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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2990-2992
BRIEF REPORT
Quantitative measure of c-abl and
p15 methylation in chronic myelogenous leukemia: biological
implications
TuDung T. Nguyen,
Ann F. Mohrbacher,
Yvonne C. Tsai,
John Groffen,
Nora Heisterkamp,
Peter
W. Nichols,
Mimi C. Yu,
Michael Lübbert, and
Peter A. Jones
From the Departments of Biochemistry & Molecular
Biology; Hematology/Oncology; Pathology; and Preventive Medicine,
USC/Norris Cancer Center, University of Southern California, Los
Angeles, CA; the Division of Hematology/Oncology,
Children's Hospital, Los Angeles, CA; and the Division of
Hematology/Oncology, the Medical University of Freiburg Medical Center,
Freiburg, Germany.
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Abstract |
We used a sensitive, quantitative bisulfite PCR assay, methylation
sensitive single nucleotide primer extension (Ms-SNuPE), to
measure methylation of the 5' CpG islands of c-abl and
p15 in chronic myelogenous leukemia (CML) patients during
progression. We found that the Pa promoter of c-abl was
methylated in 81% (17/21) of the white blood cells (WBCs) of CML
patients, which correlates with previous reports. In contrast, WBCs
from healthy donors, acute myelogenous leukemias, acute lymphocytic
leukemias, and myelodysplastic syndromes were unmethylated at the
c-abl Pa promoter locus. We also observed p15
hypermethylation in 24% (8/34) of CML cases. Methylation of the
p15 but not c-abl Pa promoters was associated with CML
progression (P = 0.047 vs 0.46), and the two events were
independently acquired. We conclude that de novo methylation of
c-abl and p15 both occur in CML, and analysis of DNA
methylation changes using the bisulfite-based MS-SNuPE assay allows
both a sensitive and quantitative assessment of these molecular events compared to other methods currently utilized.
(Blood. 2000;95:2990-2992)
© 2000 by The American Society of Hematology.
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Introduction |
Several reports have shown that the c-abl Pa
promoter is methylated in chronic myelogenous leukemia (CML), but
its significance as a prognostic indicator in the disease has been
controversial.1-3 It has also become accepted that
methylation of the 5' region of the p15 tumor suppressor
gene is not seen in CML,4 in contrast to other leukemia
types.5 The methods used to detect DNA methylation in
previous studies used the restriction endonuclease-based Southern analysis or polymerase chain reaction-based (PCR-based) assay, both of
which raise issues of sensitivity and specificity, as others have
already noted.3 We used sodium bisulfite-modified DNA
technologies to develop a sensitive and specific quantitative PCR
assay, methylation sensitive single nucleotide primer extension (Ms-SNuPE),6 to measure the methylation of both the
c-abl Pa and p15 5' regions in CML.7
Our studies demonstrate that the c-abl Pa promoter is
methylated in CML, but it is not associated with progression, and that
the p15 5' region can become hypermethylated in CML.
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Study design |
DNA was obtained from the peripheral blood and/or bone marrow of
Philadelphia chromosome-positive patients with CML who were diagnosed
at the Los Angeles/USC Medical Center, Los Angeles, CA, or the Medical
University of Freiburg Medical Center, Freiburg, Germany. We studied 34 CML patients staged by the Southwest Oncology Group (SWOG) protocol
criteria8: 17 patients were chronic, 9 patients were
accelerated, and 8 patients were in blast crisis. Controls for our
assay included DNA from the blood and bone marrow of patients without
disease (including some CD34+ enriched samples); several
leukemic cell lines (Raji, KG1a, and K562); and patients with acute
myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and
myelodysplastic syndrome (MDS). We extracted DNA from the samples as
described previously.9 The DNA was bisulfite modified and
amplified with Ms-SNuPE primers that were specific for the c-abl Pa
and p15 5' regions, and the methylation at 2 and 3 sites in each 5' region, respectively, was averaged after
quantitation (PhosphorImager; Molecular Dynamics, Sunnyvale,
CA) as described previously.6,7
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Results and discussion |
Ms-SNuPE quantitates the levels of methylation at individual CpG
sites in each DNA sample analyzed. The DNA standards (generated by
mixing SssI methylase-treated DNA with unmethylated DNA) gave the
expected methylated results for each standard by Ms-SNuPE analysis
(Figure 1). A negative control white blood
cell (WBC) DNA from a healthy donor and a positive control from the
Raji cell line also gave the expected p15 methylation in the
Ms-SNuPE assay. The assay revealed that c-abl is methylated in
CML patient samples and in the CML leukemic cell line K562, but it is
unmethylated in WBCs from healthy individuals; in non-CML cell lines;
and in patients with ALL, AML, and MDS (Figure
2A). Methylation of c-abl was found
in 10/13 (77%) patients with chronic CML, to an average extent of
41%, while 5/5 (100%) accelerated CML patients and 2/3
(67%) blast crisis CML patients were methylated to an average extent
of 48% and 29%, respectively (Figure 2A). We found that c-abl
was methylated in all stages of CML, with no statistical difference
between the 3 stages (P = .46, using a 2-sided 1-way analysis
of variance method). In addition, equal c-abl hypermethylation was detected in all phases of CML, which suggests that it does not have
prognostic significance. We also followed the methylation pattern of
c-abl during the disease progression of several CML patients,
but we did not find any change in the c-abl methylation using
either the PCR-based restriction enzyme1 method or the Ms-SNuPE method (data not shown).
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| Fig 1.
Ms-SNuPE standard curve for the p15 5'
untranslated region (UTR) showing the quantitative results obtained
from the in vitro SssI methylated DNA standards.
WBCs, which are unmethylated, are from the peripheral blood of a
healthy donor, while the Raji is a leukemic cell line that is a
positive methylated control. Methylation is a measure of the signal in
the C lane, which represents the amount of methylated DNA molecules as
a result of the [32P]dCTP incorporation. A band in the T
lane is the product of [32P]dTTP incorporation and
represents unmethylated cytosine molecules at a particular CpG site.
NDC indicates no DNA control; 1, 2, and 3 are individual cytosines in a
CpG site examined by each Ms-SNuPE primer; and T is the control for a
complete bisulfite conversion, representing a C not in a CpG
dinucleotide.
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| Fig 2.
Summaries of the c-abl Pa promoter
and p15 5' CpG island methylation levels.
(A) Summary of the c-abl Pa promoter methylation levels of 1 patient or healthy control. Each vertical bar represents the average of
2 CpG sites by Ms-SnuPE assay. CD34+ indicates a
CD34+ enriched sample from the peripheral blood of a
hematologically normal patient; WBC, white blood cell sample from a
healthy individual; CH, chronic phase of CML; AC, accelerated phase of
CML; BC, blast crisis phase of CML; ALL, acute lymphocytic leukemia;
AML, acute myelogenous leukemia; and MDS, myelodysplastic syndrome.
(B)(a) Summary of the p15 5' CpG island methylation
levels in CMLs and controls by Ms-SnuPE assay. The levels shown
represent the average of 3 CpG sites. BM indicates normal bone marrow;
WBC1 and WBC2, blood DNAs from healthy donors; CH, chronic CML; AC,
accelerated CML; and BC, blast crisis CML. (b) Southern blotting also
showed methylated p15 5' region status in the CML
patients. The following are depicted: lane 1, undigested WBC DNA; lane
2, EagI digested WBC DNA from the healthy control; lane 3, CML
accelerated samples; and lane 4, CML blast samples. The samples in
lanes 3 and 4 are methylated because a larger band appeared after
digestion with EagI, similar to the uncut DNA in lane 1.
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Earlier studies looking at c-abl methylation in CML showed a
correlation with progression, but hypermethylation was seen in 24%-68% of the CML patients in the chronic phase, while patients in
the accelerated phase and blast crisis had
hypermethylation frequencies at 73% and 80%,
respectively.1,2 These studies further demonstrated a
difference between methylation of c-abl in the early (less than
12 months after diagnosis) versus late chronic CML samples. Our results
do not reproduce these findings, perhaps because of the more specific,
sensitive, and quantitative nature of the Ms-SNuPE assay and the
differences in the CML population sampled (although there was a broad
range of both early and late chronic CML samples analyzed in our study).
Ms-SNuPE analysis of the p15 5' region in CML patients
also showed positive methylation, which contradicts the current view that CML has an unmethylated p15 region.4
Methylation of p15 in CML was detected in all stages of disease
in 8/34 (24%) patients (Figure 2Ba). The mean methylation of 3 sites
in p15 was 7% in the 17 chronic patients, 9% in the 9 accelerated samples, and 16% in the 8 blast crisis cases. The small
increase in mean methylation across the 3 stages of CML for p15
was statistically significant (P = .047). During CML
progression for more than 2 months, 1 patient also showed an increase
in p15 methylation as he progressed from chronic to blast
crisis (19% vs 45%) (less than 10% blasts for the chronic sample,
40% blasts in the blood, and t[7;11] for the blast
crisis sample). A further increase (to 58%) was observed in a second
blast crisis sample (89% blasts and t[7;11]i[17] karyotype). We
confirmed the p15 methylation detected in CML by Ms-SNuPE using both Southern analysis4 and bisulfite genomic sequencing
with our Ms-SNuPE primers (Figure 2Bb). Further studies show that
Ms-SNuPE methylation at this region of the p15 locus is often
concurrent with decreased expression of p15 at the protein
level (manuscript submitted).
Our data reveal that the c-abl Pa promoter methylation is
probably not a good marker for CML progression, while changes in the
p15 5' region methylation are found in CML and may also
play a role in the biology of this malignancy. Although most of the samples analyzed are from an unfractionated cell population,
CD34+ enriched peripheral blood from a nonleukemic control
and a healthy bone marrow sample showed unmethylated c-abl
and p15. This suggests that methylation is a change associated
with CML cells but not with hematopoietic precursors.
These findings, as well as that of earlier
work,2 determined that differences in methylation status of c-abl in the early chronic phase (grouped as
negative, low, moderate, or high methylation) were not due to the
number of blasts. All groups had similar polymorphonuclear cell (PMN) representation,2 which suggests that the c-abl and
p15 methylation in CML is not due to the various
differentiation stages of the cells that were examined.
Our results, obtained with a sensitive quantitative bisulfite-based
assay, agree with the findings of Issa et al,2 who reported
the lack of prognostic significance of c-abl methylation in
CML. Our findings also suggest that to fully determine
the role of p15 methylation in CML progression, future studies
are needed to assess the methylation status in CMLs with respect to the
type of therapy and duration. In conclusion, DNA methylation changes in
c-abl and p15 are common abnormalities in CML, and our
study shows that the use of a bisulfite-based Ms-SNuPE assay allows
both a sensitive and specific quantitative measurement of these
changes. The Ms-SNuPE assay, compared with methods that require
restriction endonucleases, might be a more quantitative method to
assess molecular changes associated with disease progression.
| |
Acknowledgment |
We would like to thank Dr William F. Benedict from the MD Anderson
Cancer Center, Houston, TX, for graciously contributing the AML and MDS samples for our studies.
| |
Footnotes |
Submitted October 4, 1999; accepted January 4, 2000.
Supported by grants from the National Cancer Institutes to J.G.
(RO1CA47456), N.H. (R01CA50248), and P.A.J. (1R01CA82422-01).
Reprints: Peter A. Jones, the Department of
Biochemistry & Molecular Biology, USC/Norris Cancer Center, University of Southern California, Los Angeles, CA; e-mail:
jones_p{at}ccnt.hsc.usc.edu
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.
| |
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Abstract
Introduction