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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Journal of Hematology & Oncology 1/2014

Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2014
Autoren:
Amy Guillaumet-Adkins, Julia Richter, Maria D Odero, Juan Sandoval, Xabi Agirre, Albert Catala, Manel Esteller, Felipe Prósper, María José Calasanz, Ismael Buño, Mi Kwon, Franck Court, Reiner Siebert, David Monk
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1756-8722-7-4) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AMA carried out the molecular studies and FC performed the bioinformatic analyses. JR, MDO, JS, ZA, AC, ME FP, MJC, IB, MK, RS provided reagents and classified patients. AMA, RS and DM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A)WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown.

Methods

To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies.

Results

Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling.

Conclusions

We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer.

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Zusatzmaterial
Additional file 1: Figure S1: (A) Confirmation of imprinted methylation at the H19-DMR in peripheral leukocytes. (B) Bisulphite PCR analysis of the WT1 promoter interval in cell lines derived from hematological cancers other than AML. (PDF 411 KB)
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Additional file 2: Table S1: Characterization of hematological neoplasms according to cytogenetic aberrations. Table S3. Specificity of AWT1/WT1 hypermethylation as a biomarker. Table S4. The PCR primer sequences used in this study. (DOCX 84 KB)
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Additional file 3: Table S2: Characterization of hematological neoplasms using WHO classification with information on age and sex of the patients (if data available). The methylation values obtained following pyrosequencing for the WT1 and AWT1 promoters are also given. (XLSX 60 KB)
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Additional file 4: Figure S2: (A) Methylation values of repetitive DNA elements in the KG1A cell lines treated with different concentrations of Decitabine. (B) The methylation values for WT1 and AWT1 promoters and (C) qRT-PCR for WT1, AWT1 and WT1-AS transcripts following Decitabine treatment. (TIFF 1 MB)
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Additional file 5: Figure S3: (A) Map of the WT1 locus showing POLII mediated ChIA-PET interactions between the GATA-2 enhancer and the WT1 promoter interval. (B) Abundance of the GATA-2 transcription factor in hematological cancer cell lines as determined by qRT-PCR. (PDF 386 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Literatur
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