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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2985-2990
Methylation of the p15INK4b Gene in Myelodysplastic
Syndromes Is Frequent and Acquired During Disease Progression
By
Bruno Quesnel,
Gaelle Guillerm,
Rodolphe Vereecque,
Eric Wattel,
Claude Preudhomme,
Francis Bauters,
Michael Vanrumbeke, and
Pierre Fenaux
From the Service des maladies du sang and the Laboratoire
d'hematologie, CHU Lille, Lille, France; and INSERM U124, Lille,
France.
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ABSTRACT |
p15INK4b gene is an inhibitor of cyclin-dependent kinase
(CDK) 4 and CDK6 whose expression is induced by transforming growth
factor (TGF) . Recent reports suggest frequent methylation of the
p15INK4b gene promoter in leukemias, and it has been
proposed that this methylation could be necessary for leukemic cells to
escape TGF regulation. We investigated the methylation status of
p15INK4b gene in 53 myelodysplastic syndromes (MDS) cases,
including nine that had progressed to acute myeloid leukemia (AML),
using a recently described sensitive method where polymerase chain
reaction (PCR) is preceded by bisulfite modification of DNA
(methylation specific PCR). p15INK4b methylation was
observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients
with greater than 10% bone marrow blasts had p15INK4b
methylation (including all nine patients who had progressed to AML) as
compared with none of MDS patients with <10% bone marrow blasts. No
correlation between karyotypic abnormalities and methylation status was
found. Patients with p15INK4b methylation had a worse
prognosis, but the prognostic significance of p15INK4b
methylation was no more found by multivariate analysis, due to its
strong correlation to the percentage of marrow blasts. In 10 MDS cases,
sequential DNA samples were available. In five of them, methylation of
the p15INK4b gene was detected at leukemic transformation,
but not at diagnosis. Our results showed that methylation of the
p15INK4b gene in MDS is correlated with blastic bone marrow
involvement and increases with disease evolution toward AML. It
suggests that proliferation of leukemic cells might require an escape
of regulation of the G1 phase of the cell cycle, and possibly of TGF
inhibitory effect.
| |
INTRODUCTION |
MYELODYSPLASTIC syndromes (MDS) are
clonal stem cell disorders characterized by ineffective hematopoiesis
leading to blood cytopenias and by a high incidence of progression to
acute myeloid leukemia (AML). MDS are heterogeneous and include
refractory anemia (RA) and refractory anemia with ring sideroblasts
(RARS), with low incidence of progression toward AML, refractory anemia
with excess blasts (RAEB), and refractory anemia with excess blasts in
transformation (RAEB-t), which often progress to AML, and chronic myelomonocytic leukemia (CMML) with an intermediate risk of
progression.1 MDS, therefore, represents an excellent model
of leukemic development with a progressive increase of blastic bone
marrow involvement, but genetic events that lead to this evolution are
still not identified. Chromosomal abnormalities have been described in
MDS and they are generally associated with resistance to chemotherapy
and short survival, but few molecular abnormalities, such as C-fms,
N-ras, and p53 mutations, have been identified in MDS.2-4
p15INK4b and p16INK4a proteins are cell cycle
regulators involved in the inhibition of G1 phase
progression.5 p15INK4b associates with
cyclin-dependent kinase (CDK) 4 and 6, and cyclin D-CDK4/6 complexes
and inhibits their kinase activities.6,7 Transforming
growth factor (TGF) inhibitory effects on normal hematopoietic
progenitors are mediated by many different mechanisms including
downregulation of CDK4 synthesis and increase in p15INK4b
gene expression and p15INK4b protein
stability.8-10 It has been previously reported that loss of
sensitivity of leukemic cell lines to growth inhibition by TGF is
correlated with inactivation of p15INK4b. Recent studies
have shown that p15INK4b gene is inactivated by 5
CpG island methylation of the promoter region in most AML and acute
lymphoblastic leukemia (ALL).11-13
To investigate the role of p15INK4b gene methylation in the
progression of MDS, we analyzed the methylation status of
p15INK4b gene in 53 MDS cases, using the recently described
methylation specific PCR (MSP) method.14 This method, which
is more sensitive than Southern Blot analysis, seemed particularly
useful in MDS, where the blastic bone marrow involvement is variable
and sometimes very low.
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MATERIALS AND METHODS |
Patients.
We investigated the methylation status of the p15INK4B gene
in 53 de novo MDS cases, including 10 RA, two RARS, five RAEB-t, 18 RAEB, nine CMML, and nine AML evolved from MDS (MDS-AML). Their characterization is summarized in Table 1.
In 10 MDS (two RAEB-t, seven RAEB, and one CMML with 26%, 23%, 15%,
12%, 12%, 11%, 7%, 9%, 8%, and 4% marrow blasts on first
analysis, respectively), sequential DNA samples were available.
Twenty-five cases of de novo AML were also studied. Genomic DNA was
extracted from mononuclear cells after bone marrow aspirates and
Ficoll-Hypaque sedimentation. T cells from a RAEB case with 17% bone
marrow blasts were isolated using magnetic Dynabeads-CD2 (Dynal, Oslo,
Norway). Controls included normal DNA obtained from healthy volunteers
after informed consent, DNA from K562 cell line (American Type Culture
Collection [ATCC], Rockville, MD, no. CCL243), which is
homozygously deleted for the p15INK4b gene, and positive
control DNA extracted from Raji cell line (ATCC, no. CCL86), previously
reported to have p15INK4B gene methylation.11
MSP.
This method, initially described by Herman et al,14
consists of two steps: modification of DNA by sodium bisulfite
converting all unmethylated, but not methylated cytosines to uracil;
and subsequent amplification with primers specific for methylated versus unmethylated DNA.
Bisulfite modification.
DNA (2 µg) in a volume of 40 µL was denatured by addition of 10 µL 1 mol/L NaOH (final concentration 0.2 mol/L) for 10 minutes at
37°C. Next, 30 µL of 10 mmol/L hydroquinone (Sigma, St Louis, MO)
and 520 µL of 3 mol/L sodium bisulfite (Sigma) at pH 5 were added and
mixed and samples were incubated under mineral oil at 50°C for 16 hours. To meet strict anaerobic conditions, all of these manipulations
were performed under nitrogen in an anaerobic chamber. Modified DNA was
purified using the Wizard purification resin and the Vacuum Manifold
(Promega, Madison, WI), and eluted into 50 µL H2O. Final
desulphonation was achieved by adding 25 µL of 1 mol/L NaOH (final
concentration 0.3 mol/L) at room temperature for 5 minutes. After
ethanol precipitation and dilution in 50 µL H2O, samples
were stored at  20°C before use.
PCR amplification and sequencing.
MSP, based on sequence differences resulting from bisulfite
modification, was performed with the primer sets designed by Herman et
al.14 Briefly, sequences of those primers recognize the
region of the p15INK4B gene, which contain frequent
cytosine to distinguish unmodified from modified DNA and CpG pairs near
the 3 end of the primers to ensure maximal discrimination
between methylated and unmethylated DNA. The multiple mismatches in
these primers allow amplification only from the intended template.
Primer sets were provided from Eurogentec (Seraing, Belgium). The PCR
reaction contained 1× PCR buffer (6.5 mmol/L MgCl2, 10 mmol/L Tris HCl, pH 9, 50 mmol/L KCl, 0.1% triton X100, 0.2 mg/mL
bovine serum albumin [BSA]), deoxynucleotide triphosphates
(dNTPs; each at 0.5 mmol/L), 20 pmol of each primer, 5%
dimethyl sulfoxide (DMSO), 1 U of Taq polymerase
(Oncor-Appligene, Gaithersburg, MD), and bisulfite modified DNA (100 to
200 ng) in a final volume of 80 µL. Reactions were hot started using
Ampliwax 100 beads (Perkin Elmer, Foster City, CA). For each reaction,
a lower layer containing PCR buffer, dNTP, DMSO, diluted in a final
volume of 30.5 µL, was pipetted in a reaction tube and AmpliWax 100 beads were added. After incubation at 85°C for 3 minutes and
25°C for 3 minutes, leading to formation of a solid wax layer, an
upper layer containing Taq polymerase, MgCL2, PCR buffer, 100 ng
modified DNA, diluted in 49.5 µL was added. Amplification were
performed in a MJ research MiniCycler thermal cycler (MJ Research,
Wattertown, MA). A first step of denaturation at 95°C
for 4 minutes then 98°C for 30 seconds, was followed by 35 cycles
of amplification (30 seconds at 95°C, 30 seconds at 60°C, 30 seconds at 72°C), and by a final 10-minute extension at 72°C.
Controls without DNA were performed for each set of PCR reactions. PCR
products (20 µL) were loaded on 2% agarose gels stained with
ethidium bromide and visualized under ultraviolet (UV) illumination
directly and with a gel scan software analysis system (Bio-Print, Marne
la Vallée, France).
To confirm the specificity of MSP, five PCR products obtained after
amplification with primer pairs specific for methylated DNA (p15M) or
unmethylated DNA (p15U) were sequenced. Ampligens purified with
Centricon-100 Concentrator columns were sequenced with the ABI Prism
dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin
Elmer) according to manufacturer's recommendations, and samples were
analyzed with the ABI Prism 310 automatic sequencer (Perkin Elmer).
Resulting sequences were compared with wild-type sequence (Genbank
accession number, S75756) using Sequence Navigator software (Perkin
Elmer).
Statistical analysis.
Comparisons were made with Fisher's exact test and the
 2 test. Multivariate analysis was performed with a Cox
model. All of these analyses were performed on SPSS 6.2 analysis
software (SPSS, Chicago, IL).
| |
RESULTS |
MSP of 25 de novo AML demonstrated p15INK4b methylation, as
shown by amplification with p15M primer pair in 18 (72%) of the
patients analyzed. Those samples could also be amplified with p15U
primer pair, due to sample contamination by nonblastic cells (whose
percentage ranged from 5% to 19% cells in bone marrow samples).
Sequencing of five p15M PCR products (from one AML and four MDS)
confirmed that all cytosines had been converted in thymidines except in CpG dinucleotides, where cytosines remained unchanged
(Fig 1). Normal unmethylated DNA from the
K562 cell line could not be amplified with p15M primer pair, but the
Raji cell line, which is methylated for the p15INK4b gene,
gave a strong amplification. Dilution of the Raji cell line showed that
detection of as little as 1 ng of methylated DNA was possible,
confirming the sensitivity of MSP (Fig 2).
All of these results were concordant with previous reports with MSP and/or p15INK4b gene methylation in
AML.11,12,14
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| Fig 1.
DNA sequence of p15M PCR product from a patient with RAEB
(upper sequence) as compared with Genbank sequence of
p15INK4b gene (lower sequence). All cytosines are converted
in thymidines except in methylated CpG dinucleotides.
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| Fig 2.
Amplification with p15M primer pair of Raji cell line
DNA, diluted in negative control DNA from a healthy volunteer. C,
amplification with p15 primer pair of a control DNA from a healthy
volunteer.
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In the 53 patients with MDS, amplification with p15M primer pair was
seen in 20 patients (Fig 3). Methylation
was exclusively seen in patients with at least 10% bone marrow blasts
(20 of 24 v 0 of 29 of those with less than 10% marrow blasts,
P < .001). In patients with at least 10% marrow blasts, 11 of 15 of those that were still in MDS phase (<30% marrow blasts)
were methylated, as compared with nine of nine of those that had
progressed to AML (>30% marrow blasts)(P = .6).
Amplification of DNA from T cells isolated from a patient with 17%
bone marrow blasts showed no evidence of methylation (Fig 3). Nine of
the 30 cases with normal karyotype or "favorable karyotype"
(isolated del5q or del20q) had p15INK4b methylation, as
compared with five of the 14 cases with other cytogenetic findings
(P = .73).
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| Fig 3.
Amplification of DNA from several MDS cases with p15M or
p15U primer pair and from T (Tc) and mononuclear bone marrow (BM) cells
isolated from a RAEB case*.
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Patients with p15INK4b gene methylation had a significantly
shorter survival than unmethylated cases (median 18 v 48 months, P = .049, log-rank test)
(Fig 4). The percentage of bone marrow blasts and French-American-British (FAB) type also had
prognostic value for survival by univariate analysis
(Table 2). In a Cox multivariate analysis,
the percentage of marrow blasts emerged as the only prognostic factor
for survival, p15INK4b methylation status being strongly
correlated with this parameter. Twelve patients (nine methylated and
three unmethylated) were treated with intensive chemotherapy. Five
patients achieved complete remission (CR), and four methylated patients
had failure; two patients achieved partial remission (PR) and one
unmethylated patient had failure.
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| Fig 4.
Actuarial survival of MDS patients according to the
p15INK4b gene methylation status (P = .049, log-rank test.)
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In 10 patients, sequential DNA samples were available. In five of them,
p15M amplification was negative at diagnosis, but became positive after
leukemic transformation. (Table 3). Four patients were found methylated on first analysis, but they already had
relatively high bone marrow blast counts (15% to 26%). The remaining
patient was unmethylated both at diagnosis and at leukemic transformation.
| |
DISCUSSION |
The progressive increase of bone marrow blast involvement in MDS
suggests that a fraction of clonal dysplastic cells loses its cell
cycle regulation and differentiation during evolution of the disease.
p16INK4a gene is frequently inactivated in cancer, by
homozygous deletion in ALL (especially T-ALL) and some solid tumors,
and by point mutation or methylation in other solid
tumors.15-17 p16INK4a deletion, point mutation,
and methylation, on the other hand, are not seen in AML and
MDS.17-19 On the contrary, methylation of CpG islands of
the p15INK4b promoter region has been recently reported to
occur at high frequency both in myeloid and lymphoid leukemias (88% of
AML and 71% of ALL), but not in lymphoma, chronic myeloid leukemia,
and in solid tumors.11,12,20
In this report, we analyzed p15INK4b gene hypermethylation
by a recently described MSP method.14 The sensitivity of
this method was 1%. This is higher than the sensitivity obtained with
Southern blot analysis, generally in the range of 5%, a technique
which also requires relatively large amounts of DNA. Such a high
sensitivity is probably important to obtain in MDS, where the
percentage of marrow blasts is often very low. Specificity of the MSP
technique, which we confirmed by direct sequencing, is probably higher
than that of classical PCR-based methylation assay, where amplification from primers flanking restriction site can be performed only if DNA
cleavage has been prevented by methylation. This latter method requires
a complete restriction, as any uncleaved DNA will be amplified by PCR
yielding a false positive result.
In this study, we observed a high incidence of p15INK4b
methylation in MDS (38%) confirming results from Uchida et
al19 (p15INK4b methylation in 16 of 32 MDS
cases analyzed). Methylation was found in bone marrow mononuclear
cells, but not in T lymphocytes from a patient with 17% bone marrow
blasts. Uchida et al observed p15INK4b methylation in 14 of
18 MDS patients with excess of blast, but not in T lymphocytes, and
only in one of 12 RA and RARS. However, their study was made by
Southern blot and PCR-based methylation assay, which appear to have
lower sensitivity and specificity, respectively, than the MSP method
used in the present work. We found p15INK4b methylation
exclusively in MDS with >10% bone marrow blasts. The high
sensitivity of MSP ruled out the possibility of a p15INK4b
methylation restricted to blast cells in MDS cases with fewer than 10%
marrow blasts. Thus, p15INK4b methylation appears to be
almost exclusively found in MDS with an excess of blasts. Precise
marrow blast counts, in MDS with an excess of marrow blasts,
particularly RAEB, were not available in the report of Uchida et al. In
our study, 10% marrow blasts was an important figure, as no cases of
p15INK4b methylation were seen below that threshold.
Interestingly, several scoring systems for prognosis in MDS have shown
that the 10% blasts threshold had an important clinical
significance.21,22 Indeed, most MDS with marrow blasts
below 5% and many of those with marrow blasts between 5% and 10%
have a relatively mild course, with unfrequent progression to AML. On
the contrary, MDS with greater than 10% blasts are generally
associated with severe blood cytopenias, very frequent transition to
AML, and short survival. Thus, the presence of p15INK4b
methylation appears to carry an important clinical significance in MDS
and had prognostic significance in univariate analysis in our study.
However, because of its high correlation with marrow blast percentage,
p15INK4b methylation lost its prognostic value for survival
in multivariate analysis, where only blast bone marrow involvement
emerged as an independent prognostic factor. The small number of
unmethylated patients treated by intensive chemotherapy precluded the
study of any correlation between response to treatment and methylation status.
By studying 10 patients where sequential DNA was available, we observed
in five patients that p15INK4b methylation was acquired
during disease progression. Four patients were methylated both at
diagnosis and at leukemic transformation, but they already had high
bone marrow blast count at diagnosis. Only one remained unmethylated
both at diagnosis and leukemic transformation. Uchida et al have also
reported progression in the methylation status in four of 10 patients
studied sequentially. All of these data support the hypothesis that
p15INK4b methylation and progression toward AML are linked
in MDS and that p15INK4b methylation is acquired during
disease progression.
Whether p15INK4b methylation in MDS is a specific event or
a simple consequence of a global hypermethylation activity remains to be determined. Indeed, hypermethylation of the calcitonin gene has also
been described in MDS, suggesting a more global increased methylation
activity in this group of disorders.23,24 However, the
calcitonin gene is close to the cell cycle inhibitor gene p57KIP2, which has been found to be
imprinted.25,26 Moreover, p15INK4b methylation
has also been reported in AML and ALL, and leukemic cell lines that are
nonsensitive to TGF inhibition generally have p15INK4b
methylation. Finally, Uchida et al in MDS, and other groups in AML and
ALL, did not find any methylation of the p16INK4a gene,
which is adjacent to the p15INK4b gene on 9p21 chromosome.
These findings strongly suggest that p15INK4b inactivation
is important and perhaps necessary for the proliferation and/or
differentiation block of marrow blast cells in myeloid malignancies.
Selective methylation of the p15INK4b gene could be a
specific mechanism of immature bone marrow cells to escape to G1 phase
regulation by TGF inhibitory effect.
| |
FOOTNOTES |
Submitted October 9, 1997;
accepted December 9, 1997.
Supported by the Ligue Contre le Cancer (Comité du Nord and
Comité du Pas de Calais), the Association de Recherche sur le Cancer, and The Centre Hospitalier Universitaire of Lille, France.
Address reprint requests to Bruno Quesnel, MD, PhD,
Service des maladies du sang, CHU Lille, 1 Place de Verdun, 59037 Lille, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract