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Negative Effect of DNA Hypermethylation on the Outcome of Intensive Chemotherapy in Older Patients with High-Risk Myelodysplastic Syndromes and Acute Myeloid Leukemia following Myelodysplastic Syndrome

Authors' Affiliations: ¹ Division of Haematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge; ² Division of Haematology, Department of Medicine and ³ Department of Pathology, Radiumhemmet, Karolinska University Hospital, Solna, Stockholm, Sweden; ⁴ Department of Haematology, Aarhus University Hospital, Aarhus, Denmark; ⁵ Department of Haematology, Linköping University Hospital, Linköping, Sweden; ⁶ Department for Haematology and Coagulation Disorders, Malmö University Hospital, Malmö, Sweden; ⁷ Medical Clinic, Department of Haematology, University Hospital of Norrland, Umeå, Sweden; ⁸ Department of Haematology, Rigshospitalet, Copenhagen, Denmark; ⁹ Department of Medicine, Örebro University Hospital, Örebro, Sweden; ¹⁰ Haematopoietic Stem Cell Laboratory and Department of Haematology, Lund University Hospital, Lund, Sweden; ¹¹ Department of Haematology, Sahlgrenska University Hospital, Göteborg, Sweden; ¹² Division of Haematology, Department of Medicine, Sundsvall Hospital, Sundsvall, Sweden; ¹³ Department of Haematology, Ullevål University Hospital, Oslo, Norway; and ¹⁴ Department of Haematology, Uppsala Academic Hospital, Uppsala, Sweden

Requests for reprints: Eva Hellström-Lindberg, Department of Medicine, Division of Haematology, Karolinska University Hospital, Karolinska Institutet, Huddinge, SE-141 86 Stockholm, Sweden. Phone: 46-8-58582506; Fax: 46-8-7748725; E-mail: eva.hellstrom-lindberg{at}ki.se.

	Abstract

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Purpose: Promoter hypermethylation of, for example, tumor-suppressor genes, is considered to be an important step in cancerogenesis and a negative risk factor for survival in patients with myelodysplastic syndromes (MDS); however, its role for response to therapy has not been determined. This study was designed to assess the effect of methylation status on the outcome of conventional induction chemotherapy.

Experimental Design: Sixty patients with high-risk MDS or acute myeloid leukemia following MDS were treated with standard doses of daunorubicin and 1-β-D-arabinofuranosylcytosine. Standard prognostic variables and methylation status of the P15^ink4b (P15), E-cadherin (CDH), and hypermethylated in cancer 1 (HIC) genes were analyzed before treatment.

Results: Forty percent of the patients achieved complete remission (CR). CR rate was lower in patients with high WBC counts (P = 0.03) and high CD34 expression on bone marrow cells (P = 0.02). Whereas P15 status alone was not significantly associated with CR rate (P = 0.25), no patient with hypermethylation of all three genes achieved CR (P = 0.03). Moreover, patients with CDH methylation showed a significantly lower CR rate (P = 0.008), and CDH methylation retained its prognostic value also in the multivariate analysis. Hypermethylation was associated with increased CD34 expression, but not with other known predictive factors for response, such as cytogenetic profile.

Conclusions: We show for the first time a significant effect of methylation status on the outcome of conventional chemotherapy in high-risk MDS and acute myelogenous leukemia following MDS. Provided confirmed in an independent study, our results should be used as a basis for therapeutic decision-making in this patient group.

The mechanisms of action of azacytidine are not known in detail, but it is clear that this drug can induce hypomethylation and reexpression of silenced genes; it can also induce a broad spectrum of apoptotic pathways (13–17).¹⁶

Unless allogeneic stem cell transplantation could be done, few patients with MDS will be cured. Patients with intermediate-2 and high-risk MDS (8) have a median survival of <1.5 years and are usually considered to be candidates for chemotherapy in lower or higher doses. Induction chemotherapy may lead to complete remission (CR) in 40% to 60% of patients; however, median remission duration is generally less than 1 year (18–25). We have previously shown a 43% CR rate in a cohort of 93 patients with RAEB-t and acute myelogenous leukemia (AML) following MDS with a median age of 72 years (range 35-90 years; ref. 24). Median duration of CR was 11.3 months. Our study showed that induction chemotherapy is potentially successful also in older patients, but that therapeutic approaches aiming to prolong remission are highly warranted.

Hypercellular bone marrow, high-risk karyotype, elevated lactate dehydrogenase (LDH), high WBC counts, and age have, in various combinations, been associated with a lower probability for CR (18, 20–22, 24). Interestingly, these variables did not predict duration of CR in the above-described Nordic study. The role of methylation status for outcome of induction chemotherapy in MDS and AML is yet to be determined.

In the present study, we show that methylation status has a significant effect on the outcome of induction chemotherapy, and, among known risk factors, only relates to the percentage blasts and of CD34⁺ cells in the bone marrow.

	Materials and Methods

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Patients. Consecutive patients with intermediate-2 and high-risk MDS, chronic myelomonocytic leukemia (CMML) with >10% marrow blasts, and patients with AML following an established MDS or mixed MDS/myeloproliferative disease (26), who were considered eligible for at least one cycle of standard induction chemotherapy, were included in the protocol. Patients were excluded if they were considered candidates for potentially curative regimens, including AML-like induction chemotherapy followed by intensive consolidation chemotherapy or allogeneic stem cell transplantation. Thus, the study was designed mainly for older patients or patients with multiple risk factors. The study was approved by the national ethical committees, and medical product agencies of Sweden, Norway, and Denmark. All patients gave their informed consent.

Study design. Initial diagnosis as well as verification of CR was assessed at a central hematopathology unit (Department of Pathology at Karolinska University Hospital Solna, Stockholm, Sweden). For diagnosis and classification, WHO criteria were applied (26). Bone marrow cellularity was assessed on bone marrow biopsies and percentage of blasts in bone marrow smears was determined by counting 500 cells in representative areas of the smears. Flow cytometry was done on bone marrow aspirates that were sent by overnight transport. Percentages of CD34⁺ cells were determined in nonseparated bone marrow samples after four-color direct immunofluorescence staining with CD13FITC/CD11bPE/CD45PECy5/CD34APC and acquisition on FACSCalibur equipped with Cell Quest software program (Becton Dickinson). Cytogenetic analyses were done locally at each center by standard techniques and the findings were classified according to the International Prognostic Scoring System (IPSS) into good, intermediate, and poor prognostic subgroups (8). Induction chemotherapy consisted of a standard DA 2+7 regimen: daunorubicin 60 mg/m² i.v. days 1 to 2 and cytarabine 150 mg/m² s.c. days 1 to 7. Patients who did not respond to the first course were given a second course if judged medically appropriate. Patients obtaining CR were eligible for a maintenance study of low-dose azacytidine, but no patients received standard consolidation chemotherapy. The criteria for CR were <5% bone marrow blasts, stable hemoglobin >100 g/L, WBC >1.5 x 10⁹/L with normal differential count, and platelets >100 x 10⁹/L. These criteria were identical with those in the previous Nordic study (24) to allow a comparison of CR rates and effect of pretreatment variables.

Bone marrow sampling. Bone marrow for methylation analysis was sampled at registration. To avoid the effect of local separation and storage factors, bone marrow was shipped by DHL using a <24 h service to a central laboratory for isolation of mononuclear cells (MNC) and CD34⁺ cells as previously described (27). Methylation analysis on all samples obtained before start of treatment was done during one serial experiment.

Cell isolation. MNC were isolated from bone marrow by density gradient technique through Lymphoprep (Axis Sield). Where the yield of MNC was sufficient, CD34⁺ cells were purified by magnetic cell sorting using the CD34 Progenitor Cell Isolation kit (MACS; Miltenyi Biotec) according to the manufacturer's guidelines. The cells were stored as pellet at –80°C.

DNA isolation and bisulfite modification. Genomic DNA was isolated from MNC and CD34⁺ cells using the QIAmp DNA mini kit (Qiagen) according to the manufacturer's guidelines. The amount of DNA obtained was measured by spectrophotometer (ND-1000, NanoDrop Technologies). The DNA was further modified with sodium bisulfite as previously described (28). In brief, 2.5 µg of DNA from each sample were denatured in NaOH for 15 min at 37°C followed by the incubation in sodium bisulfite at 55°C for 16 h. Thereafter, DNA were recovered using the GeneClean II kit (Qbiogene), desulfonated in NaOH, and precipitated in ethanol.

Polymerase chain reaction. PCR specific for bisulfite-reacted P15, CDH, and HIC promoters was carried out in a final volume of 25 µL containing 50 to 100 ng of bisulfite modified DNA, 1x TEMPase PCR Buffer (Bie & Berntsen AS), 0.2 mmol/L deoxyribonucleotide triphosphate (ABgene), 0.5 µmol/L each of forward and reverse primers (5), and 0.75 unit of TEMPase Hot Start DNA polymerase (Bie & Berntsen). PCR was done in a PX2 Thermal cycler (Thermo Electron Corporation). The polymerase was activated by incubation at 95°C for 15 min followed by 40 cycles of 95°C for 30 s, 55°C (P15 and HIC) or 48°C (CDH) for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. PCR results were examined by electrophoresis in a 2.5% agarose gel.

Denaturing gradient gel electrophoresis. Fifteen to 20 µL of the PCR product were loaded onto a 10% denaturant/6% polyacrylamide-70% denaturant/12% polyacrylamide double gradient gel (29). A fully methylated control (SssI) and an unmethylated control (peripheral blood lymphocytes) were also loaded on each gel. Gels were run at 160 V for 270 min in 1x Tris acetate/EDTA buffer kept at a constant temperature of 58°C (P15) or 55°C (CDH and HIC). After electrophoresis, gels were stained in 1x Tris acetate/EDTA buffer containing ethidium bromide (2 µg/mL) and photographed under UV transillumination. Samples were scored as methylated when bands or smears were present on the gels in the area below the band corresponding to unmethylated DNA as shown in Fig. 1 and as reported by previous publications (5, 29).

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Promoter methylation detected by bisulfite-denaturing gradient gel electrophoresis (DGGE). PBL, peripheral blood lymphocytes from a healthy individual (unmethylated); SssI, in vitro methylated DNA (fully methylated); patient 1 (methylated); patient 2 (unmethylated); patient 3 (methylated). Patients were judged methylated when bands or smears were observed in the area below the band corresponding to unmethylated control.

	Results

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Patients. A total of 60 patients with a median age of 68 years (54-83 years) were enrolled in the clinical study. Baseline characteristics are shown in Table 1 . Seventeen had MDS (RAEB-1 or RAEB-2), 6 had CMML, and 37 had AML following a well-documented phase of MDS or mixed MDS/myeloproliferative disease. All CMML patients had myelodysplastic rather than myeloproliferative features, and were scored according to IPSS. All patients with AML belonged to the WHO subcategory "AML with trilineage dysplasia with preceding MDS." Chromosome analysis was successful in 52 patients. Patients were divided into the three cytogenetic risk groups defined by IPSS (8). Twenty-five patients had a good prognosis karyotype (normal or isolated –Y, del5q or del20q), 17 had a poor prognosis karyotype (3 aberrations or chromosome 7 abnormalities), and 10 had an intermediate karyotype (not fulfilling the criteria for good or poor). Table 1 shows cytogenetic risk groups in the different WHO subgroups. The percentage of patients with poor cytogenetics was 40%, 0%, and 35% in patients with MDS, CMML, and AML, respectively. All patients started induction chemotherapy and were evaluated according to intention to treat.

Twenty-six of 47 (55%) patients showed hypermethylation of P15; 9 of 41 (22%) showed hypermethylation of HIC; and 15 of 41 (37%) showed hypermethylation of CDH. Nineteen patients showed hypermethylation of P15 plus one other gene, and six patients showed hypermethylation of all three genes. All three genes were evaluable for methylation status in 41 patients. Hypermethylation of HIC and CDH was mostly observed in patients with P15 hypermethylation; only three P15-negative patients had CDH or HIC hypermethylation. Hence, hypermethylation of P15 seems to be an earlier event than hypermethylation of CDH and HIC also in our study (5).

Methylation status in relation to other prognostic factors. Eighteen of 28 (64%) MDS-AML showed P15 hypermethylation, compared with 6 of 13 (46%) and 2 of 6 (33%), respectively, in the RAEB and CMML subgroups. These differences were not statistically significant (Table 3 ). More patients with MDS-AML were methylated on P15 plus at least one other of the genes than in the "non–AML group" (RAEB and CMML; P = 0.02). Overall, there was no difference in methylation status between patients with good and intermediate cytogenetic risk group according to IPSS. Cases with hypermethylation of P15 plus one other gene had slightly more often a high-risk karyotype (P = 0.13). P15 hypermethylation was significantly associated with CD34 expression (P = 0.04). Patients with P15 plus one other gene hypermethylated showed both higher number of CD34⁺ cells and bone marrow blasts (P = 0.01 and P = 0.007, respectively). There was no association between methylation status and other known risk factors such as WBC count, marrow cellularity, and LDH levels. Hypermethylation of all three genes was associated with higher age (P = 0.02).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Percentage of CR in patients with or without methylation of P15, CDH, HIC, P15 plus at least one other gene, or of all three genes.

	Discussion

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The aim of this prospective clinical trial was to assess the effect of DNA hypermethylation on outcome of induction chemotherapy in patients with high-risk MDS and MDS-AML, and to investigate the covariation between methylation patterns and other known risk factors. Because hypomethylating agents are available for patients with MDS, it is important to address how these therapeutic options should be used in relation to conventional chemotherapy.

The observed frequencies of methylation of the three genes corresponded well with the findings of others, which suggest that the denaturing gradient gel electrophoresis method is stable and with acceptable interlaboratory variation (5). Confirming the results from a previous study by Aggerholm et al. (5), studying methylation of the same genes with the same method, there was no difference in methylation status between MNC and CD34⁺ cells. Furthermore, we conclude that only a few patients were methylated in CDH or HIC without showing P15 methylation. Hence, P15 methylation seem to be an earlier event in the course of the disease, but whether it is necessary for subsequent hypermethylation of the other genes in MDS remains to be shown.

The CR rate in the present study was 40%, which compares well with other studies on similar patient cohorts (18–22, 24, 25). In those studies, various combinations of pretreatment factors indicative of more advanced or proliferative disease had an effect on CR rate. Our study is first to define CD34 expression, assessed by flow cytometry, as a more important predictor of response than stage of disease, marrow blast percentage, IPSS cytogenetic risk group, and LDH level (P = 0.02). In fact, CD34 expression was a stronger predictor of response than WBC in the multivariate analysis. The novelty of this finding could probably be explained by the fact that immunophenotyping, although suggested as a predictive test for survival (30), rarely had been included as a pretreatment variable in clinical trials of high-risk MDS and MDS-AML. Previous studies have suggested a negative prognostic effect of surface expression of CD34 on survival in MDS and AML and also a relation between resistance to chemotherapy and CD34 expression (30–33). Our data strongly supports the conclusion that immunophenotyping is an important predictive tool in MDS and MDS-AML undergoing induction chemotherapy.

Interestingly, and in line with the previous Nordic study (24), age did not affect the outcome of chemotherapy. It is clear that biological features of the tumor cell population have a greater effect on outcome than age in itself, and that age should not be used as a standard component to predict the response to moderate induction chemotherapy.

The present study is the first to investigate the effect of promoter methylation status on the outcome of chemotherapy in elderly high-risk MDS and MDS-AML. DNA hypermethylation was not related to peripheral blood values, LDH levels, or morphologic features, indicating that it is not associated to the degree of inefficient hematopoiesis. Instead, methylation of more than one gene was associated with CD34 expression and, to a lesser extent, with percentage of marrow blasts. Clinical studies have suggested that azacytidine and decitabine have at least as good effect in patients with high-risk karyotype as in those with low- and intermediate-risk karyotypes (9, 10). In our material, patients with methylation of P15 plus one other gene had a somewhat higher incidence of high-risk cytogenetics than other patients, which would support this observation. Interestingly, however, patients with three methylated genes showed no particular cytogenetic pattern, indicating that cytogenetics and methylation status are at least partly separated phenomenon.

Although P15 methylation has been suggested as an important biological finding in MDS (5, 7, 34), hypermethylation of this gene alone showed no correlation with other variables and did not effect CR rate in our material. Instead, hypermethylation of more than one gene had a stronger effect on response rate than cytogenetics and blast percentage. These results correspond well with the findings of Aggerholm et al. (5), showing a prognostic effect on survival of multiple gene hypermethylations, but not P15 methylation alone in low-risk MDS. CDH is a gene involved in cell adhesion and has an invasion-suppressor function (35). The specific finding of CDH hypermethylation as a particularly poor prognostic factor deserves follow-up in a larger patient material, but cannot, from the present study, be used to draw extensive conclusions. It is, however, difficult to neglect the fact that none of the six patients with all three genes hypermethylated and few of those with two hypermethylated genes responded to conventional chemotherapy. This finding corresponds well with the growing evidence that promoter hypermethylation plays an important role for drug resistance in cancer treatment (36). On the other hand, CR was achieved in more than 50% of patients without hypermethylation, which suggests that induction chemotherapy indeed is a valuable therapeutic option in this patient group. A prospective study investigating the relation between methylation status, assessed by several methods, cDNA expression profiling, and outcome of therapy in MDS would certainly add to the scientific knowledge in this field.

Azacytidine and decitabine have been shown to prolong time to death or leukemic transformation in MDS (9, 11) and is widely used in countries where they have a label. However likely that they will constitute an important addition to the therapeutic arsenal for MDS in the future, there is still a lot to learn about their mode of action and which patients are most likely to respond to therapy. Also, there is potentially much to gain by combining these agents with conventional chemotherapy. Achieving CR is usually a prerequisite for cure in high-risk myeloid disease. In this study, we show a strong negative effect of hypermethylation on the outcome of induction treatment. Whether pretreatment with hypomethylating agents could lead to a better response to conventional chemotherapy may be worth analyzing in a prospective study.

	Footnotes

 
Grant support: Nordic Cancer Union 4796-B03-01XAAN, Swedish Cancer Society 3689-B04-10XAC, and an unrestricted grant from Pharmion Corporation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹⁵ L. Shen. DNA methylation in myelodysplastic syndrome: biomarkers to predict prognosis and responses to epigenetic therapy. EHA Scientific Workshop on the Role of Epigenetics in Hematological Malignancies; 2007 Feb 9-11, 2007; Mandelieu, France..

¹⁶ R. Khan, et al. Mitochondrial involvement in 5-azacytidine-induced apoptosis. Abstract. 11th Congress of the EHA; 2006 June 15-18; Amsterdam, the Netherlands..

Received 5/15/07; revised 7/20/07; accepted 8/22/07.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Singal R, Ginder GD. DNA methylation. Blood 1999;93:4059–70.[Free Full Text]
2. Toyota M, Issa JPJ. Epigenetic changes in solid and hematopoietic tumors. Semin Oncol 2005;32:521–31.[CrossRef][Medline]
3. Lehmann U, Brakensiek K, Kreipe H. Role of epigenetic changes in hematological malignancies. Ann Hematol 2004;83:137–52.[CrossRef][Medline]
4. Esteller M. Profiling aberrant DNA methylation in hematologic neoplasms: a view from the top of the iceberg. Clin Immunol 2003;109:80–8.[CrossRef][Medline]
5. Aggerholm A, Holm MS, Guldberg P, Olesen LH, Hokland P. Promoter hypermethylation of P15ink4b, HIC1, CDH1 and ER is frequent in myelodyplastic syndrome and predicts poor prognosis in early stage patients. Eur J Haematol 2006;76:23–32.[CrossRef][Medline]
6. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-aza-2'-deoxycytidine (decitabine) treatment. Blood 2002;100:2957–64.[Abstract/Free Full Text]
7. Tien HF, Tang JH, Tsay W, et al. Methylation of the P15(ink4b) gene in myelodysplastic syndrome: it can be detected early at diagnosis or during disease progression and is highly associated with leukaemic transformation. Br J Hematol 2001;112:148–54.[CrossRef][Medline]
8. Greenberg P, Cox C, LeBeau MM, et al. International scoring system for evaluating the prognosis in myelodysplastic syndromes. Blood 1997;89:2079–88.[Abstract/Free Full Text]
9. Silverman LR, Demakos EP, Peterson BL, et al. Randomizes controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the Cancer and Leukemia Group B. J Clin Oncol 2002;20:2429–40.[Abstract/Free Full Text]
10. Wijermans P, Lübbert M, Verhoef G, et al. Low dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol 2002;18:956–62.
11. Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes; results of a phase III randomized study. Cancer 2006;106:1794–803.[CrossRef][Medline]
12. Kornblith AB, Herndon II JE, Silverman LR, et al. Impact of azacitidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol 2002;20:2441–52.[Abstract/Free Full Text]
13. Guo Y, Engelhardt M, Wider D, Abdelkarim M, Lübbert M. Effects of 5-aza-2'-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells. Leukemia 2006;20:115–21.[CrossRef][Medline]
14. Berg T, Guo Y, Abdelkarim M, Fliegauf M, Lübbert M. Reversal of p15/INK4b hypermethylation in AML1/ETO-positive and -negative myeloid leukemia cell lines. Leukemia Res 2007;31:497–506.[CrossRef][Medline]
15. Schmelz K, Sattler N, Wagner M, Lübbert M, Dörken B, Tamm I. Induction of gene expression by 5-aza-2'-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms. Leukemia 2005;19:103–11.[Medline]
16. Schmelz K, Wagner M, Dörken B, Tamm I. 5-aza-2'-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia. Int J Cancer 2005;114:683–95.[CrossRef][Medline]
17. Khan R, Aggerholm A, Hokland P, Hassan M, Hellström-Lindberg E. A pharmacodynamic study of 5-azacytidine in the P39 cell line. Exp Hematol 2006;34:35–43.[Medline]
18. De Witte T, Suciu S, Peetermans M, et al. Intensive chemotherapy for poor prognosis myelodysplasia (MDS) and secondary acute myeloid leukemia (sAML) following MDS of more than 6 months duration. A pilot study by the Leukemia Cooperative Group of the European Organization for Research and Treatment in Cancer (EORTC-LCG). Leukemia 1995;9:1805–11.[Medline]
19. Ganser A, Heil G, Seipelt G, et al. Intensive chemotherapy with idarubicine, ara-C, etoposide and m-AMSA followed by immunotherapy with interleukin-2 for myelodysplastic syndromes and high-risk Acute Myeloid Leukemia (AML). Ann Hematol 2000;79:30–5.[CrossRef][Medline]
20. Hofmann WK, Heil G, Wiebe S, et al. Intensive chemotherapy with idarubicine, cytarabine, etoposide and G-CSF priming in patients with advanced myelodyplastic syndrome and high-risk acute myeloid leukemia. Ann Hematol 2004;83:498–503.[Medline]
21. Kantarjian H, O'Brien S, Cortes J, et al. Results of intensive chemotherapy in 998 patients age 65 years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome. Cancer 2006;106:1090–98.[CrossRef][Medline]
22. Wattel E, de Botton S, Laï JL, et al. Long-term follow-up of de novo myelodysplastic syndromes treated with intensive chemotherapy: incidence of long-term survivors and outcome of partial responders. Br J Haematol 1997;98:983–91.[CrossRef][Medline]
23. Kantarjian H, Beran M, Cortes J, et al. Long-term follow-up results of the combination of topotecan and cytarabine and other intensive chemotherapy regimens in myelodysplastic syndrome. Cancer 2006;106:1099–109.[CrossRef][Medline]
24. Hast R, Hellström-Lindberg E, Ohm L, et al. No benefit from adding GM-CSF to induction chemotherapy in transforming myelodysplastic syndromes: better outcome in patients with less proliferative disease. Leukemia 2003;17:1827–33.[CrossRef][Medline]
25. Oosterveld M, Muus P, Suciu S, et al. Chemotherapy only compared to chemotherapy followed by transplantation in high risk myelodysplastic syndrome and secondary acute myeloid leukemia; two parallel studies adjusted for various prognostic factors. Leukemia 2002;16:1615–21.[CrossRef][Medline]
26. Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization classification of tumours. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2001.
27. Nilsson L, Åstrand-Grunström I, Andersson K, et al. Involvement and functional impairment of the CD34⁺ CD38^– hematopoietic stem cell pool in myelodysplastic syndromes with trisomy 8. Blood 2002;100:259–67.[Abstract/Free Full Text]
28. Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR-test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet 1997;5:94–8.[Medline]
29. Cremonesi L, Firpo S, Ferrari M, Righetti PG, Gelfi C. Double-gradient DGGE for optimized detection of DNA point mutations. Biotechniques 1997;22:326–30.[Medline]
30. Pagnucco G, Giambanco C, Gervasi F. The role of flow cytometric immunophenotyping in myelodysplastic syndromes. Ann N Y Acad Sci 2006;1089:383–94.[CrossRef][Medline]
31. Van Rhenen A, Feller N, Kelder A, et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005;11:6520–7.[Abstract/Free Full Text]
32. Junghanss C, Waak M, Knopp A, et al. Multivariate analyses of prognostic factors in acute myeloid leukemia: relevance of cytogenetic abnormalities and CD34 expression. Neoplasma 2005;52:402–10.[Medline]
33. Repp R, Schaekel U, Helm G, et al. Immunophenotyping is an independent factor for risk stratification in AML. Cytometry B Clin Cytometry 2003;53:11–9.
34. Christiansen DH, Andersen MK, Pedersen-Bjergaard J. Methylation of P15^INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia 2003;17:1813–9.[CrossRef][Medline]
35. Mareel M, Bracke M, van Roy F. Cancer metastasis: negative regulation by an invasion-suppressor complex. Cancer Detect Prev 1995;19:451–64.[Medline]
36. Glasspool RM, Teodoridis JM, Brown R. Epigenetics as a mechanism driving polygenic clinical drug resistance. Br J Cancer 2006;94:1087–92.[CrossRef][Medline]

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