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Blood, 1 November 2002, Vol. 100, No. 9, pp. 3432-3433
CORRESPONDENCE
To the editor:
Lack of p21CIP1 DNA methylation in
acute lymphocytic leukemia
Recently, Roman-Gomez et al1 reported an incidence
of p21CIP1 methylation of 41% in 124 patients
with acute lymphocytic leukemia (ALL). Most importantly, they observed
that p21CIP1 methylation was an independent
predictor of poor prognosis both in adults and children with this disease. To follow these observations, we have analyzed the methylation status
of p21CIP1 in a cohort of patients with ALL who
were previously studied for methylation of multiple
genes.2,3 We studied a total of 31 patients (19 male;
median age, 39 years [range, 7-77 years]; 6 Philadelphia (Ph)
chromosome positive). For methylation analysis, we used widely accepted
methods based on bisulfite modification of DNA because these assays are
sensitive and have a low rate of false positivity. We used 2 different
bisulfite polymerase chain reaction (PCR) methods to assess
methylation of 3 different regions in or in close proximity to
the area studied by Roman-Gomez et al. Primer location is shown in
Figure 1. The methylation status of region 1 was analyzed using the combined
bisulfite restriction analysis assay (COBRA).4 Regions 2 and 3 were analyzed using the more sensitive methylation-specific PCR
assay (MSP).5 A positive control consisting of genomic DNA
methylated in vitro by SssI methylase was used in
all these assays to verify the validity of the procedures. Using these
2 techniques, DNA methylation was not observed in any of the patients
studied in any of the 3 regions analyzed (Figure 1). In contrast,
methylation of many other genes, including ER,
MDR1, THBS1, THBS2, p15,
p73, Myf3, c-abl, and
CD10, was observed previously in some of these
cases.2 To analyze p21CIP1
expression status, we have performed reverse transcriptase
(RT)-PCR analysis in 8 cases. In contrast to the results
reported by Roman-Gomez et al, all the cases studied had evidence of
p21CIP1 RNA expression (Figure 1).
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| Figure 1.
Methylation and expression analysis of
p21CIP1 in acute lymphocytic leukemia.
(A) Map of the p21CIP1 CpG island. Each vertical
mark indicates a CpG pair. The arrow indicates the initiation of the
transcription start site. Region 1 indicates de location of COBRA
primers. Region 2 of MSP1 primers, and region 3 of the MSP 2 primers. Note that region 3 corresponds to the area studied by
Roman-Gomez et al. (B) Examples of COBRA (region 1), MSP1 (region 2),
and MSP2 (region 3) assays. SssItr indicates the methylated
positive control. The arrow indicates the restricted methylated band. M
indicates methylation-specific MSP reactions, U, unmethylated
reactions. (C) RT-PCR reactions. + indicates reactions with reverse
transcriptase; -, without.
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Our results are in contrast with those of Roman-Gomez et al. No
other investigator has reported evidence of
p21CIP1 methylation in ALL or other leukemias.
Indeed, Kikuchi et al6 did not find evidence of
p21CIP1 methylation in 19 neoplastic cell lines,
including 6 of hematopoietic origin. It is possible that the patients
studied by Roman-Gomez et al are markedly different from patients in
the United States, as geographic variation in methylation
patterns has recently been reported.7 However, we suggest
that the differences observed reside in the technique used by
Roman-Gomez et al to assess methylation. In their experiments, they
digested DNA with a methylation-sensitive restriction enzyme followed
by PCR amplification. Unrestricted PCR-amplified fragments thus
represented methylated alleles. Because all cases had evidence
of an amplified (methylated) band, samples were considered methylated
based on a normalized mean ratio of p21CIP1 to
 -actin PCR amplification. This method is prone to false-positive results because unmethylated but incompletely digested DNA may amplify
and give a positive reading. The authors did not show controls, nor did
they confirm the methylation status of p21CIP1
by Southern blot analysis or bisulfite-based methods.8
These 2 last methods are considered more reliable. In summary, we have found no evidence of p21CIP1
methylation in our cohort of patients. Other investigators have
reported similar findings in an extensive study of neoplastic cell
lines.6 We believe that Roman-Gomez et al should
reanalyze their samples using either Southern blot or bisulfite-based
methods to confirm their important observations.
LanLan Shen, Yutaka Kondo, Jean-Pierre Issa, and Guillermo Garcia-Manero
Correspondence: Guillermo Garcia-Manero, Department of Leukemia,
University of Texas MD Anderson Cancer Center, Box 428, 1515 Holcombe
Blvd, Houston, TX 77030; e-mail:
ggarciam{at}mdanderson.org
References
1.
Roman-Gomez J, Castillejo JA, Jimenez A, et al.
5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia.
Blood.
2002;99:2291-2296[Abstract/Free Full Text].
2.
Garcia-Manero G, Daniel J, Smith T, et al.
DNA methylation of multiple promoter associated CpG islands in adult acute lymphocytic leukemia.
Clin Cancer Res.
2002;8:2217-2224[Abstract/Free Full Text].
3.
Garcia-Manero G, Bueso-Ramos C, Daniel J, Williamson J, Kantarjian HM, Issa JP.
DNA methylation patterns at relapse in adult acute lymphocytic leukemia.
Clin Cancer Res.
2002;8:1897-1903[Abstract/Free Full Text].
4.
Xiong Z, Laird PW.
COBRA: a sensitive and quantitative DNA methylation assay.
Nucleic Acids Res.
1997;25:2532-2534[Abstract/Free Full Text].
5.
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB.
Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Proc Natl Acad Sci U S A.
1996;93:9821-9826[Abstract/Free Full Text].
6.
Kikuchi T, Toyota M, Itoh F, et al.
Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors.
Oncogene.
2002;21:2741-2749[CrossRef][Medline]
[Order article via Infotrieve].
7.
Shen L, Ahuja N, Shen Y, et al.
DNA methylation and environmental exposures in human hepatocellular carcinoma.
J Natl Cancer Inst.
2002;94:755-761[Abstract/Free Full Text].
8.
Clark SJ, Harrison J, Paul CL, Frommer M.
High sensitivity mapping of methylated cytosines.
Nucleic Acids Res.
1994;22:2990-2997[Abstract/Free Full Text].
Response:
Hypermethylation of the p21 gene in acute
lymphoblastic leukemia
Shen et al analyzed the methylation status of the p21
gene in a cohort of ALL patients using methods based on bisulfite
modification of DNA. They found lack of p21 methylation in
their patients. This result is in disagreement with our recent
report.1 However, the study by Shen et al contains a
number of weak points and several problems. First, their study included 31 acute lymphocytic leukemia
(ALL) patients (probably selected by the availability of
preserved DNA or other unknown reasons), which is a very small number
of patients when the goal is to reach a definitive conclusion regarding the frequency of this molecular event. By comparison, our study included 124 consecutive ALL patients. It has been suggested that there are important geographic variations in the methylation patterns of several malignancies. For example, in our series of 150 consecutive Spanish ALL patients (manuscript in preparation), we have observed methylation of the E-cadherin and p73 genes in
19% and 18% of patients respectively, whereas E-cadherin
methylation has been observed in 53% of patients in the United States
and in 76% of patients in Australia.2-3 By the same
token, p73 methylation has been detected in 32% of patients
in the United States.4-5 However, we think that these
differences do not depend only on geographic variations but also on the
premature release in high-impact journals of a plethora of descriptive
methylation studies based on small and very selected groups of
patients. These preliminary reports have reached conclusions that have
been accepted by the scientific community as absolute truths when
they are probably very far from the real incidence of this
molecular finding. Second, Southern blot methylation analysis using a U64 probe
(encompassing p21 promoter from nucleotide [nt] - 571 to + 518) revealed the same results obtained after DNA digestion
with HpaII followed by polymerase chain reaction
(PCR). Moreover, normal bone marrow and peripheral blood
showed incomplete methylation patterns, with bands corresponding to
both methylated and unmethylated states. Similar results have been
obtained analyzing peripheral lymphocytes by Chen et al.6
Therefore, it is surprising that Shen et al did not find p21
methylation when even healthy individuals show a partially methylated
status. The p21 gene has a complex promoter and while some
of its CpG sites are methylated, other HpaII sites that span
the CpG-rich region of this gene (ie,  1004, 62, +15, +93, +288, and
+553) are unmethylated in controls and ALL patients. Therefore, a
careful choice of the primers for the methylation-specific PCR
(MSP) method is necessary in order to obtain reliable results.
A bad choice will lead to underestimation of the methylation pattern of
the p21 gene. Third, Shen et al suggest that our method is prone to false-positive
results because unmethylated but incompletely digested DNA may amplify
and give a false-positive reading. To avoid this problem, we had
already examined 30 healthy individuals (a similar number of controls
to the patients reported by Shen et al) and did not find
hypermethylation in any of them. Moreover, we have extended our
p21 methylation analysis to other hematologic malignancies (manuscript submitted) and no p21 hypermethylation was
observed among 179 chronic-phase chronic acute myeloid leukemia
(CML) patients, 55 acute stage CML patients, or 72 acute
myelogeneous leukemia (AML) patients. Therefore, it is
improbable that false-positive results should appear only in ALL
patients and not in more than 300 patients affected by other types of
leukemias. It is more likely that this result seems to indicate some
kind of technical problem in the method used by Shen et al. In fact, it
is well recognized that study of the methylation status of the
CDKI genes belonging to the KIP family is sometimes
difficult and needs very sensitive technology. For example, it is
necessary to perform the amplification with enzymes that permit raising
the annealing temperature up from 68°C to 70°C with an extension
temperature more than 74°C. Thus, special DNA polymerases must be
used (ie, Pfu DNA polymerase) instead of conventional
Taq DNA polymerase. Another sensitive method employed for
amplification of KIP family is nested PCR (ie,
p57KIP2). Finally, Shen et al studied p21 expression in only 8 patients using reverse transcriptase (RT)-PCR. They found
evidence of p21 mRNA in all the cases. This result is not
surprising because, in any case, most of ALL patients show p21
expression regardless of the methylation status of the gene.
However, hypermethylated patients disclosed decreased levels of
p21 transcripts as assessed by a semiquantitative RT-PCR
method. Therefore, a quantitative method is mandatory in these
circumstances. The qualitative RT-PCR performed by Shen et al is
unsatisfactory for this purpose. Moreover, in addition to ALL,
decreased levels of p21 have been recently reported in other
hematologic tumors.7-8 For all of these reasons, the title "Lack of
p21CIP1 DNA methylation in acute
lymphoblastic leukemia" is both adventurous and premature. We
recommend that Shen et al revise some aspects of their technology and
patient selection.
Jose Roman-Gomez, Juan A. Castillejo, Antonio Torres, and Antonio Jimenez
Correspondence: Jose Roman-Gomez, Hematology Department, Reina
Sofia Hospital, 14004 Cordoba, Spain; e-mail:
peperosa{at}teleline.es
References
1.
Roman-Gomez J, Castillejo JA, Jimenez A, et al.
5' CpG island hypermethylation is associated with transcriptional silencing of the p21 CIP1/WAF1/SDI1 gene and confers poor prognosis in acute lymphoblastic leukemia.
Blood.
2002;99:2291-2296[Abstract/Free Full Text].
2.
Corn PG, Smith BD, Ruckdeschel ES, Douglas D, Baylin SB, Herman JG.
E-cadherin expression is silenced by 5' CpG island methylation in acute leukemia.
Clin Cancer Res.
2000;6:4243-4248[Abstract/Free Full Text].
3.
Melki JR, Vincent PC, Brown RD, Clark SJ.
Hypermethylation of E-cadherin in leukemia.
Blood.
2000;95:3208-3213[Abstract/Free Full Text].
4.
Kawano S, Miller CW, Gombart AF, et al.
Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation.
Blood.
1999;94:1113-1120[Abstract/Free Full Text].
5.
Corn PG, Kuerbitz SJ, van Noesel MM, et al.
Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5'CpG island methylation.
Cancer Res.
1999;15:3352-3356.
6.
Chen B, He L, Savell VH, Jenkins JJ, Parhan DM.
Inhibition of the interferon-gamma/signal transducer and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region.
Cancer Res.
2000;60:3290-3298[Abstract/Free Full Text].
7.
Cobo F, Martinez A, Pinyol M, et al.
Multiple cell cycle regulator alterations in Richter's transformation of chronic lymphocytic leukemia.
Leukemia.
2002;16:1028-1034[CrossRef][Medline]
[Order article via Infotrieve].
8.
Halta Y, Koeffler HP.
Role of tumor suppressor genes in the development of adult T cell leukemia/lymphoma.
Leukemia
2002;16:1069-1085[CrossRef][Medline]
[Order article via Infotrieve].
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