 12. 2018 Accepted: 19. 3. 2019.
Assuntos
Cromossomos Humanos/genética , Cromotripsia , Dano ao DNA , Neoplasias/genética , Neoplasias/patologia , HumanosRESUMO
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.
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Análise Mutacional de DNA/métodos , Genes p53/genética , Leucemia Linfocítica Crônica de Células B/genética , Europa (Continente) , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2-16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patient's age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.
Assuntos
Hidroxiureia/administração & dosagem , Janus Quinase 2/genética , Mutação/genética , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Progressão da Doença , Feminino , Frequência do Gene/efeitos dos fármacos , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation. MATERIAL AND METHODS: Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350-450 µm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293. RESULTS: Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350-450 µm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone. CONCLUSIONS: We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment.Key words: hematooncology - leukemia - hydrogel - stromal cells This work was supported by grant COST CZ LD15144 "Cellular and acellular grounds for regeneration of bones and teeth" awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 3. 2017Accepted: 26. 3. 2017.
Assuntos
Linfócitos B/patologia , Hidrogéis/química , Leucemia Linfocítica Crônica de Células B/patologia , Alicerces Teciduais , Proliferação de Células , Técnicas de Cocultura , Células HEK293 , HumanosRESUMO
Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated.
Assuntos
Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos T/imunologia , Idoso , Antígenos de Neoplasias , Linfócitos T CD8-Positivos/imunologia , Microambiente Celular , Rearranjo Gênico do Linfócito T , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
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.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Adulto , Idoso , Feminino , Genes p53 , Humanos , Leucemia Linfocítica Crônica de Células B/classificação , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Modelos de Riscos ProporcionaisRESUMO
High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases , Humanos , Camundongos , Mutação , Controle de QualidadeRESUMO
Urinary bladder carcinoma contributes to 4% of newly diagnosed oncological diseases in the Czech Republic. Biomarkers for its early non-invasive detection are therefore highly desirable. Urine seems to be an ideal source of such biomarkers due to the content of cell-free nucleic acids, especially microRNAs (miRNAs).To find potential biomarkers among miRNAs in urine supernatant, we examined in total 109 individuals (36 controls and 73 bladder cancer patients) in three phases. In the first - discovery - phase, microarray cards with 381 miRNAs were used for miRNA analysis of 13 controls and 46 bladder cancer patients. In the second - verification - phase, the results of this first phase were verified on the same groups of subjects by single-target qPCR assays for the selected miRNAs. For the third - validation - phase, new independent samples of urine supernatant (23 controls and 27 bladder cancer patients) were analyzed using single-target qPCR assays for 13 verified in the previous phase. The results of all phases were normalized to miR-191, miR-28-3p, and miR-200b, which were selected as suitable for our study by the qBase+®.We found that miR-125b, miR-30b, miR-204, miR-99a, and miR-532-3p are significantly down-regulated in patients' urine supernatant. In our experiments, the analysis of miR-125 levels provided the highest AUC (0.801) with 95.65% specificity and 59.26% sensitivity, the analysis of miR-99a lead to AUC (0.738) with 82.61% specificity and 74.07% sensitivity. We demonstrate that levels of these miRNAs could potentially serve as promising diagnostic markers for the non-invasive diagnostics of bladder cancer.
Assuntos
Biomarcadores Tumorais/urina , MicroRNAs/urina , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Bexiga Urinária/patologiaRESUMO
In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.
Assuntos
Antígenos CD/metabolismo , Citometria de Fluxo/normas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Neoplasia Residual/diagnóstico , Adolescente , Adulto , Terapia Combinada , Europa (Continente) , Feminino , Seguimentos , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Estadiamento de Neoplasias , Neoplasia Residual/genética , Neoplasia Residual/metabolismo , Prognóstico , Adulto JovemRESUMO
BACKGROUND: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ânodular with its extensive-nodularity variant, and anaplastic/âlarge-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5-â10% of all cases occur in connection with hereditary genetic syndromes. CASE: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ânodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations. CONCLUSION: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ânodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/âdeletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.
Assuntos
Neoplasias Cerebelares/genética , Mutação em Linhagem Germinativa , Meduloblastoma/genética , Proteínas Repressoras/genética , Feminino , Humanos , LactenteRESUMO
Chronic lymphocytic leukemia is the most common leukemia in Western countries affecting particularly elderly adults. Despite the constantly improving therapy options, chronic lymphocytic leukemia is still an incurable disease owing to considerable clinical and bio-logical heterogeneity. Pathogenesis of chronic lymphocytic leukemia is not fully understood; however, aberrant antigenic stimulation, apoptosis deregulation and microenvironmental interactions play a crucial role in disease development. The most important molecular prognostic markers with clinical relevance include mutation status of heavychain immunoglobulin genes (IGHV), presence of cytogenetic aberrations and TP53 and ATM gene mutations. Recent implementation of next generation sequencing technologies has enabled more accurate analysis of both wellestablished and novel potential prognostic markers. The most relevant candidates are mutations in SF3B1, NOTCH1 and BIRC3 genes, which are now intensively studied with respect to their clinical importance. The other examined molecular mechanisms of chronic lympho-cytic leukemia pathogenesis include deregulation of Bâcell receptor signalization and abnormal regulation of gene expression by microRNA. The precise characterization of molecular abnormalities improves the risk stratification of chronic lymphocytic leukemia patients, which could possibly benefit from new treatment approaches.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Biomarcadores , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/etiologia , Mutação , Prognóstico , Microambiente TumoralRESUMO
Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Coortes , Dano ao DNA , Análise Mutacional de DNA , Doxorrubicina/farmacologia , Citometria de Fluxo , Deleção de Genes , Genoma Humano , Histonas/metabolismo , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.
Assuntos
Linfócitos B/metabolismo , Evolução Clonal/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Evolução Clonal/efeitos dos fármacos , Células Clonais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva , Estudos Retrospectivos , Transdução de Sinais , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismoRESUMO
Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.
Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Idoso , Citogenética , Análise Mutacional de DNA , Europa (Continente) , Feminino , Deleção de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Recidiva , Ribonucleoproteína Nuclear Pequena U2/genética , Fatores de Tempo , Proteína Supressora de Tumor p53/genéticaRESUMO
INTRODUCTION: Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time-consuming and require experienced laboratory staff. METHODS: To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. RESULTS: Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n = 20) or after fludarabine-based therapy (n = 22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r = 0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. CONCLUSION: Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.
Assuntos
Citometria de Fluxo/métodos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Neoplasia Residual/diagnóstico , Adulto , Idoso , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transplante HomólogoRESUMO
BACKGROUND: Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. PATIENTS AND METHODS: In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. RESULTS: Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. CONCLUSION: Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.
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Bandeamento Cromossômico , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 13 , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Hibridização in Situ Fluorescente , Interleucina-2/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos , Células Tumorais CultivadasRESUMO
Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.