RESUMEN
FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.
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Benzotiazoles/farmacología , Dasatinib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Metabolismo Energético , Duplicación de Gen , Técnicas de Silenciamiento del Gen , Glucólisis , Humanos , Fosforilación , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Imidazoles/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Niacinamida/análogos & derivados , Pirazoles/farmacología , Piridazinas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Imidazoles/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Terapia Molecular Dirigida/métodos , Mutación , Niacinamida/farmacología , Niacinamida/uso terapéutico , Cultivo Primario de Células , Pirazoles/uso terapéutico , Piridazinas/uso terapéuticoRESUMEN
OBJECTIVES: The 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology reviewed the role of genetic testing in the diagnosis of hematopoietic neoplasms, including non-acute leukemia myeloid malignancies. METHODS: The workshop panel assigned 98 submitted cases to the category of non-acute leukemia myeloid neoplasms, of which 13 were selected for oral presentation. RESULTS: Data from both conventional karyotyping and genetic sequencing had important impact on diagnosis, classification, and prognostication. However, some cases had genetic results that appeared discordant from the morphology and/or clinical features. Thus, the workshop underscored the need for careful management of genetic data by the pathologist and clinician, in the context of other findings. CONCLUSIONS: The workshop cases highlighted the significance of genetic aberrations in the diagnosis and treatment of non-acute leukemia myeloid neoplasms. Many genetic data have already been incorporated in the most recent World Health Organization classification, and undoubtedly they will factor increasingly in future classifications.
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Pruebas Genéticas/métodos , Síndromes Mielodisplásicos/diagnóstico , Trastornos Mieloproliferativos/diagnóstico , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patologíaRESUMEN
A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.
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Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/fisiología , Factor 1 de Transcripción de Unión a Octámeros/fisiología , Animales , Médula Ósea/patología , Trastornos de Fallo de la Médula Ósea/etiología , Trastornos de Fallo de la Médula Ósea/genética , Factor de Transcripción CDX2/biosíntesis , Factor de Transcripción CDX2/genética , Transformación Celular Neoplásica/genética , Islas de CpG , Metilación de ADN , Progresión de la Enfermedad , Fluorouracilo/toxicidad , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Leucemia Experimental/genética , Leucemia Experimental/prevención & control , Leucemia Mieloide Aguda/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones Endogámicos C57BL , Factor 1 de Transcripción de Unión a Octámeros/deficiencia , Proteínas de Fusión Oncogénica/fisiología , Regiones Promotoras Genéticas , Quimera por RadiaciónRESUMEN
Copy number variants (CNVs) and copy neutral loss of heterozygosity (CN-LOH) represent important types of genomic abnormalities in cancer. Genomic DNA microarray serves as the current gold standard method for detecting genome-wide CNVs and CN-LOH. However, as next-generation sequencing (NGS) is widely used to detect gene variants in clinical testing, the ability of NGS to detect CNVs and CN-LOH has also been demonstrated. This chapter describes a protocol for detecting genome-wide large somatic CNVs and CN-LOH using a single nucleotide polymorphism (SNP) sequencing backbone. When combined with a targeted gene mutation panel, this strategy allows for simultaneous detection of somatic gene mutations and genome-wide CNVs and CN-LOH.
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Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pérdida de Heterocigocidad , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Análisis de Datos , Genómica/métodos , Humanos , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. EXPERIMENTAL DESIGN: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). RESULTS: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. CONCLUSIONS: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.
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Núcleo Celular/metabolismo , Citoplasma/metabolismo , Mielofibrosis Primaria/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Biomarcadores , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Biología Computacional/métodos , Citoplasma/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Ratones , Terapia Molecular Dirigida , Mutación , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/etiología , Factores de Transcripción STAT/metabolismo , TranscriptomaRESUMEN
CONTEXT.: B-cell lymphomas exhibit balanced translocations that involve immunoglobulin loci and result from aberrant V(D)J recombination, class switch recombination, or somatic hypermutation. Although most of the breakpoints in the immunoglobulin loci occur in defined regions, those in the partner genes vary; therefore, it is unlikely that 2 independent clones would share identical breakpoints in both partners. Establishing whether a new lesion in a patient with history of lymphoma represents recurrence or a new process can be relevant. Polymerase chain reaction (PCR)-based clonality assays used in this setting rely only on evaluating the length of a given rearrangement. In contrast, next-generation sequencing (NGS) provides the exact translocation breakpoint at single-base resolution. OBJECTIVE.: To determine if translocation breakpoint coordinates can serve as a molecular fingerprint unique to a distinct clonal population. DESIGN.: Thirty-eight follicular lymphoma/diffuse large B-cell lymphoma samples collected from different anatomic sites and/or at different time points from 18 patients were analyzed by NGS. For comparison, PCR-based B-cell clonality and fluorescence in situ hybridization studies were performed on a subset of cases. RESULTS.: IGH-BCL2 rearrangements were detected in all samples. The breakpoint coordinates on derivative chromosome(s) were identical in all samples from a given patient, but distinct between samples derived from different patients. Additionally, 5 patients carried a second rearrangement also with conserved breakpoint coordinates in the follow-up sample(s). CONCLUSIONS.: Breakpoint coordinates in the immunoglobulin and partner genes can be used to establish clonal relatedness of anatomically/temporally distinct lesions. Additionally, an NGS-based approach has the potential to detect secondary translocations that may have prognostic and therapeutic significance.
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Dermatoglifia del ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/genética , Reordenamiento Génico , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Hibridación Fluorescente in Situ , Proteínas Proto-Oncogénicas c-bcl-2/genética , Translocación GenéticaRESUMEN
The life expectancy of patients with chronic phase chronic myeloid leukemia on tyrosine kinase inhibitor therapy now approaches that of the general population. Approximately 60% of patients treated with second generation tyrosine kinase inhibitors achieve a deep molecular response, the prerequisite for a trial of treatment-free remission. Those patients unlikely to achieve deep molecular response may benefit from more intensive therapy up front. To identify biomarkers predicting deep molecular response we performed transcriptional profiling on CD34+ progenitor cells from newly diagnosed chronic phase chronic myeloid leukemia patients treated with nilotinib on a prospective clinical trial. Using unsupervised and targeted analytical strategies, we show that gene expression profiles are similar in patients with and without subsequent deep molecular response. This result is in contrast to the distinct expression signature of CD34+ chronic phase chronic myeloid leukemia patients failing to achieve a cytogenetic response on imatinib and suggests that deep molecular response to second-generation tyrosine kinase inhibitors is governed by the biology of more primitive chronic myeloid leukemia cells or extrinsic factors.
RESUMEN
AIMS: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). METHODS: For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. RESULTS: We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. CONCLUSIONS: We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Trastornos Mieloproliferativos/genética , Femenino , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.
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Leucemia Mieloide Aguda/diagnóstico , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Translocación Genética , Humanos , Leucemia Mieloide Aguda/genética , Plásmidos , Estándares de ReferenciaAsunto(s)
Enfermedad de Hodgkin/patología , Linfocitos T Colaboradores-Inductores/patología , Adulto , Complejo CD3/metabolismo , Tamaño de la Célula , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/metabolismo , Humanos , Masculino , Factor de Transcripción PAX5/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0-11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.
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Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Metilación de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Nucleofosmina , Empalme del ARN/genética , CohesinasRESUMEN
OBJECTIVES: We describe a rare case of a male child with X-linked lymphoproliferative disease type 1 (XLP1) who presented with Burkitt lymphoma (BL) when he was 6 years old, achieved a complete response to therapy, and developed a second BL after seven years. METHODS: Diagnostic H&E stained slides and ancillary studies were reviewed for both lymphomas. B-cell clonality by PCR and SNP array studies were performed on both specimens. RESULTS: Both lymphomas were Epstein-Barr virus (EBV) negative. Flow cytometry showed λ light chain restriction in the initial BL and κ light chain restriction in the subsequent BL. B-cell clonality testing indicated that the two lymphomas are not clonally related. SNP array analysis of the second BL showed genomic changes that were not present in the first BL. CONCLUSIONS: These results confirm that these two tumors represent unrelated BLs. Pathologists and clinicians should be aware that second lymphomas in XLP1 patients may represent new neoplasms rather than late relapses.
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Linfoma de Burkitt/patología , Trastornos Linfoproliferativos/complicaciones , Neoplasias Primarias Secundarias/patología , Adolescente , Biomarcadores de Tumor/análisis , Linfoma de Burkitt/genética , Niño , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Masculino , Neoplasias Primarias Secundarias/genéticaRESUMEN
The distinction between chronic eosinophilic leukemia, not otherwise specified and idiopathic hypereosinophilic syndrome largely relies on clonality assessment. Prior to the advent of next-generation sequencing, clonality was usually determined by cytogenetic analysis. We applied targeted next-generation sequencing panels designed for myeloid neoplasms to bone marrow specimens from a cohort of idiopathic hypereosinophilic syndrome patients (n=51), and assessed the significance of mutations in conjunction with clinicopathological features. The findings were further compared with those of 17 chronic eosinophilic leukemia, not otherwise specified patients defined by their abnormal cytogenetics and/or increased blasts. Mutations were detected in 14/51 idiopathic hypereosinophilic syndrome patients (idiopathic hypereosinophilic syndrome/next-generation sequencing-positive) (28%), involving single gene in 7 and ≥2 in 7 patients. The more frequently mutated genes included ASXL1 (43%), TET2 (36%), EZH2 (29%), SETBP1 (22%), CBL (14%), and NOTCH1 (14%). Idiopathic hypereosinophilic syndrome/next-generation sequencing-positive patients showed a number of clinical features and bone marrow findings resembling chronic eosinophilic leukemia, not otherwise specified. Chronic eosinophilic leukemia, not otherwise specified patients showed a disease-specific survival of 14.4 months, markedly inferior to idiopathic hypereosinophilic syndrome/next-generation sequencing-negative (P<0.001), but not significantly different from idiopathic hypereosinophilic syndrome/next-generation sequencing-positive (P=0.117). These data suggest that targeted next-generation sequencing helps to establish clonality in a subset of patients with hypereosinophilia that would otherwise be classified as idiopathic hypereosinophilic syndrome. In conjunction with other diagnostic features, mutation data can be used to establish a diagnosis of chronic eosinophilic leukemia, not otherwise specified in patients presenting with hypereosinophilia.
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Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Síndrome Hipereosinofílico/genética , Leucemia/genética , Mutación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Examen de la Médula Ósea , Diagnóstico Diferencial , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Síndrome Hipereosinofílico/mortalidad , Síndrome Hipereosinofílico/patología , Síndrome Hipereosinofílico/terapia , Hibridación Fluorescente in Situ , Estimación de Kaplan-Meier , Cariotipo , Leucemia/patología , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Estados Unidos , Adulto JovenAsunto(s)
Azacitidina/uso terapéutico , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Azacitidina/efectos adversos , Biomarcadores Farmacológicos/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/mortalidad , Leucemia Mielomonocítica Crónica/patología , Masculino , Persona de Mediana Edad , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon-level duplications, including so-called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof-of-concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.
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Variaciones en el Número de Copia de ADN , Neoplasias Hematológicas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Neoplasias/genética , Femenino , Humanos , Masculino , NucleofosminaRESUMEN
Detection of BCR-ABL1 mutations that confer resistance to tyrosine kinase inhibitors is important for management of patients with t(9;22);BCR-ABL1-positive (Ph+) leukemias. Testing is often performed using Sanger sequencing (SS) which has relatively poor sensitivity. Given the widespread adoption of next generation sequencing (NGS), we sought to reevaluate the testing in the context of NGS methods. We developed an NGS-based BCR-ABL1 mutation test on the Ion Torrent Personal Genome Machine (PGM) to test for resistance mutations, primarily in the kinase domain in BCR-ABL1. We analyzed 508 clinical samples from patients with Ph+ leukemias. In a subset of these samples (n = 97), we conducted a comparison of the NGS results to a classical SS-based test. NGS facilitated detection of low-level mutations (<20 % allele frequency) that were not detectable by SS. In a subset of cases with multiple mutations, NGS was also able to determine if two mutations were on the same molecule (compound) or on separate molecules (polyclonal) but this was limited by the distance between mutated positions and by the effects of apparent distance-dependent PCR recombination. We found 22 compound mutations that centered on one or two key residues including two novel compound mutants: Q252H/Y253H and F311Y/F359I. The advantages of NGS make it a superior method for inventorying BCR-ABL1 resistance mutations. However, data analysis may be complicated by short read lengths and the effects of PCR recombination.
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Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Mutación/genética , Inhibidores de Proteínas Quinasas , Análisis de Secuencia de ADN/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Frecuencia de los Genes/genética , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
BACKGROUND: T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). OBJECTIVE: We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. METHODS: Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. RESULTS: Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. LIMITATIONS: Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. CONCLUSION: TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.