RESUMEN
Treatment with hypomethylating agents such as decitabine, which results in overall response rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy. However, there still exists a lack of prognostic and predictive molecular biomarkers that enable selection of patients who are likely to benefit from epigenetic therapy. Here, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα, and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331) to identify potential biomarkers for patients receiving hypomethylating therapy. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, patients harboring DNMT3A R882 mutations showed a non-significant association towards shorter overall survival (hazard ratio (HR) 2.15, 95% confidence interval (CI) 0.91-5.12, p = 0.08). Promoter DNA methylation analyses using pyrosequencing also revealed a non-significant association towards shorter overall survival of patients with higher levels of methylation of ERα (HR 1.50, CI 0.97-2.32, p = 0.07) and OLIG2 CpG4 (HR 1.52, CI 0.96-2.41, p = 0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR 1.76, CI 1.01-3.06, p = 0.05 and HR 1.67, CI 0.91-3.08, p = 0.10, respectively). In contrast, none of the investigated genetic and epigenetic markers was associated with response to treatment. Additional to the previously reported adverse prognostic clinical parameters such as patients' age, reduced performance status, and elevated lactate dehydrogenase levels, DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status, may prove to be molecular markers in older AML patients prior to hypomethylating therapy.
Asunto(s)
Biomarcadores de Tumor/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis Genética/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutación/genética , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/análogos & derivados , Azacitidina/uso terapéutico , ADN Metiltransferasa 3A , Decitabina , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Nucleofosmina , Tasa de Supervivencia/tendencias , Resultado del TratamientoRESUMEN
Aberrant DNA methylation and concomitant transcriptional silencing of death-associated protein kinase 1 (DAPK1) have been demonstrated to be key pathogenic events in chronic lymphocytic leukemia (CLL). In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, the presence of elevated DNA methylation levels has been a matter of continued controversy. Several studies demonstrated highly variable frequencies of DAPK1 promoter methylation by the use of methylation-specific PCR (MSP). By quantitative high-resolution assessment, we demonstrate that aberrant DNA methylation is an extremely rare event in this region. We observed elevated levels just in one out of 246 (0.4%) AML patients, all 42 MDS patients were unmethylated. In conclusion, we present a refined DAPK1 methylation analysis in a large representative patient cohort of AML and MDS patients proofing almost complete absence of elevated DNA methylation. Our results highlight the importance of quantitative measurements for translational research questions on primary patient specimens, particularly.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/metabolismo , Células de la Médula Ósea , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Estudios de Cohortes , Metilación de ADN , Proteínas Quinasas Asociadas a Muerte Celular , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patología , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARß2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARß2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARß2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARß2 was not necessarily required for the differentiation effect, and pharmacological RARß2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARß2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARß2 gene silencing by DNA methylation.