RESUMO
Multiple myeloma (MM) cells from 1 out of 20 patient expressed high basal levels of membrane B-cell maturation antigen (BCMA, TNFRSF17, CD269), which was not upregulated by gamma-secretase inhibitor, suggesting a defective BCMA shedding by gamma-secretase. Genetic analyses of the patient's bone marrow DNA showed no mutations within the BCMA coding region, but rather partial deletion of PSEN1 and amplification of PSEN2, which encode alternative catalytic units of gamma-secretase. Altogether the data suggest that pt#12 MM cells express high and dysregulated BCMA with no shedding, due to genetic alterations of one or more gamma-secretase subunits.
Assuntos
Mieloma Múltiplo , Humanos , Antígeno de Maturação de Linfócitos B , Secretases da Proteína Precursora do Amiloide , Medula Óssea/químicaRESUMO
Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Nucleofosmina/genética , Adulto , Éxons , Feminino , Fusão Gênica , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Hematológicas , Leucemia Linfocítica Crônica de Células B , Humanos , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Viés , Biomarcadores Tumorais/genéticaRESUMO
Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6-targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor-resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide "proof of principle" that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the "CDK6 dependence" of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.
Assuntos
Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 6 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Genes cdc , Humanos , Camundongos , Estrutura Molecular , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
Assuntos
Leucemia Eritroblástica Aguda/genética , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Transcriptoma , Adulto , Animais , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Eritroblastos/metabolismo , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/deficiência , Fator de Transcrição GATA1/genética , Técnicas de Introdução de Genes , Heterogeneidade Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA-Seq , Quimera por Radiação , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/fisiologia , Sequenciamento do Exoma , Adulto JovemRESUMO
We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab')2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.
Assuntos
Deleção de Genes , Neutrófilos/imunologia , Receptores de IgG/genética , Células Cultivadas , Expressão Gênica , Humanos , Imunoglobulina G/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Neutrófilos/metabolismo , Fagocitose , RNA Mensageiro/genética , Receptores de IgG/imunologiaRESUMO
Secondary acute myeloid leukemia (sAML) after myelodysplastic or myeloproliferative disorders is a high-risk category currently identified by clinical history or specific morphological and cytogenetic abnormalities. However, in the absence of these features, uncertainties remain to identify the secondary nature of some cases otherwise defined as de novo AML. To test whether a chromatin-spliceosome (CS) mutational signature might better inform the definition of the de novo AML group, we analyzed a prospective cohort of 413 newly diagnosed AML patients enrolled into a randomized clinical trial (NILG AML 02/06) and provided with accurate cytogenetic and molecular characterization. Among clinically defined de novo AML, 17.6% carried CS mutations (CS-AML) and showed clinical characteristics closer to sAML (older age, lower white blood cell counts and higher rate of multilineage dysplasia). Outcomes in this group were adverse, more similar to those of sAML as compared to de novo AML (overall survival, 30% in CS-AML and 17% in sAML vs 61% in de novo AML, P<0.0001; disease free survival, 26% in CS-AML and 22% in sAML vs 54% of de novo AML, P<0.001) and independently confirmed by multivariable analysis. Allogeneic transplant in first complete remission improved survival in both sAML and CS-AML patients. In conclusion, these findings highlight the clinical significance of identifying CS-AML for improved prognostic prediction and potential therapeutic implications. (NILG AML 02/06: ClinicalTrials.gov Identifier: NCT00495287).
Assuntos
Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Idoso , Cromatina/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/genética , Prognóstico , Estudos Prospectivos , SpliceossomosRESUMO
Early recognition of Ph-like acute lymphoblastic leukemia cases could impact on the management and outcome of this subset of B-lineage ALL. To assess the prognostic value of the Ph-like status in a pediatric-inspired, minimal residual disease (MRD)-driven trial, we screened 88 B-lineage ALL cases negative for the major fusion genes (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1 and KTM2Ar) enrolled in the GIMEMA LAL1913 front-line protocol for adult BCR/ABL1-negative ALL. The screening - performed using the BCR/ABL1-like predictor - identified 28 Ph-like cases (31.8%), characterized by CRLF2 overexpression (35.7%), JAK/STAT pathway mutations (33.3%), IKZF1 (63.6%), BTG1 (50%) and EBF1 (27.3%) deletions, and rearrangements targeting tyrosine kinases or CRLF2 (40%). The correlation with outcome highlighted that: i) the complete remission (CR) rate was significantly lower in Ph-like compared to non-Ph-like cases (74.1% vs 91.5%, p=0.044); ii) at time point 2 (TP2), decisional for transplant allocation, 52.9% of Ph-like cases vs 20% of non-Ph-like were MRD-positive (p=0.025); iii) the Ph-like profile was the only parameter associated with a higher risk of being MRD-positive at TP2 (p=0.014); iv) at 24 months, Ph-like patients had a significantly inferior event-free and disease-free survival compared to non-Ph-like patients (33.5% vs 66.2%, p=0.005 and 45.5% vs 72.3%, p=0.062, respectively). This study documents that Ph-like patients have a lower CR rate, EFS and DFS, as well as a greater MRD persistence also in a pediatric-oriented and MRD-driven adult ALL protocol, thus reinforcing that the early recognition of Ph-like ALL patients at diagnosis is crucial to refine risk-stratification and to optimize therapeutic strategies.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Doença Aguda , Adulto , Intervalo Livre de Doença , Humanos , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , PrognósticoAssuntos
Automação , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas , Humanos , Projetos Piloto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Biblioteca Gênica , Microfluídica/métodos , Microfluídica/instrumentação , Região Variável de Imunoglobulina/genéticaRESUMO
Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5-10% of cases of acute myeloid leukemia. CEBPA-double-mutated cases usually bear biallelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multidimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multidimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for large-scale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação , Fenótipo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Medula Óssea/patologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Análise por Conglomerados , Análise Citogenética , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
Allogeneic stem cell transplantation (alloHSCT) in first complete remission (CR1) remains the consolidation therapy of choice in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The prognostic value of measurable levels of minimal residual disease (MRD) at time of conditioning is a matter of debate. We analyzed the predictive relevance of MRD levels before transplantation on the clinical outcome of Ph+ ALL patients treated with chemotherapy and imatinib in 2 consecutive prospective clinical trials. MRD evaluation before transplantation was available for 65 of the 73 patients who underwent an alloHSCT in CR1. A complete or major molecular response at time of conditioning was achieved in 24 patients (37%), whereas 41 (63%) remained carriers of any other positive MRD level in the bone marrow. MRD negativity at time of conditioning was associated with a significant benefit in terms of risk of relapse at 5 years, with a relapse incidence of 8% compared with 39% for patients with MRD positivity (P = .007). However, thanks to the post-transplantation use of tyrosine kinase inhibitors (TKIs), disease-free survival was 58% versus 41% (P = .17) and overall survival was 58% versus 49% (P = .55) in MRD-negative compared with MRD-positive patients, respectively. The cumulative incidence of nonrelapse mortality was similar in the 2 groups. Achieving a complete molecular remission before transplantation reduces the risk of leukemia relapse even though TKIs may still rescue some patients relapsing after transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Adolescente , Adulto , Idoso , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Mesilato de Imatinib/uso terapêutico , Pessoa de Meia-Idade , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Valor Preditivo dos Testes , Prognóstico , Recidiva , Indução de Remissão , Prevenção Secundária , Análise de Sobrevida , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
Developing optimal radiation-free central nervous system prophylaxis is a desirable goal in acute lymphoblastic leukemia, to avoid the long-term toxicity associated with cranial irradiation. In a randomized, phase II trial enrolling 145 adult patients, we compared intrathecal liposomal cytarabine (50 mg: 6/8 injections in B-/T-cell subsets, respectively) with intrathecal triple therapy (methotrexate/cytarabine/prednisone: 12 injections). Systemic therapy included methotrexate plus cytarabine or L-asparaginase courses, with methotrexate augmented to 2.5 and 5 g/m(2) in Philadelphia-negative B- and T-cell disease, respectively. The primary study objective was the comparative assessment of the risk/benefit ratio, combining the analysis of feasibility, toxicity and efficacy. In the liposomal cytarabine arm 17/71 patients (24%) developed grade 3-4 neurotoxicity compared to 2/74 (3%) in the triple therapy arm (P=0.0002), the median number of episodes of neurotoxicity of any grade was one per patient compared to zero, respectively (P=0.0001), and even though no permanent disabilities or deaths were registered, four patients (6%) discontinued intrathecal prophylaxis on account of these toxic side effects (P=0.06). Neurotoxicity worsened with liposomal cytarabine every 14 days (T-cell disease), and was improved by the adjunct of intrathecal dexamethasone. Two patients in the liposomal cytarabine arm suffered from a meningeal relapse (none with T-cell disease, only one after high-dose chemotherapy) compared to four in the triple therapy arm (1 with T-cell disease). While intrathecal liposomal cytarabine could contribute to improved, radiation-free central nervous system prophylaxis, the toxicity reported in this trial does not support its use at 50 mg and prompts the investigation of a lower dosage. (clinicaltrials.gov identifier: NCT-00795756).
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Citarabina/administração & dosagem , Profilaxia Pós-Exposição/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Adulto , Idoso , Feminino , Humanos , Injeções Espinhais , Lipossomos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with Myelodysplastic syndromes (MDS). For many years, the selection of patients to allogeneic HSCT has been largely based on use of the International Prognostic Scoring System-Revised (IPSS-R). However, the recent broader application of next generation sequencing in clinical practice provided an abundance of molecular data and led to the introduction of molecular prognostic scores as IPSS-Molecular (IPSS-M). In this paper, we retrospectively analyzed the outcomes of 57 consecutive MDS patients treated with allogeneic HSCT in our center. Re-stratification from IPSS-R to IPSS-M occurred in almost half of patients. The application of IPSS-M to our cohort demonstrated a stronger prognostic separation compared to IPSS-R and improved the C-index. Very high-risk IPSS-M patients showed worse outcomes following HSCT compared to high-risk patients. This study provides data supporting the need of integrating molecular information in the transplant decision making of patients with MDS. This allows an earlier and better identification of patients to whom the transplant should be advised.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Síndromes Mielodisplásicas , Humanos , Síndromes Mielodisplásicas/terapia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Síndromes Mielodisplásicas/diagnóstico , Estudos Retrospectivos , Transplante de Células-Tronco Hematopoéticas/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Prognóstico , Idoso , Adulto , Tomada de Decisão Clínica , Transplante Homólogo , Sequenciamento de Nucleotídeos em Larga Escala , Adulto JovemRESUMO
Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.
Assuntos
Exoma/genética , Regulação Leucêmica da Expressão Gênica/genética , Testes Genéticos/métodos , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Evolução Fatal , Feminino , Humanos , Cariotipagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Masculino , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sobrevida , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic tumor, characterized by several genetic alterations, that constitutes 15% of pediatric and 25% of adult ALL. While with current therapeutic protocols children and adults' overall survival (OS) rates reach 85-90% and 40-50%, respectively, the outcome for both pediatric and adult T-ALL patients that relapse or are refractory to induction therapy, remains extremely poor, achieving around 25% OS for both patient groups. About 60% of T-ALL patients show increased NOTCH1 activity, due to activating NOTCH1 mutations or alterations in its ubiquitin ligase FBXW7. NOTCH signaling has been shown to contribute to chemotherapy resistance in some tumor models. Hence, targeting the NOTCH1 signaling pathway may be an effective option to overcome relapsed and refractory T-ALL.Here, we focused on the therapeutic activity of the NOTCH1-specific monoclonal antibody OMP-52M51 in combination either with drugs used during the induction, consolidation, or maintenance phase in mice xenografts established from pediatric and adult relapsed NOTCH1 mutated T-ALL samples. Interestingly, from RNAseq data we observed that anti-NOTCH1 treatment in vivo affects the purine metabolic pathway. In agreement, both in vitro and in vivo, the greatest effect on leukemia growth reduction was achieved by anti-NOTCH1 therapy in combination with antimetabolite drugs. This result was further corroborated by the longer life span of mice treated with the anti-NOTCH1 in combination with antimetabolites, indicating a novel Notch-targeted therapeutic approach that could ameliorate pediatric and adult T-ALL patients outcome with relapse disease for whom so far, no other therapeutic options are available.
RESUMO
Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).
Assuntos
Linfoma de Burkitt , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Humanos , Criança , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Pediatric-inspired chemotherapy is the standard of care for younger adults with Philadelphia chromosome-negative acute lymphoblastic leukemia/lymphoma (Ph- ALL/LL). In LAL1913 trial, the Gruppo Italiano Malattie EMatologiche dell'Adulto added pegaspargase 2000 IU/m2 to courses 1, 2, 5, and 6 of an 8-block protocol for patients aged from 18 to 65 years, with dose reductions in patients aged >55 years. Responders were risk stratified for allogeneic hematopoietic cell transplantation (HCT) or maintenance per clinical characteristics and minimal residual disease (MRD). Of 203 study patients (median age, 39.8 years), 91% achieved a complete remission. The 3-year overall survival, event-free, and disease-free survival (DFS) rates were 66.7%, 57.7%, and 63.3%, respectively, fulfilling the primary study end point of a 2-year DFS >55%. Although based on the intention-to-treat, the DFS being 74% and 50% in the chemotherapy (n = 94) and HCT (n = 91) assignment cohorts, respectively, a time-dependent analysis proved the value of HCT in patients who were eligible (DFS HCT 70% vs no HCT 26%; P <.0001). In multivariate analysis, age and MRD were independent factors predicting DFS rates of 86% (age ≤ 40 and MRD-negative), 64%-65% (MRD-positive or age > 40) and 25% (age > 40 and MRD-positive); P < .0001. Grade ≥2 pegaspargase toxicity was mainly observed at course 1, contributing to induction death in 2 patients but was rare thereafter. This program improved outcomes of patients with Ph- ALL/LL aged up to 65 years in a multicenter national setting. This trial was registered at www.clinicaltrials.gov as #NCT02067143.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Adulto , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Intervalo Livre de Doença , Indução de Remissão , Doença AgudaRESUMO
BACKGROUND: The identification of the JAK2V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. DESIGN AND METHODS: We set up a novel molecular method for the identification of the JAK2V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. RESULTS: This assay detects, within 1 hour, the JAK2V617F mutation down to an allele burden of 0.1-0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. CONCLUSIONS: Allele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2V617F mutation in patients with chronic myeloproliferative neoplasms.
Assuntos
Bioensaio , DNA/análise , Janus Quinase 2/genética , Mutação/genética , Transtornos Mieloproliferativos/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Alelos , Estudos de Casos e Controles , Doença Crônica , DNA/genética , Humanos , Transtornos Mieloproliferativos/genética , Células Tumorais CultivadasRESUMO
The prognostic significance of CD20 expression in acute lymphoblastic leukemia has been investigated in children and adults but is still a subject of debate. The aim of our study was to correlate CD20 expression with clinical-biological characteristics and outcome in 172 Philadelphia chromosome negative patients prospectively treated in a multicenter trial introducing the molecular evaluation of minimal residual disease for therapeutic purposes. We considered 20% as the threshold for CD20 positivity. Complete remission rate, minimal residual disease negativity rate at weeks 10, 16 and 22, and disease-free and overall survival were similar among CD20-positive and -negative patients, even considering minimal residual disease results and related therapeutic choices. Our study failed to demonstrate any prognostic significance for CD20 expression in Philadelphia chromosome negative acute lymphoblastic leukemia. This conclusion is supported for the first time by a comparable minimal residual disease response rate among CD20-positive and -negative and positive patients.
Assuntos
Antígenos CD20/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Adulto , Idoso , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasia Residual , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
The therapeutic role of mediastinal radiotherapy and stem cell transplantation (SCT) in lymphoblastic lymphoma (LL) remains controversial. In a risk-oriented design, we adopted a flexible treatment program in which (1) patients with persistent mediastinal abnormality, evaluated by post-induction computed chest tomography, received mediastinal irradiation; and (2) those with persistence of minimal residual disease (MRD), evaluated by MRD analysis of the bone marrow, underwent SCT. Twenty-eight out of 30 patients (T-lineage, n = 24; B-lineage, n = 6) achieved a complete response. Of 21 patients with mediastinal mass, 13 (62%) achieved a complete response after chemotherapy alone, while 6 (28.5%) required additional irradiation. Eleven patients were evaluated for MRD: 6 were negative and 5 positive. On the basis of MRD findings and clinical risk characteristics, 14 patients underwent SCT, 13 received maintenance chemotherapy, and 1 had local radiotherapy. Five patients relapsed. Among the 14 non-irradiated patients with T-LL, the mediastinal recurrence rate was only 7%. After a median follow-up of 3.9 years, 21 patients who responded were alive without recurrence (75%). The projected 5-year survival, disease-free survival, and relapse rate were 72%, 77%, and 18%, respectively. This program induced high remission and survival rates, indicating the feasibility and the benefits potentially associated with a selective, response-oriented policy of mediastinal irradiation and a concurrent MRD-based strategy to assign adult LL patients to SCT.