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1.
Leuk Res ; 89: 106288, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31924585

RESUMO

TP53 gene defects represent the most unfavorable prognostic factor in chronic lymphocytic leukemia (CLL). Although recently introduced small-molecule B-cell receptor signalling inhibitors have revolutionized CLL treatment, data for ibrutinib still point to impaired prognosis for TP53-affected patients. Among cancer-associated TP53 mutations, missense substitutions predominate and typically result in a high mutated-p53 protein level. Therefore, rescuing the p53 tumor suppressor function through specific small molecules restoring p53 wild-type (wt) conformation represents an attractive therapeutic strategy for cancer patients with TP53 missense mutations. We tested the effect of mutated-p53 reactivating molecule PRIMA-1MET in 62 clinical CLL samples characterized for TP53 mutations and p53 protein level. At the subtle PRIMA-1MET concentrations (1-4 µM), most samples manifested concentration-dependent viability decrease and, conversely, apoptosis induction, with the response being similar in both the TP53-mutated and TP53-wt groups, as well as in the TP53-mutated samples with p53 protein stabilization and without it. PRIMA-1MET was able to reduce mutated p53 protein in a proportion of TP53-mutated CLL samples, and this reduction correlated with a significantly stronger viability decrease and apoptosis induction than samples with stable p53 levels. CLL cells are mostly sensitive to PRIMA-1MET apart from those with stable mutated p53.

2.
Klin Onkol ; 32(Supplementum2): 51-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409081

RESUMO

BACKGROUND: Deleterious mutations in the BRCA1 and BRCA2 genes account for a considerable proportion of dominantly inherited breast and ovarian cancer susceptibility. The laboratory interpretation has always been dependent on the information available at the time of the report conclusion. The aim of this study has been to review the results from the BRCA testing at Masaryk Memorial Cancer Institute (MMCI). PATIENTS AND METHODS: Patients with suspected hereditary predisposition to breast/ovarian cancer, belonging to 7,400 families, were referred by genetic counsellors for BRCA1 and BRCA2 mutation testing at the MMCI from 1999 to the beginning of 2018. Various methods have been used over 20 years of laboratory practice - starting with the Protein Truncation Test and Heteroduplex Analysis via the High Resolution Melting analysis and Sanger sequencing up to Next Generation Sequencing. RESULTS: BRCA1 and BRCA2 mutation screening resulted in the identification of 1,021 families with a germline high-risk BRCA1 mutation and 497 families carrying a high-risk BRCA2 mutation, representing a mutation detection rate of 20.5%. A broad spectrum of unique mutations classified as pathogenic or likely pathogenic has been detected in both genes - 124 in the BRCA1 and 123 in the BRCA2 gene. Other sequence variants (96 unique variants in the BRCA1 and 126 in the BRCA2 gene) have been revised and classified as benign or likely benign. The other 82 unique variants remain classified as of uncertain significance mainly due to a lack of information for inclusion in other groups. All the results are summarised in the tables, including the reasons for their classification. CONCLUSION: The clinical classification of rare sequence variants identified in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling. Here we present an overview of BRCA mutation frequencies in our region and the retrospective evaluation and eventually reclassification of previously reported rare variants in light of recent findings.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença , Neoplasias Ovarianas/genética , Institutos de Câncer , República Tcheca , Feminino , Testes Genéticos , Humanos , Mutação
3.
Leuk Res ; 81: 75-81, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054420

RESUMO

The impact of genetic aberrations on rituximab-based therapeutic regimens has been intensely studied in chronic lymphocytic leukemia (CLL). According to the current consensus chemoimmunotherapy consisting of rituximab and DNA-damaging drugs is not suitable for patients with TP53 defects. In our study, we focused on CLL patients with an intact TP53 gene and investigated four recurrently mutated genes in CLL, genomic aberrations by FISH, and IGHV status with the aim of analyzing their impact on progression-free survival (PFS) after front-line therapy with FCR (fludarabine, cyclophosphamide, rituximab) or BR (bendamustine, rituximab) regimens. Using next-generation sequencing, we analyzed 120 patients treated with FCR and 57 patients treated with BR at a university hospital. We used a 10% cut-off for variant allele frequency and recorded the following mutation frequencies in the pre-therapy samples: ATM 23%, SF3B1 20%, NOTCH1 19% and BIRC3 11%. The data on cytogenetic aberrations (11q22, 13q14, trisomy 12) and IGHV mutation status were also considered in PFS analyses. In univariate analyses, we observed a negative impact of BIRC3 mutations and 11q22 deletion in both regimens, while the unmutated IGHV status was associated with a significantly shorter PFS only in the FCR-treated cohort. In a multivariate analysis, only deletion 11q22 in both regimens, and the unmutated IGHV in the FCR cohort maintained an independent association with the reduced PFS. Notably, sole 11q22 deletion, without an ATM mutation on the other allele, manifested the shortest PFS of all analyzed markers. Deletion 11q22 and IGHV status predict PFS in previously untreated CLL patients.

4.
Haematologica ; 104(12): 2443-2455, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30975914

RESUMO

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.

5.
Leuk Lymphoma ; 60(6): 1420-1428, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30626249

RESUMO

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through ATM or TP53 defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of ATM and TP53 mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated ATM and TP53 alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated TP53 gene was associated with the significantly reduced PFS and OS and the same output was observed when ATM and TP53 defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.

6.
Methods Mol Biol ; 1881: 63-81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350198

RESUMO

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfocítica Crônica de Células B/genética , Proteína Supressora de Tumor p53/genética , Alelos , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Genes Reporter/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Células Neoplásicas Circulantes/patologia , Saccharomyces cerevisiae/genética , Transfecção/instrumentação , Transfecção/métodos
7.
Ann Hematol ; 98(2): 423-435, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30368590

RESUMO

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.


Assuntos
Processamento Alternativo , Citidina Desaminase , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B , Modelos Biológicos , Proteínas de Neoplasias , Trissomia , Idoso , Animais , Cromossomos Humanos Par 12/enzimologia , Cromossomos Humanos Par 12/genética , Simulação por Computador , Citidina Desaminase/biossíntese , Citidina Desaminase/química , Citidina Desaminase/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Trissomia/genética , Trissomia/patologia
8.
Leukemia ; 33(2): 403-414, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30111844

RESUMO

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Dano ao DNA/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , MicroRNAs/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Ciclofosfamida/administração & dosagem , Dano ao DNA/genética , Feminino , Seguimentos , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Antígenos de Linfócitos B/genética , Proteínas Repressoras/genética , Rituximab/administração & dosagem , Transdução de Sinais , Taxa de Sobrevida , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados
9.
Blood ; 132(22): 2389-2400, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30213873

RESUMO

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.


Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Folicular/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Humanos , Linfoma Folicular/diagnóstico , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional , Regulação para Cima
10.
Hum Mutat ; 39(4): 515-526, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29280214

RESUMO

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Neoplasias Ovarianas/genética , Sítios de Splice de RNA , Simulação por Computador , DNA Complementar , Éxons/genética , Feminino , Variação Genética , Humanos , Mutação , RNA Mensageiro/genética
13.
Blood ; 128(12): 1609-13, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27480113

RESUMO

Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.


Assuntos
Antígenos CD20/química , Quimiocina CXCL12/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores CXCR4/metabolismo , Antígenos CD20/genética , Antígenos CD20/metabolismo , Quimiocina CXCL12/genética , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Receptores CXCR4/genética , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima
14.
Oncotarget ; 7(38): 62091-62106, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27556692

RESUMO

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.


Assuntos
Linfócitos B/citologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Nucleosídeos/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Citarabina/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Camundongos Transgênicos , Mitose , Mutação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados
15.
Leuk Lymphoma ; 56(11): 3198-206, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25827173

RESUMO

Xenograft models represent a promising tool to study the pathogenesis of hematological malignancies. To establish a reliable and appropriate in vivo model of aggressive human B-cell leukemia and lymphoma we xenotransplanted four p53-mutated cell lines and one ATM-mutated cell line into immunodeficient NOD/SCID IL2Rγ-null mice. The cell lines MEC-1, SU-DHL-4, JEKO-1, REC-1, and GRANTA-519 were transplanted intraperitoneally or subcutaneously and the engraftment was investigated using immunohistochemistry and flow cytometry. We found significant differences in engraftment efficiency. MEC-1, JEKO-1 and GRANTA-519 cell lines engrafted most efficiently, while SU-DHL-4 cells did not engraft at all. MEC-1 and GRANTA-519 massively infiltrated organs and the whole intraperitoneal cavity showing very aggressive growth. In addition, GRANTA-519 cells massively migrated to the bone marrow regardless of the transplantation route. The MEC-1 and GRANTA-519 cells can be especially recommended for in vivo study of p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma, respectively.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante Heterólogo
16.
Am J Hematol ; 90(5): 417-21, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25645263

RESUMO

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high-dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab-dexamethasone (O-Dex) combination in relapsed or refractory CLL. The trial was an open-label, multicenter, nonrandomized, Phase II study. The O-Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2-6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1-4 and 15-18; Cycles 1-6). The O-Dex regimen was given until best response, or a maximum of six cycles. Thirty-three patients (pts) were recruited. Twenty-four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression-free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3-5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O-Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101).


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Dexametasona/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Idoso , Anticorpos Monoclonais Humanizados , Esquema de Medicação , Quimioterapia Combinada/métodos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética
17.
Tumour Biol ; 36(5): 3371-80, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25527155

RESUMO

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.


Assuntos
Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único
18.
Haematologica ; 100(3): 370-6, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25480502

RESUMO

Next-generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re-sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features (unmutated IGHV, n=137; IGHV3-21 subset #2, n=51) were sequenced on the HiSeq 2000 and data were analyzed using well-established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/180 (63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/177 (84%) of all mutations. We selected 155 mutations for Sanger validation (variant allele frequency, 10-99%) and 93% (144/155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11-27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/82 (94%) mutations. In summary, this study demonstrates that targeted next-generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand-alone test without the need for confirmation by Sanger sequencing.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Linfocítica Crônica de Células B/diagnóstico , Mutação , Proteínas de Neoplasias/genética , Alelos , Expressão Gênica , Frequência do Gene , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Receptor Notch1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
20.
Exp Hematol ; 42(10): 867-74.e1, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24970561

RESUMO

Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 µg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Genes p53 , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados , Antígenos CD20/efeitos dos fármacos , Antígenos de Neoplasias/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Antígenos CD55/imunologia , Antígenos CD59/imunologia , Proteínas do Sistema Complemento/imunologia , Meios de Cultura , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rituximab , Soro , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/fisiologia
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