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Prepublished online as a Blood First Edition Paper on February 13, 2003; DOI 10.1182/blood-2002-08-2466.
NEOPLASIA
From the Department of Leukemia, University of Texas
M.D. Anderson Cancer Center, Houston, TX; and the First Department of
Internal Medicine and the Department of Molecular Biology, Cancer
Research Institute, Sapporo Medical University, Sapporo,
Japan.
P57KIP2 is a cyclin-dependent kinase inhibitor silenced in a
variety of human malignancies. DNA methylation of a region surrounding the transcription start site of p57KIP2 was found in acute lymphocytic leukemia (ALL)-derived cell lines. Methylation of this region correlated with gene silencing, and treatment of methylated/silenced cell lines with 5-aza-2'-deoxycytidine resulted in gene re-expression. P57KIP2 was methylated in 31 (50%) of 63 patients with newly diagnosed ALL, and in 11 (52%) of 21 patients with relapsed ALL. In 5 of them
(25%), methylation was acquired at relapse. No association was
observed between methylation of p57KIP2 alone and clinical-biologic characteristics studied, including overall survival (OS) or
disease-free survival. Methylation of multiple genes in a cell-cycle
regulatory pathway composed of p73, p15, and p57KIP2 occurred in 22%
of Philadelphia chromosome (Ph)-negative patients.
Ph-negative patients with methylation of 2 or 3 genes of this pathway
had a significantly worse median OS compared with those with
methylation of 0 or 1 gene (50 vs 467 weeks, respectively;
P = .02). Our results indicate that p57KIP2 is
frequently methylated in adult patients with ALL, and that inactivation of a pathway composed of p73, p15, and p57KIP2 predicts for poor prognosis in Ph-negative patients.
(Blood. 2003;101:4131-4136) Aberrant methylation of promoter-associated
cytosine guanine pair (CpG) islands (DNA methylation) is an
epigenetic modification of DNA frequently observed in human
malignancies,1 including leukemias.2 This
phenomenon is particularly prevalent in acute lymphocytic leukemia
(ALL) where it has been shown that 80% of patients have methylation of
at least one promoter-associated CpG island both at initial
presentation3 and at relapse.4
P57KIP2 is located on chromosome 11p15, a region frequently deleted in
human cancers that has been associated with the Beckwith-Wiedemann syndrome.5-7 It encodes a cyclin-dependent kinase
inhibitor (CDKI) that belongs to the CIP/KIP family and is
considered a putative tumor suppressor gene. Evidence for this stands
from experiments demonstrating that p57KIP2 inhibits cell
transformation,8 and that lack of p57KIP2 results in
altered cell differentiation and proliferation in mice.7
Decreased expression of p57KIP2 has been observed in different
epithelial malignancies,9-12 but p57KIP2 mutations are
rare.13 This suggests that epigenetic alterations, including DNA methylation, may have a role in aberrant silencing of
p57KIP2. Recently, several groups have shown that p57KIP2 is aberrantly
methylated in hematopoietic malignancies.14,15 In particular, Kikuchi et al14 have shown that a region in
close proximity to the transcriptional start site ( To study the prevalence and clinical and biologic implications of
p57KIP2 methylation in ALL, we have analyzed its methylation status in
a cohort of adult ALL patients whose methylation patterns have been
previously characterized.3 Our results indicate that aberrant methylation of p57KIP2 is a frequent phenomenon in adult ALL
both at initial presentation and at relapse in adult ALL, and that
methylation of a cell-cycle regulatory pathway involving p73, p15,
and p57KIP2 identified a subgroup of patients with Philadelphia chromosome (Ph)-negative disease with poor prognosis.
Cell lines
Patients
DNA extraction and bisulfite modification Pretreatment samples consisted of cell suspensions obtained from bone marrow aspiration procedures. Median percentage of leukemic blasts was 89% (range, 22%-99%), indicating that the cell population analyzed is representative of the predominant malignant leukemic cell clone.DNA was extracted from these samples and cell lines using standard phenol-chloroform methods. DNA from relapsed samples, and paired initial presentation samples, was extracted from paraffin-embedded bone marrow biopsies. Prior to DNA extraction, these samples were deparaffinazed using xylene and ethanol followed by digestion with proteinase K. After extraction, DNA was modified with bisulfite. Bisulfite induces deamination of unmethylated cytosines converting unmethylated CpG sites to uracil guanine pair (UpG) without modifying methylated sites. Bisulfite treatment of genomic DNA was performed as described.18 DNA (2 µg) was used. DNA was denatured in 0.2 N NaOH at 37°C for 10 minutes and incubated with 3 M Na-bisulfite at 50°C for 16 hours. DNA was then purified using the Wizard cleanup system (Promega, Madison, WI) and desulfonated with 0.3 N NaOH at 25°C for 5 minutes. DNA was then precipitated with ammonium acetate and ethanol, washed with 70% ethanol, dried, and resuspended in H2O. Further information can be obtained from http://www3.mdanderson.org /leukemia/methylation.19 Methylation analysis To analyze the methylation status of p57KIP2, we have used 2 methods. First, we analyzed all cell lines and patient samples with bisulfite polymerase chain reaction (PCR) followed by restriction enzyme digestion (combined bisulfite restriction analysis; COBRA).20 This method allows the quantification of the methylation level, that is the ratio of methylated (restricted) to unmethylated (unrestricted) PCR products. PCR reactions were carried in 50 µL reactions. In each reaction, 2 µL bisulfite-treated DNA was used, as well as 1.25 mM deoxynucleoside triphosphate, 6.7 mM MgCl2, 5 µL PCR buffer, 1 nmol primers, and 1 unit Taq polymerase. Table 2 summarizes PCR conditions, primer sequences, and GenBank sequence numbers. Figure 1A shows a map of the p57KIP2 promoter-associated CpG island indicating the position of the primers used in relation to the transcription start site. PCR products were digested with EcoRI or HhaI and were separated in nondenaturing polyacrylamide gels. Gels were stained with ethidium bromide. The proportion of methylated versus unmethylated product (digested vs undigested, respectively) was quantitated by densitometric analysis, determining the level of methylation. Densitometric analysis was performed using a BioRad Geldoc 2000 digital analyzer (Hercules, CA) equipped with the Quantity One version 4.0.3 software (BioRad, Hercules, CA).
To confirm the COBRA results we performed bisulfite sequencing in unselected cases. Bisulfite PCR fragments were cloned into pCR2 vector using a TOPO-cloning kit (Invitrogen, San Diego, CA). DNA sequencing was performed using an ABI377 automated sequencer (Applied Biosystems, Foster City, CA). Methylation analysis of p21CIP121 and p14ARF was performed using COBRA. Conditions are summarized in Table 2. Methylation analysis of p15 and p73 has been described elsewhere.3 Analysis of gene expression Gene expression was analyzed using reverse transcriptase (RT)-PCR. Total RNA (5 µg) was treated for 30 minutes with DnaseI, which was inactivated with DNA Free Kit (Ambion, Austin, TX). Reverse transcription was performed using Superscript II (Life Technologies, San Diego, CA), and it was followed by PCR using conditions summarized in Table 2. Glyceraldehyd-3-phospate dehydrogenase (GAPDH) was used as control as described.22 Of these reactions, 10-µL aliquots were run in 2% agarose gels and stained with ethidium bromide.Statistical methods To analyze association of methylation between p57KIP2 and clinical biologic characteristics, we used the Spearman rank correlation coefficient. Variables analyzed included age, sex, white blood cell count (WBC), hemoglobin levels, platelet count, albumin levels, 2-microglobulin levels,
cytogenetics, immunophenotype, and systemic and central nervous system
(CNS) risk at presentation.17 Complete remission (CR)
rate, disease-free survival (DFS), and overall survival (OS) have been
defined elsewhere.17 Estimates of 5-year DFS and OS were
based on the Kaplan-Meier method, and differences were tested using the
log-rank test. To perform this analysis a sample was considered
methylated for p57KIP2 or p15 if the level of methylation was 15% or
higher. For p73 we used methylation-specific PCR.3,23 For this gene, a sample was considered methylated if a band was observed in the methylated PCR reaction. We used a
t test for dependent samples to compare methylation of
paired matched samples at initial presentation and at relapse. As in a
previous study,4 methylation was considered stable if it was higher or lower than 5% both at initial presentation and
at relapse. Increased methylation was considered if it was lower than
5% at initial presentation, and higher than that at relapse. Decreased
methylation was considered if it was higher than 5% at initial
presentation and lower than that at relapse.
Methylation of p57KIP2 in ALL-derived cell lines The following 8 cell lines have been studied here: Jurkat, CCRF-CEM, BALL1, CCRF-HSB2, Malt4, SupT1, PEER, and TALL1 (Figure 1B). There were 2 cell lines, Jurkat and BALL1, that had no methylation of p57KIP2. The other 6 cell lines were methylated. Median methylation level was 45.8% (range, 6%-95%). Of these 6 cell lines, 4 were densely methylated (Malt4, SupT1, PEER, and TALL1), median methylation level 65.5% (range, 28%-95%). Methylation results were confirmed by bisulfite sequencing (Figure 2). There were 4 densely methylated cell lines (TALL1, PEER, SupT1, and Malt4) that had methylation of the majority of CpG sites in the 12 alleles evaluated, whereas the 2 partially methylated cell lines, CCRF-HSB2 and CCRF-CEM, showed variable methylation.
As demonstrated by RT-PCR analysis, methylation was strongly associated with gene silencing (Figure 1C). Only 1 of the 4 densely methylated cells lines expressed p57KIP2, whereas the 2 unmethylated and the 2 partially methylated cell lines did. To further confirm that methylation of the region studied here was associated with gene silencing, we treated 2 densely methylated cell lines (SupT1 and PEER) with 5-aza-2' deoxycytidine. Exposure of these 2 cell lines to this agent resulted in restoration of p57KIP2 gene expression (Figure 1D). DNA methylation of p57KIP2 in patients with ALL We next studied p57KIP2 methylation in patients with ALL. There were 36 patients (50%) who had a p57KIP2 level of methylation higher than 5%. Mean p57KIP2 methylation was 14% (range, 0%-100%). Table 3 summarizes the range of methylation of p57KIP2. Figure 3A shows representative examples of p57KIP2 methylation at initial presentation.
To study the role of p57KIP2 methylation at relapse, we analyzed paired matched samples obtained at initial presentation and at relapse from 21 patients who initially had achieved CR. Thirteen patients (65%) had no change in the methylation status: 9 had no methylation (methylation level, < 5%) at both end points, and 4 were methylated (> 5%) at both end points. There were 5 patients (25%) who had increased methylation at relapse compared with initial presentation, and 2 patients (10%) had demethylation at relapse (P = .39). Figure 3B shows representative examples of p57KIP2 methylation at initial presentation and at relapse. To confirm the results obtained with COBRA, we performed bisulfite
sequencing of 9 cases (Figure 4). As in
cell lines, we found an excellent concordance between COBRA and
bisulfite sequencing results. There were 2 completely
unmethylated cases who had methylation of none of the CpG sites
involved, whereas a case that was completely methylated by COBRA was
found to have all CpG sites analyzed methylated.
We also studied the methylation status of p21CIP1, another member of the CIP/KIP family, and p14ARF. Analyzed were 30 patients, none of whom had evidence of either p21CIP121 or p14ARF methylation (Figure 3C-D). Using a methylation level of 15% or methylation-specific PCR (MSP) positivity, p15 and p73 were methylated in 16 patients, respectively. No association was found between methylation of p73, p15, and p57KIP2. There were 33 patients (45%) who had methylation of 0 genes; 28 (39%), of 1 gene; 8 (11%), of 2 genes; and 3 (4%), of 3 genes. Correlation between methylation of p57KIP2 and gene expression in ALL patients To assess the impact of p57KIP2 methylation on gene expression, we performed RT-PCR using RNA extracted from cell suspensions obtained from bone marrow aspiration procedures obtained at the time of initial presentation. Analyzed were 11 patients. Of the 6 patients with methylated p57KIP2, only 1 expressed p57KIP2. In contrast, 4 of 5 unmethylated cases expressed p57KIP2 (P = .03) (Figure 5).
Clinical and pathologic correlates To assess the clinical impact of p57KIP2 methylation, we have analyzed the correlation of p57KIP2 methylation with the following variables: age, sex, WBC, hemoglobin levels, platelet count, albumin levels,2-microglobulin levels, cytogenetics,
immunophenotype, and systemic and CNS risk at
presentation.17 No association was observed between any of
these variables and p57KIP2 methylation. We have also analyzed the
impact of p57KIP2 on DFS and OS. No differences were observed among
patients with or without p57KIP2 methylation.
In a previous analysis,3 we observed a trend toward a
worse prognosis in patients with methylation of p73 and p15. Because p57KIP2, p73, and p15 are all involved in cell-cycle regulation, we
decided to investigate the prognostic impact of methylation of these 3 genes in our patients. Table 4 summarizes
the impact of methylation of patients with methylation of 0 to 1 gene
or 2 to 3 genes of this triad of p57KIP2, p73, and p15 including and
excluding patients with Ph-positive disease. Although both OS and DFS
were always worse for patients with methylation of at least 2 genes,
this reached statistical significance for the subgroup of patients with
Ph-negative disease. The median OS for patients with methylation of 0 to 1 gene was 467 weeks, whereas it was 50 weeks for those with
methylation of 2 to 3 genes (P = .023) (Figure
6). This last subgroup accounted for 22%
of Ph-negative patients.
We have identified that aberrant methylation of p57KIP2 is a common abnormality in ALL. First, we showed that this abnormality is frequent in ALL-derived cell lines. Methylation of a region in close proximity to the transcription start site of p57KIP2 was associated with gene silencing. Treatment of methylated/silenced ALL cell lines with a hypomethylating agent resulted in gene re-expression. As was originally found by Kikuchi et al,14 this finding strongly supports the notion that methylation of the promoter region studied here is important for the regulation of transcriptional activity of p57KIP2. Further, we have found that methylation of p57KIP2 occurs in 50% of adult patients with ALL, with 30% of them having dense methylation (> 20%). The results obtained with the bisulfite PCR method were confirmed in a subset of patients by bisulfite sequencing. This last method allows the analysis of the methylation status of each CpG site in a given DNA sequence.18 As was shown in cell lines, methylation of the region studied here inversely correlated with the level of gene expression. This evidence strongly suggests that methylation of the region analyzed here is important for the control of p57KIP2 gene expression also in vivo. It is important to note that several lines of evidence indicate that p57KIP2 is an imprinted gene,5,6 but the role of promoter DNA methylation in the control of imprinting is unknown. Indeed, the evidence shown here and that of 2 other groups14,15 indicate that p57KIP2 is expressed in nonneoplastic tissues and argue against any role of DNA methylation in the regulation of p57KIP2 imprinting. It has been proposed that aberrant methylation of target genes may have a role in resistance/relapse mechanisms in leukemia.2 We had previously studied the methylation patterns at initial presentation and at relapse in a cohort of patients with ALL.4 Our results demonstrated that methylation patterns were stable in a majority of patients, but that a significant minority, 28%, acquired methylation changes at relapse, affecting, in particular, cell-cycle regulatory genes such as p15 and p16. Methylation of p57KIP2 followed the same pattern as described for p16 and p15,4 remaining stable in 65% of patients, increasing in 25%, and decreasing in 10%. We had previously observed a trend toward a worse prognosis for patients with methylation of both p73 and p15.3 p73 is a p53 family member that is frequently methylated and silenced in lymphoid malignancies.23,24 By acting as a p53 homologue, activation of p73 may lead toward cell-cycle arrest.25 This phenomenon is mediated in part by CDKIs, such as p57KIP2.25,26 Indeed, p57KIP2 has been shown to be up-regulated by p73.27 Based on this knowledge, we studied the impact of methylation of p73, p15, and p57KIP2 in this cohort of homogeneously treated ALL patients.17 Results of this analysis revealed that methylation of 2 or 3 genes of this triad occurs in close to 25% of patients with Ph-negative disease, and that patients with this abnormality had a very poor prognosis. It could be argued that this finding is an artifact based on arbitrary gene selection. Several facts argue against this. First is the lack of an association between methylation of these 3 genes (data not shown), and the fact that other genes, including p14ARF, p21CIP1,20 and p16,3 that are also involved in this cell-cycle regulatory pathway are not methylated in these patients. Second is the need for methylation of more than one gene to alter the prognosis of these patients. This cooperative effect could be explained by the redundancy in the function of these genes,25,26 and is also supported by the lack of a fully malignant phenotype in animal models in which these genes were individually deleted.7,28,29 This observation suggests that at least 2 of these genes are required to induce a more aggressive disease phenotype. The implications of these findings are several. First, methylation of p57KIP2 characterizes a molecular pathway prevalently altered in adult ALL patients. Second, methylation of 2 or 3 genes of this pathway may allow the identification of a subgroup of patients with Ph-negative ALL and extremely poor prognosis analogous to Ph-positive disease. It should be noted that due to the relatively small sample size, these results should be considered preliminary and need to be confirmed in a larger cohort of patients. The early identification of these patients may allow the development of targeted therapies for this subgroup of patients. These could include the incorporation of hypomethylating agents, such as 5-aza-2'deoxycytidine, to chemotherapy programs in ALL, and the introduction of early allogeneic stem cell transplantation in first complete remission for these high-risk patients, as is recommended for patients with Ph-positive disease. In summary, we have identified methylation of p57KIP2 as a common abnormality in adult patients with ALL, and a cell-cycle regulatory pathway involving p73, p15, and p57KIP2 that when methylated confers poor prognosis to patients with Ph-negative disease.
Submitted August 12, 2002; accepted January 14, 2003.
Prepublished online as Blood First Edition Paper, February 13, 2003; DOI 10.1182/blood-2002-08-2466.
G.G.-M. is the recipient of a Career Development Award from the American Society of Clinical Oncology and the Physician-Scientist Program Award. M.T. is supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.
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: Guillermo Garcia-Manero, Department of Leukemia, M.D. Anderson Cancer Center, Box 428, 1515 Holcombe Blvd, Houston, TX, 77030; e-mail: ggarciam{at}mdanderson.org.
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© 2003 by The American Society of Hematology.
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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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 H. Yang, K. Hoshino, L. Xiao, G. Rosner, and G. Garcia-Manero Aberrant DNA Methylation of a Cell Cycle Regulatory Pathway Composed of p73, p15 and P57kip2 Is Associated with Poor Prognosis in Adult Patients with Philadelphia Chromosome Negative Acute Lymphocytic Leukemia (ALL). Blood (ASH Annual Meeting Abstracts), November 16, 2005; 106(11): 481 - 481. [Abstract]
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S.-Q. Kuang, X. Ling, B. Sanchez-Gonzalez, H. Yang, and G. Garcia-Manero The Cyclin-Dependent Kinase Inhibitor p57KIP2 Functions as a Tumor Suppressor Gene in Human Leukemia. Blood (ASH Annual Meeting Abstracts), November 16, 2005; 106(11): 1604 - 1604. [Abstract]
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 N Diaz-Meyer, Y Yang, S N Sait, E R Maher, and M J Higgins Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome J. Med. Genet., August 1, 2005; 42(8): 648 - 655. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
300 to + 400
base pair [bp]) of p57KIP2 is aberrantly methylated in different
neoplastic cell lines, including several of hematopoietic origin, but
not in nonneoplastic tissues. Treatment of methylated/silenced cell lines with 5-aza-2'-deoxycytidine, an inhibitor of DNA
methyltransferases with potent hypomethylating activity,
) indicate
unmethylated CpG sites; black circles (
), methylated ones. Each row
represents an allele. The numbers in parentheses indicate the
level of methylation as determined by COBRA.
