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ABSTRACT: Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Mutación , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Masculino , Persona de Mediana Edad , Femenino , Adulto , Anciano , Transducción de Señal , Inhibidores de Proteínas Quinasas/uso terapéutico , Pronóstico , Factores de Transcripción/genética , Resultado del Tratamiento , Secuenciación de Nucleótidos de Alto Rendimiento , Adulto Joven , Anciano de 80 o más Años , Progresión de la EnfermedadRESUMEN
Overall survival of patients classified according to the European LeukemiaNet 2020 classification. Chronic phase (CP), accelerated phase (AP), blast crisis (BC), low risk (LR), intermediate risk (IR), high risk (HR).
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Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Crisis Blástica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND: Changes in DNA methylation are common events in the pathogenesis of acute myeloid leukemia (AML) and have been repeatedly reported as associated with prognosis. However, studies integrating these numerous and potentially prognostically relevant DNA methylation changes are lacking. Therefore, we aimed for an overall evaluation of these epigenetic aberrations to provide a comprehensive NGS-based approach of DNA methylation assessment for AML prognostication. RESULTS: We designed a sequencing panel targeting 239 regions (approx. 573 kb of total size) described in the literature as having a prognostic impact or being associated with AML pathogenesis. Diagnostic whole-blood DNA samples of adult AML patients divided into a training (n = 128) and a testing cohort (n = 50) were examined. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Altogether, 1935 CpGs affecting the survival (p < 0.05) were revealed in the training cohort. A summarizing value MethScore was then calculated from these significant CpGs. Patients with lower MethScore had markedly longer overall survival (OS) and event-free survival (EFS) than those with higher MethScore (p < 0.001). The predictive ability of MethScore was verified on the independent testing cohort for OS (p = 0.01). Moreover, the proof-of-principle validation was performed using the TCGA dataset. CONCLUSIONS: We showed that comprehensive NGS-based approach of DNA methylation assessment revealed a robust epigenetic signature relevant to AML outcome. We called this signature MethScore and showed it might serve as a strong prognostic marker able to refine survival probability of AML patients.
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Metilación de ADN , Leucemia Mieloide Aguda , Adulto , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Pronóstico , Epigenómica , Supervivencia sin ProgresiónRESUMEN
BACKGROUND: NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. METHODS AND RESULTS: We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. CONCLUSIONS: With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.
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Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Leucemia Mieloide Aguda/genética , Mutación/genética , Neoplasia Residual/genética , Proteínas Nucleares/genética , Nucleofosmina , Plásmidos/genéticaRESUMEN
BACKGROUND: Multiple studies have reported the prognostic impact of DNA methylation changes in acute myeloid leukemia (AML). However, these epigenetic markers have not been thoroughly validated and therefore are still not considered in clinical practice. Hence, we aimed to independently verify results of selected studies describing the relationship between DNA methylation of specific genes and their prognostic potential in predicting overall survival (OS) and event-free survival (EFS). RESULTS: Fourteen studies (published 2011-2019) comprising of 27 genes were subjected to validation by a custom NGS-based sequencing panel in 178 newly diagnosed non-M3 AML patients treated by 3 + 7 induction regimen. The results were considered as successfully validated, if both the log-rank test and multivariate Cox regression analysis had a p-value ≤ 0.05. The predictive role of DNA methylation was confirmed for three studies comprising of four genes: CEBPA (OS: p = 0.02; EFS: p = 0.03), PBX3 (EFS: p = 0.01), LZTS2 (OS: p = 0.05; EFS: p = 0.0003), and NR6A1 (OS: p = 0.004; EFS: p = 0.0003). For all of these genes, higher methylation was an indicator of longer survival. Concurrent higher methylation of both LZTS2 and NR6A1 was highly significant for survival in cytogenetically normal (CN) AML group (OS: p < 0.0001; EFS: p < 0.0001) as well as for the whole AML cohort (OS: p = 0.01; EFS < 0.0001). In contrast, for two studies reporting the poor prognostic effect of higher GPX3 and DLX4 methylation, we found the exact opposite, again linking higher GPX3 (OS: p = 0.006; EFS: p < 0.0001) and DLX4 (OS: p = 0.03; EFS = 0.03) methylation to a favorable treatment outcome. Individual gene significance levels refer to the outcomes of multivariate Cox regression analysis. CONCLUSIONS: Out of twenty-seven genes subjected to DNA methylation validation, a prognostic role was observed for six genes. Therefore, independent validation studies are necessary to reveal truly prognostic DNA methylation changes and to enable the introduction of these promising epigenetic markers into clinical practice.
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Biomarcadores de Tumor/análisis , Metilación de ADN/genética , Leucemia Mieloide Aguda/diagnóstico , Adulto , Biomarcadores de Tumor/genética , Metilación de ADN/fisiología , Femenino , Humanos , Inmunoquímica/métodos , Inmunoquímica/estadística & datos numéricos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/estadística & datos numéricos , Factores de Transcripción/genética , Resultado del Tratamiento , Estudios de Validación como AsuntoRESUMEN
Genetic mutations in acute myeloid leukaemia (AML) are assumed to occur in a sequential order; however, the predominant hierarchical roles of specific mutated genes have not been fully described. In this study, we aimed to determine the clonal involvement of the most frequent AML-associated mutations. Using a targeted sequencing panel for 18 genes, we traced changes and relative clonal contribution of mutations in 52 patients. We analysed 35 pairs of diagnosis and relapse samples, 27 pairs of primary samples and corresponding patient-derived xenografts, and 34 pairs of total leukocytes and corresponding isolated primitive cells or blast populations. In both relapse and xenografts, we observed conservation of main leukaemic clones and variability was limited to subclones with late-acquired mutations. AML evolution thus mainly involved modification of subclones while the clonal background remained unchanged. NPM1 mutations were identified as the most probable leukaemia-transformation lesion, remaining conserved in contrast to high variation of accompanying subclonal FLT3 and NRAS mutations. DNMT3A mutations represented the most stable mutations forming a preleukaemic background in most samples. Mutations in genes IDH1/2, TET2, RUNX1, ASXL1 and U2AF1 were detected both as preleukaemic and as subclonal lesions, suggesting a non-specific order of acquisition.
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Genes Relacionados con las Neoplasias , Leucemia Mieloide Aguda/genética , Mutación , Proteínas de Neoplasias/genética , Adulto , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Evolución Clonal , Células Clonales , Terapia Combinada , Femenino , Trasplante de Células Madre Hematopoyéticas , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/terapia , Leucocitos , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Trasplante de Neoplasias , Células Madre Neoplásicas , Nucleofosmina , Recurrencia , Adulto JovenRESUMEN
In this multi-centre study, we analysed the prognostic impact of mutations in 19 genes associated with myeloid malignancies in 258 newly diagnosed acute myeloid leukaemia patients (aged 19-70 years) undergoing intensive therapy. We identified five patient groups with different prognostic risks and different benefits from allogeneic hematopoietic stem cell transplantation (alloHSCT) within the intermediate cytogenetic risk group patients (n = 184). The most adverse prognosis was observed in patients with DNMT3A and FLT3-ITD co-mutation, whose survival could be significantly improved with alloHSCT. In contrast, the most favourable prognosis without any further benefit from alloHSCT was identified in patients with mutations in NPM1 or CEBPA, after exclusion of the unfavourable prognostic groups defined by mutations in DNMT3A, RUNX1 or genes from chromatin/spliceosome group. An additional analysis of 113 diagnosis-remission paired samples revealed that persistence of non-DNMT3A mutations (above 2% VAF) represented a further negative prognostic factor. The proposed model offers a possible molecular stratification and treatment guidance for intermediate cytogenetic risk group patients.
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Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Mutación , Proteínas de Neoplasias/genética , Adulto , Anciano , Aloinjertos , Progresión de la Enfermedad , Femenino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Nucleofosmina , Pronóstico , Factores de RiesgoRESUMEN
PURPOSE: This study aimed at analyzing the association of gene mutations and other acute myeloid leukemia (AML) characteristics with engraftment outcomes in immunodeficient mice and to select the engraftment outcomes that best reflect patient survival. METHODS: Mutations in 19 genes as well as leukemia- and patient-related characteristics were analyzed for a group of 47 de novo AML samples with respect to three engraftment outcomes: engraftment ability, engraftment intensity (percentage of hCD45+ cells) and engraftment latency. Leukemia-related characteristics were additionally analyzed in an extended group of 68 samples that included the 47 de novo samples, and additional 21 samples from refractory and relapsed cases. Engraftment outcomes were compared with overall and event-free survival of the patients. RESULTS: For the 47 de novo samples, no single mutation influenced engraftment, whereas the NPM1 mut /DNMT3A mut co-mutation was associated with higher engraftment ability. NPM1 mut /FLT3-ITD neg had lower engraftment intensity. Among leukemia-related characteristics, a complex karyotype was associated with higher engraftment intensity. Among patient-related characteristics, higher cytogenetic risk was associated with higher engraftment intensity, and failure to achieve clinical remission was associated with shorter engraftment latency. In the extended group of 68 samples, white blood count was associated with higher engraftment ability, and the presence of a complex karyotype was associated with higher engraftment intensity. Association with patient overall survival was seen only for engraftment intensity. CONCLUSIONS: The engraftment of AML was influenced by mutation-interactions and other AML characteristics, rather than by single mutated genes, and engraftment intensity best reflected clinical penetrance of AML.
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Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación , Adulto , Anciano , Animales , Xenoinjertos/patología , Humanos , Leucemia Mieloide Aguda/sangre , Leucocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Nucleofosmina , Trasplante Heterólogo , Adulto JovenAsunto(s)
Alelos , Biomarcadores de Tumor , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Leucemia/diagnóstico , Leucemia/etiología , Femenino , Genotipo , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia/terapia , Repeticiones de Microsatélite , Persona de Mediana Edad , Polimorfismo GenéticoAsunto(s)
Prueba de Histocompatibilidad , Leucemia Mieloide Aguda/terapia , Trasplante de Células Madre de Sangre Periférica , Anciano , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Femenino , Humanos , Quimioterapia de Inducción/métodos , Masculino , Persona de Mediana EdadAsunto(s)
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Combinada/métodos , Exones , Genotipo , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Nucleofosmina , Adulto JovenRESUMEN
The DNA methyl-transferase 3A gene (DNMT3A) is the third most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) patients (20-30 %), who belong to a group of patients with intermediate risk. About 60 % of mutations in this gene have been identified in the arginine codon R882. To date, there is no consensus on whether these mutations can be used as biomarkers for monitoring of minimal residual disease and management of preemptive AML therapy. We studied the occurrence of mutations in the DNMT3A gene in our cohort of patients and their persistence during AML treatment. Using next-generation sequencing, we identified four mutations in 11/25 of our analyzed patients--frequent R882C and R882H mutations, rare Y735S mutation, and a novel L347P mutation. Mutation R882C was detected in 5/11, R882H in 4/11 patients, and Y735S and L347P in one patient each. In 4/7 patients initially carrying mutations in the R882 codon, we found the persistence of mutations also during complete remission with, however, no correlation to AML kinetics. Our findings suggest that mutations in the DNMT3A gene can only be used as a biomarker for those AML patients in whom DNMT3A mutation is lost after therapy.
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Biomarcadores de Tumor/genética , Codón/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Leucemia Mieloide Aguda/genética , Monitoreo Fisiológico , Mutación Missense , Proteínas de Neoplasias/genética , Adulto , Anciano , Sustitución de Aminoácidos , ADN Metiltransferasa 3A , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasia ResidualRESUMEN
The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14(+) monocytes and CD66b(+) granulocytes. However, we were also able to detect NPM1 mutations in CD19(+) B cells and CD3(-)14(-)16(+)56(+) natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3(+) T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.