PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Leuk Res. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2730658
NIHMSID: NIHMS119224

Assessment of ATRX Expression in Patients With Myelodysplastic Syndromes Treated with Decitabine

Treatment with one of the DNA methyltransferase inhibitors (DNMTIs, “hypomethylating agents”), azacitidine or decitabine, is now considered standard care for patients with higher-risk myelodysplastic syndromes (MDS) (1) – especially since results were released from the AZA-001 trial (2), which demonstrated a 9-month survival advantage with azacitidine therapy compared to supportive care. However, many patients with MDS do not respond to treatment with DNMTIs as they are currently used, and adverse events are common. It is desirable to be able to predict a priori which patients are most likely to benefit from DNMTI therapy, in order to spare patients unlikely to respond from the risks and cost of treatment, but there are currently no well-established molecular methods for DNMTI response prediction.(3)

ATRX is an X-encoded chromatin-associated protein with an evolutionarily conserved Nterminal DNA methyltransferase (DNMT3-like) domain.(4) Germline mutations in ATRX cause a mental retardation-dysmorphology syndrome often associated with mild alpha thalassemia, which gave the gene its name (ATR-X syndrome: alpha thalassemia, retardation, X-linked) (5); in contrast, somatic point mutations in ATRX have been linked to acquired alpha thalassemia arising in the context of MDS, which may be present in as many as 8% of patients.(6-8) Germline mutations in ATRX are associated with widespread and diverse DNA methylation abnormalities across the genome.(9) Recent evidence suggests that the ATRX protein also has an important role in chromosome dynamics during mitosis, as ATRX-depleted cells exhibit defective sister chromatid cohesion and congression at the metaphase plate, as well as abnormal chromosome alignment. (10, 11)

Circumstantial evidence for a role of ATRX in neoplasia is increasing. In an array-based gene expression profiling study of 42 children with acute myeloid leukemia (AML) associated with somatic FLT3 mutations, the expression ratio of the transcription factor RUNX3 (formerly AML2) to ATRX predicted event-free survival (EFS).(12) In a subsequent study of 132 adults with de novo AML by another investigative group, low ATRX expression correlated with high-risk karyotype and poor clinical outcome.(13) Altered ATRX expression levels have also been reported in gene expression profiling experiments using prostate cancer primary cells (14), esophageal squamous cell carcinoma cell lines (15), chronic lymphocytic leukemia primary cells (Neil Kay, personal communication October 2007), and irradiated breast cancer cell lines (16).

Given the known effects of ATRX on global DNA methylation, the suggestive but still puzzling findings with respect to ATRX in cancer generally and myeloid neoplasia in particular, and the hypothesized mechanism of action of decitabine as a DNMTI, we studied ATRX and RUNX3 gene expression levels in MDS patients enrolled in the DACO-020 clinical trial, an open-label North American multicenter Phase 2 study of decitabine administered at a dose of 20 mg/m2/day IV daily for 5 days every 4 weeks.(17)

The clinical trial enrolled 99 consenting patients with International Prognostic Scoring System (IPSS) Intermediate-1 or higher risk MDS, who were treated at 28 different institutions. The study was approved by the Institutional Review Boards of participating institutions. RNA was isolated from patients' density centrifugation-separated peripheral blood mononuclear cells (PBMCs) prior to treatment. We confirmed RNA quality, generated cDNA, and performed real-time PCR (RT-PCR) with the Hs00230877_m1 ATRX and Hs00231709_m1 RUNX3 multiplex primer-probe sets with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) controls. Additionally, we performed brilliant cresyl blue stains to look for hemoglobin H inclusions and sequenced ATRX in all patients with inclusions (previous investigations (8) have shown that ATRX is wild-type in MDS patients without hemoglobin H inclusions.).

We were able to extract RNA of sufficient quantity and quality for analysis from PBMCs of 80 of the 99 patients enrolled in the multicenter clinical trial (81%). Clinical response data were available from all patients, and cytogenetic data from 74 patients; 38 (51%) had an abnormal karyotype. ATRX and RUNX3 expression or the ratio did not correlate with either the presence or complexity of a cytogenetic abnormality, and also did not predict clinical response to decitabine therapy (p>0.20 for all comparisons). Therefore, we were unable to confirm a correlation between ATRX or RUNX3 expression levels and either cytogenetic patterns or treatment outcomes in MDS. Only 1 patient had any Hb H inclusions, and sequencing of ATRX in this patient did not reveal any mutations.

There are several possible reasons for failure to confirm the hypothesis. The cell population studied (PBMCs) may not have allowed a true measure of ATRX/RUNX3 expression levels in the neoplastic clone. We were limited to using PBMCs because of small volumes of sample received from various sites, precluding separation of CD34+ cells, and a high rate of neutropenia among enrolled patients. It is also possible that ATRX expression has distinct effects in MDS compared to other neoplasms such as AML. These possibilities require further investigation, as the recurrent finding of ATRX expression changes in various neoplasms suggests a previously unrecognized role for the ATRX protein in cancer.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Greenberg PL, NCCNPanel National Comprehensive Cancer Network (NCCN) Clinical Practic Guidelines in Oncology: Myelodysplastic syndromes (v1. 2009) 2009. cited 2008 October 11. Available from: http://www.nccn.org/professionals/physician_gls/PDF/mds.pdf.
2. Fenaux P, Mufti GJ, Santini V, Finelli C, Giagounidis A, Schoch R, et al. Abstract 817: Azacitidine (AZA) Treatment Prolongs Overall Survival (OS) in Higher-Risk MDS Patients Compared with Conventional Care Regimens (CCR): Results of the AZA-001 Phase III Study. Blood. 2007;110(11)
3. Silverman LR, Mufti GJ. Methylation inhibitor therapy in the treatment of myelodysplastic syndrome. Nature clinical practice. 2005 Dec;2 1:S12–23. [PubMed]
4. Gibbons RJ, Bachoo S, Picketts DJ, Aftimos S, Asenbauer B, Bergoffen J, et al. Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nat Genet. 1997 Oct;17(2):146–8. [PubMed]
5. Gibbons RJ, Higgs DR. Molecular-clinical spectrum of the ATR-X syndrome. Am J Med Genet. 2000 Fall;97(3):204–12. [PubMed]
6. Gibbons RJ, Pellagatti A, Garrick D, Wood WG, Malik N, Ayyub H, et al. Identification of acquired somatic mutations in the gene encoding chromatin-remodeling factor ATRX in the alpha-thalassemia myelodysplasia syndrome (ATMDS) Nat Genet. 2003 Aug;34(4):446–9. [PubMed]
7. Steensma DP, Gibbons RJ, Higgs DR. Acquired alpha-thalassemia in association with myelodysplastic syndrome and other hematologic malignancies. Blood. 2005 Jan 15;105(2):443–52. [PubMed]
8. Steensma DP, Porcher JC, Hanson CA, Lathrop CL, Hoyer JD, Lasho TA, et al. Prevalence of erythrocyte haemoglobin H inclusions in unselected patients with clonal myeloid disorders. British journal of haematology. 2007 Nov;139(3):439–42. [PubMed]
9. Gibbons RJ, McDowell TL, Raman S, O'Rourke DM, Garrick D, Ayyub H, et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet. 2000 Apr;24(4):368–71. [PubMed]
10. Ritchie K, Seah C, Moulin J, Isaac C, Dick F, Berube NG. Loss of ATRX leads to chromosome cohesion and congression defects. J Cell Biol. 2008 Jan 28;180(2):315–24. [PMC free article] [PubMed]
11. De La Fuente R, Viveiros MM, Wigglesworth K, Eppig JJ. ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes. Dev Biol. 2004 Aug 1;272(1):1–14. [PubMed]
12. Lacayo NJ, Meshinchi S, Kinnunen P, Yu R, Wang Y, Stuber CM, et al. Gene expression profiles at diagnosis in de novo childhood AML patients identify FLT3 mutations with good clinical outcomes. Blood. 2004 Nov 1;104(9):2646–54. [PubMed]
13. Serrano E, Lasa A, Perea G, Carnicer MJ, Brunet S, Aventin A, et al. Acute myeloid leukemia subgroups identified by pathway-restricted gene expression signatures. Acta Haematol. 2006;116(2):77–89. [PubMed]
14. Coutinho-Camillo CM, Miracca EC, dos Santos ML, Salaorni S, Sarkis AS, Nagai MA. Identification of differentially expressed genes in prostatic epithelium in relation to androgen receptor CAG repeat length. Int J Biol Markers. 2006 Apr-Jun;21(2):96–105. [PubMed]
15. Bo H, Ghazizadeh M, Shimizu H, Kurihara Y, Egawa S, Moriyama Y, et al. Effect of ionizing irradiation on human esophageal cancer cell lines by cDNA microarray gene expression analysis. J Nippon Med Sch. 2004 Jun;71(3):172–80. [PubMed]
16. Roy D, Guida P, Zhou G, Echiburu-Chau C, Calaf GM. Gene expression profiling of breast cells induced by X-rays and heavy ions. International journal of molecular medicine. 2008 May;21(5):627–36. [PubMed]
17. Steensma DP, Baer MR, Slack JL, Buckstein R, Godley L, Larsen JS, et al. Abstract 1450: Preliminary Results of a Phase II Study of Decitabine Administered Daily for 5 Days Every 4 Weeks to Adults with Myelodysplastic Syndrome (MDS) Blood. 2007;110(11):1450a.