Blood, 1 January 2003, Vol. 101, No. 1, pp. 374-374
CORRESPONDENCE
To the editor:
p14ARF,
p15INK4b, and p16INK4a
methylation status in chronic myelogenous leukemia
The promoters of the p16INK4a
and p15INK4b genes located at chromosome band
9p21 are frequently silenced by CpG island methylation in hematologic
malignancies and solid tumors.1 In chronic myelogenous leukemia (CML) the situation is confused because 2 reports
have produced contradictory results on p15INK4b
methylation, possibly because of the sensitivity of the techniques used
for these studies. By use of restriction endonuclease-based Southern
blot, methylation of the 5' region of p15INK4b
was not detected on 21 samples in contrast to other leukemia types.2 In another study with the more sensitive
methylation-specific polymerase chain reaction (MSP)
technique, methylation of p15INK4b was detected
in 24% (8/34) of CML cases and was associated with progression.3
In the present report, we used MSP4 to
study p15INK4b- and
p16INK4a-promoter hypermethylation status in 76 cases of CML at various phases of the disease: chronic (50),
accelerated (11), myeloblastic transformation (5), and
complete cytogenetic remission (10) upon interferon-base
regimen5 or imatinib treatment.6 In
addition, we tested p14ARF promoter methylation,
as this study has not been described yet.
Partial p15INK4b hypermethylation was
found for 4 patients (5%), 2 in chronic phase and 2 in blast crisis
(patients 11, 12, 15, and 19 in Figure 1
), consistent with previous data on a smaller series.2 In
all cases, the intensity of the unmethylated bands greatly exceeded
that of methylated ones. While the technique is only semiquantitative,
this suggests that only a minor part of the leukemic cells was
implicated in the hypermethylation process. Patient 19 in myeloid
transformation showed slight hypermethylation of the
p14ARF promoter (Figure 1). For all the other
patients, the p14ARF promoter was unmethylated.
The p16INK4a promoter was found unmethylated for
all patients (not shown), again in agreement with Herman et al's
data.2
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| Figure 1.
Methylation status of
p15INK4b and
p14ARF analyzed by MSP. DNA was
phenol/chloroform extracted from blood. Promoter methylation was
determined by the method of MSP.4 The modified DNA was
used as a template for PCR amplification using primers specific for
either methylated or unmethylated DNA for
p14ARF,7
p15INK4b, and
p16INK4a.4 The specificity of the
PCR reaction was checked by sequencing the amplified fragments. Control
without DNA was performed for each set of PCRs. Placental DNA treated
or not in vitro with SssI methyltransferase (New England
Biolabs, Beverly, MA) was used as positive control for the
methylated or the unmethylated form, respectively. Examples are given
for patients 9 to 19. M indicates methylated form; U, unmethylated
form; P, unmethylated placental DNA; and SssI, methylated
placental DNA. The DNA standards (methylated or unmethylated) gave the
expected results.
|
|
On the basis of these data, negative regulation by
hypermethylation of p14ARF,
p15INK4b, and p16INK4a,
which contributes to the cell cycle, does not appear to be a frequent inactivating event in CML. This is in contrast with
other hematopoietic malignancies, particularly acute myelogenous
leukemia (AML), in which p15INK4b
hypermethylation is frequent.8 Perhaps more
interestingly, in myelodysplastic syndromes hypermethylation of
p15INK4b has been reported to increase with the
phase of the disease.9
Sophie Kusy, Marie Cividin, Nathalie Sorel, Françoise Brizard, François Guilhot, André Brizard, Christian Larsen, and Joëlle Roche
Correspondence: Joëlle Roche, EA 2224, IBMIG,
Université de Poitiers, 40 avenue du Recteur Pineau, 86022 Poitiers Cédex, France; e-mail:
joelle.roche{at}univ-poitiers.fr
Acknowledgments
Supported by Association pour la Recherche Contre le Cancer et
Ligue Nationale Contre le Cancer.
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