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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3144-3147
Loss of Imprinting in Disease Progression in Chronic Myelogenous
Leukemia
By
Gurvaneet S. Randhawa,
Hengmi Cui,
Janet A. Barletta,
Liora Z. Strichman-Almashanu,
Moshe Talpaz,
Hagop Kantarjian,
Albert B. Deisseroth,
Richard C. Champlin, and
Andrew P. Feinberg
From the Departments of Medicine, Oncology, and Molecular Biology & Genetics, and the Graduate Program in Human Genetics, Johns Hopkins
University School of Medicine, Baltimore, MD; and the Department of
Hematology, MD Anderson Cancer Center, Houston, TX.
 |
ABSTRACT |
The pathophysiologic role of the Philadelphia chromosome
translocation in chronic myelogenous leukemia (CML) has been known for
nearly 20 years. However, the most significant morbidity and mortality
in CML are caused by progression to blast crisis, about which
comparatively little is known at the molecular level. Genomic imprinting is a chromosomal modification leading to
parental-origin-specific gene expression in somatic cells. Recently,
we and others have described loss of imprinting (LOI) of the
insulin-like growth factor-II gene (IGF2), leading to biallelic rather
than monoallelic expression in a wide variety of solid tumors. We have
now examined the imprinting status of IGF2 in samples from CML patients
in stable phase, accelerated phase, and blast crisis. Five of six stable-phase patients showed normal imprinting, but LOI was found in
all six cases of advanced disease (three accelerated phase, three blast
crisis), which was statistically highly significant (P < .01). Thus, LOI represents a novel type of genetic alteration in CML
that appears to be specifically associated with disease progression.
| |
INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is
characterized by an expansion of myeloid cells containing the hallmark
Philadelphia chromosome translocation.1 This translocation
leads to formation of the bcr-abl fusion gene, whose
protein product is a highly active tyrosine kinase implicated in the
pathogenesis of the disease.2,3 CML has been shown to
evolve hematologically from an initial chronic phase to an accelerated
phase, finally culminating in an aggressive, fulminant blast
crisis.2,3 This evolution is characterized by the
appearance of progressively immature myeloid cells as well as the
acquisition of additional cytogenetic anomalies.3 The most
frequent genetic alterations in progression to blast crisis are
deletions and mutations of p53, occurring in 20% to 30% of patients,
and amplification of c-myc in 20% of patients.4,5 Other
events occurring more rarely include mutations in ras (6%) and
rearrangement and deletions of RB (14%) and p16 (15%).6-8
Genomic imprinting is a modification of a gene or the chromosome on
which it resides, in the gamete or zygote, leading to differential
expression of the two parental alleles of the gene in somatic cells of
the offspring. Imprinting usually involves monoallelic expression of
either the paternal or maternal allele, depending on the gene. Genomic
imprinting is thought to be important in the development of cancer
because loss of heterozygosity (LOH) in cancer often involves one of
the parental chromosomes preferentially,9 and some
imprinted genes are involved directly in the regulation of cell growth,
such as insulin-like growth factor-II (IGF2).9 Furthermore,
we and others have found loss of imprinting (LOI) in cancer, resulting
in loss of parental-origin-specific differential allele
expression.10 LOI usually leads to activation of the normally silent maternal copy of IGF2, which is expressed normally only
from the paternal allele. LOI can also lead to epigenetic silencing of
growth inhibitory genes such as p57KIP2 and
H19.11,12 Although LOI was originally observed in embryonal tumors of childhood, including Wilms' tumor, hepatoblastoma, and rhabdomyosarcoma, it has now been found in a wide variety of adult solid tumors, including those of the prostate, breast, liver, and
lung.9
Because LOI is one of the most common genetic alterations in solid
tumors but has not been examined previously in CML, we sought to
determine whether there is abnormal imprinting in CML, and if so
whether it is specifically associated with disease progression.
| |
MATERIALS AND METHODS |
Screening for informative CML patients by DNA polymerase chain reaction
(PCR).
Peripheral blood and bone marrow samples from CML patients were diluted
1:1 with phosphate-buffered saline (PBS) and layered over a
discontinuous Percoll (Pharmacia, Uppsala, Sweden)
gradient of specific gravity 1.077. The samples were centrifuged at
1,500 rpm for 7 to 10 minutes, and the white cells in the supernatant were washed with PBS and processed for DNA and RNA. For DNA extraction the cells were incubated overnight at 42°C, in a lysis buffer containing 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 25 mmol/L EDTA,
0.5% sodium dodecyl sulfate (SDS), and 0.1 mg/mL Proteinase K. The
lysate was extracted with phenol/chloroform, ethanol precipitated, and
resuspended in TE buffer. The genomic DNA was analyzed by PCR for an
Apa I polymorphism in IGF2, as previously
described.10 PCR products were electrophoresed on 3%/1%
Nuseive/agarose gels.
Imprinting analysis by reverse transcription polymerase chain
reaction (RT-PCR).
Samples from heterozygous patients were processed for total RNA using
RNAzol B (Tel-Test) following the manufacturer's
instructions. To avoid any possible contamination by genomic DNA, the
total RNA was digested with DNase I and then extracted with
phenol/chloroform. To ensure the absence of genomic DNA contamination,
all RT-PCR analysis was performed in duplicate in the presence and
absence of reverse transcriptase (AMV-RT; Promega) using primers as
described and PCR conditions as follows: 94°C for 3 minutes; 35 cycles of 94°C for 1 minute; 55°C for 1 minute; and 72°C
for 1.5 minutes followed by a 10-minute extension at
72°C.12 The samples were electrophoresed on 3%/1%
Nusieve/agarose gels.
Analysis of H19 methylation.
DNA extracted from the CML patient samples was screened for a Hha
I polymorphism upstream of the H19 gene.13 Heterozygous patients were analyzed for methylation using HpaII. To assess allele-specific methylation, the genomic DNA from informative patients
was digested with an excess of HpaII, PCR amplified, and
digested with Hha I, as described.13
| |
RESULTS |
Our primary goal was to determine whether patients with CML maintain
normal imprinting or undergo LOI. To analyze genomic imprinting of
IGF2, we first identified CML patients who were heterozygous for a
transcribed Apa I polymorphism in the IGF2 gene. Of 21 chronic-phase patients 6 were informative (heterozygous); of 7 accelerated-phase patients 3 were informative; and of 12 patients in
blast crisis, 3 were informative for the Apa I polymorphism. For example, Fig 1 lane 1 shows a patient
homozygous for the A allele (236 bp), and lanes 2, 3, and 6 show
patients homozygous for the B allele (173 bp) and thus were not
informative. In contrast, lanes 5, 6, and 7 show patients having both A
and B alleles and are thus informative for the IGF2 polymorphism.
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| Fig 1.
Analysis of DNA from CML patients for heterozygosity of
IGF2. Lane 1 shows patient homozygous for the A allele; lanes 2, 3, and
6 show patients homozygous for the B allele; and lanes 4, 5, and 7 show
heterozygotes, with both A and B alleles.
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A total of 40 patient samples were tested for the polymorphism, of
which 12 informative samples were analyzed for imprinting status by
performing RT-PCR for the Apa I polymorphism, as described in
Materials and Methods. An example of the analysis of genomic imprinting
is shown in Fig 2. Patients 4 and 10, who
were in chronic phase, showed expression of only the B allele after
RT-PCR, indicating normal monoallelic expression of IGF2 in their
leukemic cells. In contrast, patient 12 (accelerated phase) and
patients 14 and 15 (both in blast crisis) showed biallelic expression
of IGF2 (Fig 2). In 3 samples, LOI was also confirmed using exon
connection between exon 8 and exon 9. Of the 6 informative patients in
chronic phase, 5 exhibited normal monoallelic IGF2 expression and only one (patient 6) showed biallelic expression of IGF2 gene in his leukemic cells. This patient had an atypical clinical course in that he
was unresponsive to therapy, and he died 3 months after his blood
sample was obtained because of failure of engraftment after allogeneic
bone marrow transplantation.
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| Fig 2.
Analysis of Imprinting of IGF2 in CML. Normal monoallelic
expression was seen in patients 4 and 10 (chronic phase), and biallelic expression was observed in patients 12 (accelerated phase), 14, and 15 (blast crisis). All reactions were performed in duplicate in the
presence (+) and absence ( ) of reverse transcriptase.
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In contrast to most of the stable-phase patients, all three informative
patients in accelerated stage and all three informative blast crisis
patients exhibited biallelic expression of the IGF2 gene in their
leukemic cells (Table 1). The difference
between the frequency of LOI in chronic phase (1 of 6) compared with
that of accelerated phase (3 of 3) was statistically highly significant (P < .05), as was the difference between the frequency of LOI in
chronic phase and that of blast crisis (3 of 3; P < .05). The frequency of LOI in overall advanced-stage disease compared with chronic phase was even more strikingly significant (P < .01). Thus, LOI is a frequent alteration in accelerated phase and blast crisis in CML, and it appears to be specific for disease progression.
In some solid tumors, such as Wilms' tumor, LOI is associated with
abnormal methylation of a CpG island upstream of the neighboring H19
gene, which is normally methylated only on the paternal
allele.12 In other patients, LOI of IGF2 can occur
independently of methylation changes in the H19 gene.14,15
To determine whether LOI in CML was methylation-dependent or
independent, we used a PCR-based assay, which we previously described,
to detect an Hha I polymorphism within the H19 CpG island and
that distinguishes between the two alleles in heterozygous
patients.13 Four patients were heterozygous and thus
informative for the H19 Hha I polymorphism. As expected, a
chronic-phase patient (patient 10) with normal imprinting of IGF2
showed monoallelic methylation of the H19 CpG island. Of the four
informative patients, one was in accelerated phase (patient 12) and
exhibited LOI of IGF2 (Fig 2). This patient also showed normal
methylation of the H19 CpG island, ie, methylation of only one allele
(Fig 3). Thus, LOI of IGF2 occurred independent of hypermethylation of
the H19 CpG island in this patient. Two additional patients in blast
crisis (patients 17 and 18) also showed monoallelic methylation of the
H19 CpG island. Thus, it is likely that LOI of IGF2 in CML occurs by a
pathway independent of H19 methylation. Interestingly, the blast crisis
cell line K562 also displayed a monoallelic pattern of H19 methylation
(Fig 3), although it has been reported to
show hypermethylation at other loci such as the calcitonin and bcr
genes.18-20
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| Fig 3.
Analysis of H19 promoter methylation in CML. Patients 10, 12, 17, 18, and cell line K562 (K) showed normal monoallelic
methylation. Samples pretreated (+) or not pretreated () with
HpaII, PCR amplified and then digested with Hha I,
which detects the H19 polymorphism.
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| |
DISCUSSION |
In summary, we found that six of six patients with advanced CML, three
in blast crisis and three in accelerated phase, showed loss of
imprinting (LOI) of IGF2, but only one of six patients in chronic phase
exhibited LOI. This patient was somewhat unusual in that he was
refractory to treatment and died 3 months after the sample was taken.
The high frequency of LOI in advanced disease is more frequent than
that of other genetic alterations previously observed.4-8
These results are important for two reasons. First, they extend our
observation of abnormal imprinting in cancer to this common leukemia in
adults. In addition, these results suggest a specific relationship of
LOI to disease progression. Recently, it was shown that IGF2 was
normally imprinted in blood and in early hematopoietic precursors and
that LOI occurs in 50% of acute myelogenous leukemia patients,
although relationship to disease progression was not determined.19 We plan to examine LOI prospectively in a
larger series of patients to determine whether it will serve as a
useful prognostic marker for disease progression in CML. It is
intriguing in this regard that the one stable-phase patient with LOI
was refractory to therapy. Finally, even though LOI was independent of
H19 methylation, alterations in DNA methylation at other loci have been
described in CML progression.16-18 Thus, it will be
important to examine other imprint-specific sites of DNA methylation,
such as within the IGF2 gene itself.14 Regardless of the
mechanism of LOI in CML, little is known about the molecular basis of
progression to blast crisis, and thus the data reported here provide a
new direction for further study.
How might loss of imprinting be functionally related to disease
progression in CML? We and others have identified a large (approximately 1 Mb) genomic domain of 11p15 that includes
at least eight imprinted genes,9 several of which are
predicted to have an effect on cell growth, including the following:
IGF29,10; H199,10;
p57KIP2, a cyclin-dependent kinase
inhibitor11,20; and TSSC3 (the human homologue of mouse
apoptosis-related gene TDAG51).21,22 We have also found
that loss of imprinting can affect one or more of these genes
simultaneously within tumors.9,11,20-22 Our observation of
LOI of IGF2 in CML thus leads to the following questions:
- Do any of the other genes within this complex show altered imprinting
in CML?
- What is the consequence of altered imprinting on the expression of
these genes in advanced stages of CML? Preliminary analysis does not
suggest a significant increase in IGF2 expression in patients with
advanced CML (data not shown), although other tumors with LOI also do
not show a dramatic increase in IGF2 expression,23 even
though LOI is associated with predisposition to
malignancy,24 and it appears to be a necessary step in
tumor progression.25 Thus, deregulated expression of IGF2,
rather than overall expression per se, may be important, or other genes
within this cluster may be more important in the pathophysiology of
CML.
- What is the relationship between LOI and other epigenetic changes in
CML, specifically altered DNA methylation? At least six CpG
dinucleotide-rich "CpG islands" have been reported within this
cluster.10,12-14,26 Because altered DNA methylation at
other chromosomal sites is a feature of CML progression,18
it will be important to determine whether altered methylation of this imprinted domain also plays a role in CML.
Regardless of the answers to these questions, the data provided here
that show that LOI is an important marker for disease progression, and
it may serve as an important new prognostic marker for predicting
response to therapy. This strong association itself suggests a
biologically important role for LOI in CML.
| |
FOOTNOTES |
Submitted December 18, 1997;
accepted February 18, 1998.
G.S.R. and H.C. contributed equally to this work.
Supported by Grant Nos. CA49639 and CA65145 from the National
Institutes of Health.
Address reprint requests to Andrew P. Feinberg, MD, MPH, Johns Hopkins
University School of Medicine, 1064 Ross, 720 Rutland Ave, Baltimore,
MD 21205.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Richard Jones for generously providing some of the samples,
Leslie Calvert for technical assistance, and Pam Hill for preparing the
manuscript.
| |
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Abstract
Introduction
Abstract