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NEOPLASIA
From the Johns Hopkins Oncology Center, Baltimore, MD;
the University of New Mexico School of Medicine, Albuquerque, NM; the
Southwest Oncology Group Statistical Center, Seattle, WA; and the
Leukemia Department, University of Texas at MD Anderson Cancer Center,
Houston, TX.
Aberrant methylation of multiple CpG islands has been described in
acute myeloid leukemia (AML), but it is not known whether these are
independent events or whether they reflect specific methylation
defects in a subset of cases. To study this issue, the
methylation status of 14 promoter-associated CpG islands was analyzed
in 36 cases of AML previously characterized for
estrogen-receptor methylation (ERM). Cases with methylation
density of 10% or greater were considered positive. Seventeen cases
(47%) were ERM+ while 19 cases were
ERM Acute myeloid leukemia (AML) is a hematopoietic
malignancy that frequently exhibits nonrandom chromosomal
translocations.1 Cytogenetic studies reveal a wide variety
of translocations involving specific subtypes of the disease, and
structurally altered genes play important roles in cell proliferation,
differentiation, and gene transcription.2 However,
oncogenes and tumor-suppressor genes that are frequently altered in
solid tumors, such as p53 and K-RAS, are
infrequently mutated in AML.3,4 In addition to these
genetic changes, several studies indicate that epigenetic changes, such
as aberrant DNA methylation, can also play important roles in the
progression of a wide variety of human neoplasms.5,6 In
particular, hypermethylation of promoter-associated CpG-rich regions,
termed CpG islands, can result in gene silencing that is clonally
propagated through mitosis by the action of DNA-methyltransferase enzymes. Such methylation-associated silencing plays a physiological role in X-chromosome inactivation7 and
imprinting8 and a pathological role in silencing
tumor-suppressor genes in neoplasia. Silencing of methylated CpG
islands appears to result from the acquisition of a closed chromatin
structure.9 For selected genes, allele-specific
methylation suggests that this process can be an alternative mechanism
to mutations or deletions in tumor-suppressor gene inactivation in
human neoplasms.6
In AML, several genes, including p15,10
MDR1,11 ER,12 and
HIC1,13 have been shown to be inactivated by
methylation. Methylation patterns may have clinical implications in
that ER methylation was found to be associated with improved
survival.14 However, it is unclear whether such
ER methylation is prognostic because of estrogen receptor
function or whether it reflects a distinct pathway of tumorigenesis in
AML, with distinct expression profiles of genes that could influence
prognosis, such as MDR1. Indeed, it has been reported that
AML cases have frequent methylation of multiple genes
simultaneously.15 To study this issue further, we have
analyzed the methylation status of 14 separate loci in a series of AML
cases previously characterized for ER methylation. We found
remarkable concordance of methylation for all the genes analyzed,
suggesting the existence of distinct methylator pathways in this disease.
Samples
All but one of the cases selected here had previously been studied for
estrogen receptor (ER) methylation (ERM) by Southern blot
analysis. The cases were selected as follows: 8 cases were selected
because of high levels of ERM (greater than 90%); 8 cases were chosen
on the basis of low ERM (lower than 5%); and 20 additional unselected
cases were included in the study. Normal bone marrow samples were also
obtained from bone marrow transplant donors. Informed consent was
obtained from all patients before the specimens were collected
according to institutional guidelines. Genomic DNA and RNA were
extracted by means of standard procedures.
Bisulfite polymerase chain reaction
Reverse transcriptase PCR We used 6 µg of total RNA to synthesize complementary DNA (cDNA) using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) according to the manufacturer's protocol. To amplify cDNA, PCR was performed in a 50-µL reaction mixture containing 1 µL cDNA, 1 × PCR buffer (Life Technologies), 1.25 mM dNTP, 1.5 mM MgCl2, primers, and 1 U Taq polymerase (Life Technologies). Sequences of the PCR primers used are as follows. p15F: 5'-TGGGGGCGGCAGCGATGAG-3; p15R: 5'-AGGTGGGTGGGGGTGGGAAAT-3'; SDC4F: 5-CCTCTAGATAACCATATCCCTGAGA-3'; SDC4R: 5'-CCTAATGTCCACCCTTCAAAAT-3'. To verify the integrity of messenger RNA (mRNA), the GAPDH gene was amplified by means of the following primers: GAPDHF: 5'-CGGAGTCAACGGATTGGTCGTAT-3' and GAPDHR 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. After PCR, 10 µL of the samples were electrophoresed in 1.5% agarose gels and visualized by ethidium bromide staining. All reactions included negative controls where reverse transcriptase was omitted.Statistical considerations For each gene analyzed, the methylation frequency was defined as the proportion of subjects in whom the methylation density equaled or exceeded 10%; the selection of this criterion is described in the "Results" section. Since the distributions of methylation densities were unlikely to be Gaussian, correlation among methylation densities of the various genes was measured by means of Spearman rank order correlation coefficient (RS). Associations between quantitative clinicopathological characteristics and the number of methylated genes were examined by means of ordinary least squares regression analysis.
Samples and genes analyzed The 36 selected AML cases (17 females, 19 males) ranged in age from 19 to 69 years (median, 39 years). All but 1 had previously been characterized for ERM by Southern blot.14 To allow the analysis of multiple genes in each sample, we used a bisulfite-PCR approach that requires considerably less DNA than Southern blotting. We first determined the methylation status of ER using bisulfite-PCR. Of 36 cases analyzed, 20 (56%; 95% confidence interval, 38%-72%) showed methylation of ER, ie, methylation density of 2% or greater. Among these 20, the ER methylation density ranged from 8% to 61% (median, 44%). There was a strong correlation between ER methylation density as determined by bisulfite-PCR and by Southern blot analysis (RS = 0.75, P < .0001). However, the values obtained by bisulfite-PCR were generally lower than those previously determined by Southern blot analysis. This most likely relates to a lower sensitivity of bisulfite-PCR because the reaction requires 2 adjacent CpGs to be simultaneously methylated, while Southern blot analysis requires 1 of 2 CpGs to be methylated. In addition, the sites analyzed by PCR are slightly different from those analyzed by Southern blot, which probably partly explains the lack of a perfect correlation between the 2 determinations.Methylation of multiple CpG Islands in AML To examine whether ERM reflected genome-wide aberrant CpG-island methylation, we studied the methylation status of 14 additional loci (Table 1; examples in Figure 1). In normal bone marrow, no significant (2% or greater) methylation was observed in any of the genes analyzed (Figure 1 and data not shown). The proportion of patients with significant methylation of individual CpG islands varied from 0 of 36 (0%) of the cases for MLH1 to 31 of 36 (86%) for PITX2 (Table 2). Of the 14 additional CpG islands analyzed, MLH1 was unmethylated in all samples; 9 CpG islands (CACNA1G, MINT1, MINT2, p16INK4A, THBS1, p15INK4B, PTC1A, PTC1B, and MDR1) were methylated relatively infrequently (range: 6% to 36% of patients); and 4 CpG islands (MYOD, SDC4, GRP37, and PITX2) were methylated relatively frequently (range: 61% to 86% of patients). The pairwise correlations between methylation densities of the genes examined (including ER, but omitting MLH1) were measured by means of RS. All of the correlation coefficients were positive, ranging from 0.04 to 0.83, suggesting that methylation affects multiple genes in a subset of the cases. For example, ER methylation correlated positively with methylation of all other genes (R = 0.53, P = .0008, for p15; R = 0.34, P = .04, for p16; R = 0.40, P = .017, for THBS1; R = 0.55, P = .0005, for PTC1A; R = 0.36, P = .031, for PTC1B; R = 0.59, P = .0002, for MDR1; R = 0.38, P = .023, for CACNA1G; R = 0.32, P = .058, for MINT1; R = 0.36, P = .030, for MINT2; R = 0.43, P = .0087, for SDC4; R = 0.69, P = .0001, for GPR37; R = 0.57, P = .0003, for MYOD; and R = 0.56, P = .0004, for PITX2).
Hypermethylator phenotypes in AML The strong concordance between different methylation events described above suggested the presence of hypermethylator phenotypes in AML, as described in colon cancer. In order to begin defining a classification of AML based on methylation profiling, we grouped the cases according to the number of loci methylated in each case, using a threshold of 10% or greater for all loci. The use of a threshold is suggested by the fact that, in colon cancer, the presence of a hypermethylator phenotype is characterized by both more frequent methylation and more dense methylation.19 As shown in Table 2, the infrequently methylated loci (ie, the first 9 loci) seldom had methylation densities of 2% to 9.9%. Such intermediate densities were much more common among the 4 frequently methylated genes. Overall, only 36 of 540 methylation densities were between 2% and 10% and are therefore affected by the use of a threshold.Using the 10% threshold, we found that the proportion of
methylated genes ranged from 0 of 15 loci to 13 of 15 loci. As Table 3 shows, 16 patients (44%) had
methylation of fewer than 3 genes (including 1 patient with none),
while 13 (36%) and 7 (19%) had methylation of 3 to 7 and 8 to 13 genes, respectively. Moreover, the mean methylation density of the
methylated genes tended to increase with the number of methylated
genes, from a median of 22% in the 15 patients with 1 or 2 methylated
genes, to 61% in the 7 patients with 8 to 13 methylated genes (Table
3, Figure 2). These data suggest the
presence of a hypermethylator phenotype in AML that affects both
methylation frequency and density.
This analysis raised the possibility that the correlations between the methylation of ER and the 13 additional loci could have been due largely to the group of 7 cases with particularly dense and frequent methylation. To address this issue, we repeated the correlation analyses excluding these 7 cases. Of the remaining 29 cases, none were methylated at CACNA1G, MINT2, PTC1-A, or PTC1-B. However, there were still at least marginally statistically significant correlations between methylation densities of ER and each of the following: p15 (RS = 0.42, P = .023); MDR1 (RS = 0.40, P = .030); PITX2 (RS = 0.33, P = .083); GPR37 (RS = 0.58, P = .0010); and MyoD (RS = 0.38, P = .045). Clinicopathological features of hypermethylation in AML The clinical features of all 36 cases grouped into 3 methylation categories are shown in Table 4. Age, white blood count (WBC), absolute peripheral blast count, MRK16 expression, and cyclosporin A (CsA)-inhibited DiOC2 efflux appear to decrease with increasing methylation, while marrow blast percentage tends to increase with increasing methylation. However regression analysis of each variable in relation to the number of genes PCR+ for methylation indicates that the association between age and methylation is statistically significant (2-tailed P = .0040) (Figure 3), with older patients tending to have fewer methylated genes. None of the other patient or disease characteristics are significantly associated with methylation. However, in view of the small sample size, the absence of significant associations should be viewed as inconclusive. The inverse correlation between age and methylation is consistent with our prior study of ER methylation.14 Centrally reviewed karyotypes were available for only 7 of these patients and did not reveal clustering of abnormalities in specific groups. The number of cases studied is too low to allow meaningful correlations with outcome. Nevertheless, it is interesting to note that the group of 7 cases with high levels of methylation had a strikingly poor complete response rate after induction chemotherapy (29%, compared with 62% and 63% in the other groups), despite the relatively young age of the patients (median, 27 years). Definitive estimation of correlations between methylation category and therapeutic results will have to await larger studies.
Correlation between hypermethylation and differential expression of multiple genes To study whether the aberrant methylation detected in AML actually correlates with gene silencing, the expression status of p15INK4B and SDC4 was determined by reverse transcriptase (RT) PCR for 15 cases with or without methylation of these loci. Previous data had demonstrated differential ER gene expression in these same cases.14 Representative results for p15 and SDC4 are shown in Figure 4. The tumors that showed aberrant methylation of p15 did not express mRNA or expressed significantly lower levels compared with unmethylated cases. Similarly, SDC4 was expressed weakly or not at all in methylated cases. Expression of MDR1 was previously determined for 29 of the 36 cases by various methods, including immunohistochemistry and functional analyses.20 MRK16 expression tended to be lower among patients with high levels of MDR1 methylation. The 5 patients with MDR1 methylation densities greater than 40% were all MRK16 , ie, had D-values lower than 0.15. Similarly, of the 5 patients with moderate or bright MRK16 expression
(D greater than 0.20), 4 were PCR for MDR1
methylation. However there were discrepant cases. Of 17 patients with
no MDR1 methylation (0% methylation density), 10 were
MRK16, suggesting that mechanisms other than methylation
may prevent MRK16 expression. Also 1 patient with 33% MDR1
methylation density expressed MRK16 at a high level (D = 0.50),
perhaps reflecting tumor heterogeneity in this patient.
In the present study, we examined the methylation status of multiple CpG islands in a series of AML patients with or without methylation of ER. Our results indicate that ER methylation occurs concomitantly with aberrant methylation of other loci. On the basis of the number of genes methylated, AML cases can be divided into 3 groups that differ substantially in the frequency, nature, and extent (density) of hypermethylation events. These results suggest that CpG-island methylation is related to specific methylation defects in subsets of AMLs, rather than that methylation of each individual island represents a random event followed by selection for the affected cell. It should be emphasized that this study was based on a group of 36 previously untreated AML patients with comparatively young ages (median 39 years, with only 1 patient over 65 years old) and high WBCs (median 37.7 × 109/L [37 700/µL]). These selection factors may affect the generalizability of these results. Moreover, since these patients were selected in part on the basis of their ERM results, their distribution of methylation density and frequency may not be representative of the more general population of AML patients. In addition, the number of patients studied here did not allow us to make definitive correlations between methylation and clinico-pathologic correlates. These issues must now be addressed in studies that include larger numbers of patients. Several studies now point to the existence of hypermethylator phenotypes in various human neoplasms. In colorectal cancer,21 gastric cancer,22 and pancreatic cancer,23 a profound hypermethylator phenotype termed CpG-island methylator phenotype was described in subsets of cases, and was shown to involve such genes as p16INK4A, MLH1, and THBS1. Apparently similar methylator phenotypes were described in breast,24 brain, bladder, and prostate cancers.25 Preliminary data suggest the presence of such hypermethylator phenotypes in hepatocellular carcinomas and acute lymphoblastic leukemia. Overall, then, hypermethylator phenotypes have been observed in most of the major types of human cancers. Because hypermethylation frequently correlates with differential gene expression compared with unmethylated tumors (and normal tissues), these data point to the existence of subsets of cancers with markedly different expression profiles. In AML, the different methylation categories appear to identify clinically distinct groups. The inverse correlation between methylation and age is particularly interesting because much previous work has indicated that, in epithelial cells, CpG-island methylation actually increases with age.26 One possible explanation for this finding is that methylation reflects specific carcinogenic insults that lead to neoplastic transformation. Indeed, previous studies have suggested different methylation profiles in experimental lung tumors induced by various carcinogens,27 and preliminary studies suggest correlations between CpG-island methylation and specific carcinogenic exposures in hepatocellular carcinomas. If this hypothesis is correct, it would suggest that AML in the elderly is a substantially different disease from AML in young people; this is supported by observations of vastly different cytogenetic changes and clinical courses in these patients. Our results suggest then that methylation could be useful in the further molecular classification of AML in the hope of identifying clinically distinct subgroups. The cause of hypermethylation in AML remains unclear. One proposed mechanism of this is up-regulation of DNA methyltransferase. Melki et al28 reported an elevated level of expression of DNA methyltransferase in acute leukemias, but were unable to relate it to degrees of aberrant methylation.15 These results were consistent with other studies that showed aberrant methyltransferase activity in various types of tumors,6 but little evidence of correlation between either mRNA levels or enzyme activity and CpG island methylation.29 Other factors, such as loss of trans-acting factors protecting CpG island,30 aberrant recruitment of DNA-methyltransferases to CpG islands,31 or alterations in demethylase gene/activity,32 may also participate in aberrant CpG-island methylation. Finally, as mentioned above, hypermethylation may reflect specific carcinogenic insults. Of special relevance to AML, radiation exposures have been associated with high rates of ER methylation in rodent models of lung cancer.27 Given that CpG-island methylation is deregulated globally in subsets of
AML, it becomes difficult to assign functional significance to each of
these events. Indeed, while it has been argued that hypermethylation in
cancer suggests a tumor-suppressor role for the affected
genes,10 many genes affected by hypermethylation in AML
are not expressed in hematopoietic cells, and even genes with clear-cut
oncogene function have been shown to be hypermethylated in selected
cases. Thus, it is probably more accurate to think of hypermethylation
in neoplasia as a deregulated process akin to microsatellite
instability, whereby many loci are affected but only a few have truly
functional significance. It is also worth noting that, although the
frequency of methylation of SDC4, GPR37,
PITX2, and MYOD was not different in
ERM+ and ERM One gene that may have functional significance in AML is
p15/INK4B. AML is one of the few neoplasms that show methylation of p15/INK4B.10 The p15/INK4B gene
plays an important role in transforming growth factor
Submitted August 9, 2000; accepted January 3, 2001.
Supported by a Translational Research Grant from the Leukemia and Lymphoma Society of America, a Research Grant from the American Cancer Society, and National Institutes of Health grants CA-38926 and CA-32102 to the Southwest Oncology Group. M.T. is a postdoctoral fellow of the Japan Society for the Promotion of Science.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jean-Pierre Issa, The MD Anderson Cancer Center, Box 428, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: jpissa{at}mdanderson.org.
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G. Garcia-Manero Modifying the Epigenome as a Therapeutic Strategy in Myelodysplasia Hematology, January 1, 2007; 2007(1): 405 - 411. [Abstract] [Full Text] [PDF]
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G. Garcia-Manero, H. M. Kantarjian, B. Sanchez-Gonzalez, H. Yang, G. Rosner, S. Verstovsek, M. Rytting, W. G. Wierda, F. Ravandi, C. Koller, et al. Phase 1/2 study of the combination of 5-aza-2'-deoxycytidine with valproic acid in patients with leukemia Blood, November 15, 2006; 108(10): 3271 - 3279. [Abstract] [Full Text] [PDF]
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A. Kieusseian, J. Chagraoui, C. Kerdudo, P.-E. Mangeot, P. J. Gage, N. Navarro, B. Izac, G. Uzan, B. G. Forget, and A. Dubart-Kupperschmitt Expression of Pitx2 in stromal cells is required for normal hematopoiesis Blood, January 15, 2006; 107(2): 492 - 500. [Abstract] [Full Text] [PDF]
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K. N. Bhalla Epigenetic and Chromatin Modifiers As Targeted Therapy of Hematologic Malignancies J. Clin. Oncol., June 10, 2005; 23(17): 3971 - 3993. [Abstract] [Full Text] [PDF]
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R. van Doorn, W. H. Zoutman, R. Dijkman, R. X. de Menezes, S. Commandeur, A. A. Mulder, P. A. van der Velden, M. H. Vermeer, R. Willemze, P. S. Yan, et al. Epigenetic Profiling of Cutaneous T-Cell Lymphoma: Promoter Hypermethylation of Multiple Tumor Suppressor Genes Including BCL7a, PTPRG, and p73 J. Clin. Oncol., June 10, 2005; 23(17): 3886 - 3896. [Abstract] [Full Text] [PDF]
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D. C. Malins, K. M. Anderson, N. L. Polissar, G. K. Ostrander, E. T. Knobbe, V. M. Green, N. K. Gilman, and J. L. Spivak Models of granulocyte DNA structure are highly predictive of myelodysplastic syndrome PNAS, April 6, 2004; 101(14): 5008 - 5011. [Abstract] [Full Text] [PDF]
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S. Zheng, X. Ma, L. Zhang, L. Gunn, M. T. Smith, J. L. Wiemels, K. Leung, P. A. Buffler, and J. K. Wiencke Hypermethylation of the 5' CpG Island of the FHIT Gene Is Associated with Hyperdiploid and Translocation-Negative Subtypes of Pediatric Leukemia Cancer Res., March 15, 2004; 64(6): 2000 - 2006. [Abstract] [Full Text] [PDF]
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M. T. Voso, A. Scardocci, F. Guidi, G. Zini, A. Di Mario, L. Pagano, S. Hohaus, and G. Leone Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes Blood, January 15, 2004; 103(2): 698 - 700. [Abstract] [Full Text] [PDF]
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A. F. List, J. Vardiman, J.-P. J. Issa, and T. M. DeWitte Myelodysplastic Syndromes Hematology, January 1, 2004; 2004(1): 297 - 317. [Abstract] [Full Text] [PDF]
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M. Daskalakis, T. T. Nguyen, C. Nguyen, P. Guldberg, G. Kohler, P. Wijermans, P. A. Jones, and M. Lubbert Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment Blood, September 26, 2002; 100(8): 2957 - 2964. [Abstract] [Full Text] [PDF]
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G. Garcia-Manero, J. Daniel, T. L. Smith, S. M. Kornblau, M.-S. Lee, H. M. Kantarjian, and J.-P. J. Issa DNA Methylation of Multiple Promoter-associated CpG Islands in Adult Acute Lymphocytic Leukemia Clin. Cancer Res., July 1, 2002; 8(7): 2217 - 2224. [Abstract] [Full Text] [PDF]
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G. Garcia-Manero, C. Bueso-Ramos, J. Daniel, J. Williamson, H. M. Kantarjian, and J.-P. J. Issa DNA Methylation Patterns at Relapse in Adult Acute Lymphocytic Leukemia Clin. Cancer Res., June 1, 2002; 8(6): 1897 - 1903. [Abstract] [Full Text] [PDF]
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J. Roman-Gomez, J. A. Castillejo, A. Jimenez, M. G. Gonzalez, F. Moreno, M. d. C. Rodriguez, M. Barrios, J. Maldonado, and A. Torres 5' CpG island hypermethylation is associated with transcriptional silencing of the p21CIP1/WAF1/SDI1 gene and confers poor prognosis in acute lymphoblastic leukemia Blood, April 1, 2002; 99(7): 2291 - 2296. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
.058 for each gene). Hypermethylation of
MYOD1, PITX2, GPR37, and
SDC4 was frequently found in AML (47% to 64% of
patients). For each of these genes as well, methylation density was
positively correlated with ERM density (correlation coefficients 0.43 to 0.69, P 
indicates lanes where reverse transcriptase was omitted (negative
controls).
(TGF-