Ana Valencia

Impact of adjunct cytogenetic abnormalities for prognostic stratification in patients with myelodysplastic syndrome and deletion 5q.

This cooperative study assessed prognostic factors for overall survival (OS) and risk of transformation to acute myeloid leukemia (AML) in 541 patients with de novo myelodysplastic syndrome (MDS) and deletion 5q. Additional chromosomal abnormalities were strongly related to different patients’ characteristics. In multivariate analysis, the most important predictors of both OS and AML transformation risk were number of chromosomal abnormalities (P<0.001 for both outcomes), platelet count (P<0.001 and P=0.001, respectively) and proportion of bone marrow blasts (P<0.001 and P=0.016, respectively). The number of chromosomal abnormalities defined three risk categories for AML transformation (del(5q), del(5q)+1 and del(5q)+⩾2 abnormalities) and two for OS (one group: del(5q) and del(5q)+1; and del(5q)+⩾2 abnormalities, as the other one); with a median survival time of 58.0 and 6.8 months, respectively. Platelet count (P=0.001) and age (P=0.034) predicted OS in patients with ‘5q−syndrome’. This study demonstrates the importance of additional chromosomal abnormalities in MDS patients with deletion 5q, challenges the current ‘5q−syndrome’ definition and constitutes a useful reference series to properly analyze the results of clinical trials in these patients.

Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia.

Activation of the Wnt signaling pathway has been implicated recently in the pathogenesis of leukemia. We studied the function of epigenetic regulation of the Wnt pathway and its prognostic relevance in acute myelogenous leukemia (AML). We used a methylation-specific polymerase chain reaction approach to analyze the promoter methylation status of a panel of Wnt antagonists including sFRP1, sFRP2, sFRP4, sFRP5, DKK1 and DKK3. Aberrant methylation of Wnt antagonists was detected in four AML cell lines and in up to 64% of AML marrow samples. Treatment of the cell lines with 5-aza-2′-deoxycytidine induced reexpression of methylated Wnt antagonists and inactivation of the Wnt pathway by downregulating the Wnt pathway genes cyclin D1, TCF1 and LEF1 and reducing nuclear localization of β-catenin. In a subgroup of patients 60 years and younger with newly diagnosed AML and intermediate-risk cytogenetics, abnormal methylation of Wnt antagonists was associated with decreased 4-year relapse-free survival (28 vs 61%, respectively, P=0.03). Our results indicate a function of the epigenetic regulation of the Wnt pathway in predicting relapse in a subgroup of AML patients.

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. Methodology/Principal Findings We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. Conclusions/Significance Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.

Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia

Background The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia. Design and Methods We analyzed the prevalence and prognostic impact of EVI1 over-expression in a series of 476 patients with acute myeloid leukemia, and investigated the epigenetic modifications of the EVI1 locus which could be involved in the transcriptional regulation of this gene. Results Our data provide further evidence that EVI1 over-expression is a poor prognostic marker in acute myeloid leukemia patients less than 65 years old. Moreover, we found that patients with no basal expression of EVI1 had a better prognosis than patients with expression/over-expression (P=0.036). We also showed that cell lines with over-expression of EVI1 had no DNA methylation in the promoter region of the EVI1 locus, and had marks of active histone modifications: H3 and H4 acetylation, and trimethylation of histone H3 lysine 4. Conversely, cell lines with no expression of EVI1 have DNA hypermethylation and are marked by repressive trimethylation of histone H3 lysine 27 at the EVI1 promoter. Conclusions Our results identify EVI1 over-expression as a poor prognostic marker in a large, independent cohort of acute myeloid leukemia patients less than 65 years old, and show that the total absence of EVI1 expression has a prognostic impact on the outcome of such patients. Furthermore, we demonstrated for the first time that an aberrant epigenetic pattern involving DNA methylation, H3 and H4 acetylation, and trimethylation of histone H3 lysine 4 and histone H3 lysine 27 might play a role in the transcriptional regulation of EVI1 in acute myeloid leukemia. This study opens new avenues for a better understanding of the regulation of EVI1 expression at a transcriptional level.

Epigenetic regulation of the non‐canonical Wnt pathway in acute myeloid leukemia

Wnt5a is a member of the Wnt family of proteins that signals through the non‐canonical Wnt/Ca2+pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter–exon 1 was analyzed by methylation‐specific PCR and sequencing in eleven AML‐derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5‐Aza‐2’‐deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non‐methylated group. Disease‐free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML. (Cancer Sci 2009)

A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia

Chromosomal translocations in hematological malignancies often result in novel fusion chimeric genes. We report a case of acute myeloid leukemia with a clonal translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. The gene fused to NUP98 (nucleoporin 98) was detected by comparative genomic hybridization array as the retinoid acid receptor gamma gene (RARG). The involvement of RARG in a chimeric fusion transcript has not been reported previously in human leukemia.

Distinct Signal Transduction Abnormalities and Erythropoietin Response in Bone Marrow Hematopoietic Cell Subpopulations of Myelodysplastic Syndrome Patients

Purpose: Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. Experimental Design: Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34+, CD45+, or CD71+CD45− bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. Results: In CD71+CD45− cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71+CD45− cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34+ and CD45+ MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34+ cells. Conclusions: Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71+CD45− cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations. Clin Cancer Res; 18(11); 3079–89. ©2012 AACR.

Rapid Detection of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia Using High-Resolution Melting Analysis

The most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations.

Lack of RPS14 promoter aberrant methylation supports the haploinsufficiency model for the 5q- syndrome

To the editor: In the past few decades several groups have tried to identify the putative gene(s) responsible for the development of 5q- syndrome, a specific clinical, morphologic, and cytogenetic myelodysplastic syndrome (MDS) subtype characterized by a defect in erythroid differentiation.[1][1],[

Hypermethylation of Wnt antagonist gene promoters and activation of Wnt pathway in myelodysplastic marrow cells

We observed aberrant gene methylation of Wnt antagonists: sFRP1, sFRP2, sFRP4, sFRP5 and DKK1 in marrow cells of 55 MDS cases. Methylation of Wnt antagonist genes was associated with activation of the Wnt signaling pathway, consistent with the up-regulation of the Wnt downstream genes TCF1 and LEF1. Azacitidine exposure induced demethylation of Wnt-antagonist gene promoters and reduction of the non-phosphorylated β-catenin (NPBC) which is prevalent during Wnt pathway inactivation. Presence of ≥5% of bone marrow blasts was associated with methylation of sFRP1 and DKK1 and with methylation of more than two of the five Wnt antagonist genes.