Amplification or duplication of chromosome band 21q22 with multiple copies of the AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML

Abstract

Amplification or duplication of the AML1 gene at chromosome band 21q22 was detected by FISH using a locus-specific probe in three out of 171 unselected patients with therapy-related myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1 signals were located tandemly on derivative chromosomes, in one patient on a dic(9;21) and in the the other patient on a derivative chromosome 18 made up of interchanging layers of material from chromosomes 9, 14, 18, and 21. In the third patient three single supernumerary copies of AML1 were located on derivatives of chromosomes 19 and 21. All three patients were older, had previously received therapy with alkylating agents without topoisomerase II inhibitors, had complex karyotypes including abnormalities of chromosomes 5 or 7, and presented acquired point mutations of the TP53 gene. No point mutations of the AML1 gene were observed. The results support a pivotal role of impaired TP53 function in the development of gene amplification or duplication in t-MDS and t-AML.

Full text

Amplification or duplication of chromosome band 21q22 with multiple copies of the
AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML
MK Andersen
1
, DH Christiansen
1
and J Pedersen-Bjergaard
1
1
Department of Clinical Genetics, Section of Hematology/Oncology, The Juliane Marie Center, Rigshospitalet, Copenhagen,
Denmark
Amplification or duplication of the AML1 gene at chromosome
band 21q22 was detected by FISH using a locus-specific probe
in three out of 171 unselected patients with therapy-related
myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1
signals were located tandemly on derivative chromosomes, in
one patient on a dic(9;21) and in the the other patient on a
derivative chromosome 18 made up of interchanging layers of
material from chromosomes 9, 14, 18, and 21. In the third
patient three single supernumerary copies of AML1 were
located on derivatives of chromosomes 19 and 21. All three
patients were older, had previously received therapy with
alkylating agents without topoisomerase II inhibitors, had
complex karyotypes including abnormalities of chromosomes
5 or 7, and presented acquired point mutations of the TP53
gene. No point mutations of the AML1 gene were observed. The
results support a pivotal role of impaired TP53 function in the
development of gene amplification or duplication in t-MDS and
t-AML.
Leukemia (2005) 19, 197–200. doi:10.1038/sj.leu.2403612
Published online 23 December 2004
Keywords: AML1 amplification; t-MDS; t-AML; mutation of TP53;
alkylating agents
Introduction
The AML1 gene located at chromosome band 21q22 encodes
one of the two subunits of the human core binding factor (CBF),
which regulates the expression of several genes essential to
normal hematopoiesis.
1
In acute leukemia three types of
abnormality of AML1 have been observed. Most frequently,
AML1 is disrupted by recurrent reciprocal chromosome
translocations with formation of new chimeric oncogenes with
a pivotal role in leukemogenesis.
2,3
The second type of
abnormality is point mutations of AML1, observed in de novo
myelodysplasia (MDS) and acute myeloid leukemia (AML),
4
and
in therapy-related MDS (t-MDS) significantly associated with
previous therapy with alkylating agents, with deletion or loss
of chromosome arm 7q, and with subsequent progression to
t-AML.
5
The third type of abnormality of AML1 in leukemia is
amplification or duplication of the unrearranged gene. This
phenomenon has been observed in approximately 2% of patients
with childhood acute lymphoblastic leukemia (ALL),
6–9
but to our knowledge only in seven cases of de novo MDS
and AML.
10–15
In these seven patients the mutational status of
TP53 was not examined. In t-MDS and t-AML a similar
amplification or duplication of chromosome band 11q23
including the unrearranged MLL gene has been observed in
17% of the patients closely associated with mutations of the
TP53 gene.
16
As also AML1 amplification could be more
common in t-MDS and t-AML than in de novo disease, we
performed FISH for amplification or duplication of AML1 in 171
cases of t-MDS and t-AML, and examined positive cases for
mutations of TP53.
Materials and methods
Patients and cytogenetics
Bone marrow cells of 171 unselected patients with t-MDS/t-AML
were obtained at diagnosis and stored in methanol–acetic acid
at 201C. The G-banded karyotypes of 128 of the patients have
been published,
17
whereas the karyotypes of the remaining 43
cases are unreported.
FISH analyses
FISH analyses were performed as recommended by the
manufacturer using a locus-specific TEL/AML1 probe (Vysis
(Downers Grove, IL, USA), and various whole-chromosome and
centromere-specific probes (Vysis). M-FISH was performed in
cases I–III (Spectra Vision probes, Vysis). Signals were visualized
by an epiflourescence microscope (Zeiss Axioscop, Oberko-
chen, Germany), and images captured by the Quips Smart
Capture FISH Imaging Software (Vysis) and Applied Imaging
Cytovision Workstation (Newcastle, UK). Cases were classified
as AML1 amplification or duplication, if, per metaphase cell,
two or more signals were located on the same chromosome arm,
or if two or more extra single signals of AML1 were detected on
different chromosome arms. Cases with extra copies of AML1
solely due to gain of whole extra chromosomes 21 were not
considered.
Mutations of TP53 and AML1
Mutations of TP53 were searched for by direct sequencing of
TP53 exons 2–10, and mutations of AML1 were searched for by
direct sequencing of exons 3–8 as previously described.
5,18.
Results and discussion
Three out of 171 unselected patients (1.7%) with t-MDS and
t-AML presented 4–6 copies of AML1 per cell confirming that
amplification of AML1 is a rather rare phenomenon in myeloid
malignancies.
10–15
Patient characteristics, karyotypes, results of
FISH, and mutation status of TP53 and AML1 of the three cases
are shown in Table 1.
Patient I disclosed four or five apparently normal copies of
AML1 all located on a dic(9;21) (Figure 1a). Using M-FISH and
whole-chromosome painting probes it was shown that material
Received 13 September 2004; accepted 28 October 2004; Published
online 23 December 2004
Correspondence: Dr MK Andersen, Department of Clinical Genetics,
Section of Hematology/Oncology, Rigshospitalet 4052, Blegdamsvej
9, 2100 Copenhagen, Denmark; Fax: þ 45 35 45 25 77; E-mail:
mka@rh.dk
The study was supported by grants from the Danish Cancer society
Leukemia (2005) 19, 197–200
& 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00
www.nature.com/leu

Table 1 Characteristics of three patients with t-MDS and t-AMLand amplification or duplication of AML1
Case
no.
Age/sex Primary tumor Type of treatment for
primary tumor and
duration (months)
Time to
development
of t-MDS/t-
AML (months)
FAB-subtype of
MDS/AML
Survival
(months)
Karyotype of bone marrow cells Number of AML1
signals per cell
Mutation status of
TP53 and AML1
I 61/M Mb. Hodgkin MOPP/ABV (5) 40 RA 12 45,XY,del(7)(q11q22 or q22q32), 5–6 TP53:
St. IIA dic(9;21)(21pter-21q22::9?p24- 734G-A
9?p13::21q?21-21q22::9?p24- AML1:
9?p13::21q?21-21q22::9?p24- No point mutation
9?p13::21q?21-21q22::9?p24-
9?p13::21q?21-21q22::9p24-9qter),
del(20)(q11)[26]
II 62/M Wegeners
granulomatosis
CTX (33)
MTX (8)
Chlorambucil (12)
108
72
48
M1/M2 1 43B47,X,-Y,dic(5;17)(q11;p11),
dic(5;17)(q11;p11)t(13;17)(q?14;q?),
-7,der(9)t(9;12)(q34;q?13),+del(10)(p?q?),
-12,i(13)(q10),der(13;21)(q10;q10),
der(18)t(14;18)(q?;p?)t(14;21)(q?24;q?11),
der(18)t(9;18)(?;p11)t(9;14)t(9;21)[cp25]
3–7 TP53:
IVS5-2 A-C
AML1:
No point mutation
III 63/M Non-Hodgkin’s
lymphoma St. IB
CHOP (4)
CHOP (3)
BEAM + PSCT
R-DHAP (5)
86
33
30
19
RA 4 44B47,XY,der(7)t(7;13)(?;?),-9,
der(17)t(7;17)(p12;p11),der(19)t(9;19)(?;?),
der(19)t(19;21)(?;q11),+21,der(21)
(21qter-21q11::19?::21p11-
21qter)[cp8]/46,XY[17]
4–5 TP53:
731G-T
AML1:
No point mutation
MOPP/ABV ¼ mechlorethamine, vincristine, procarbacine, prednisone/doxorubicin, bleomycin, vinblastine; CHOP ¼ cyclophosphamide, doxorubicin, vincristine, prednisone; BEAM ¼ lomustine,
etoposide, cytosine-arabinoside, melphalan; PSCT ¼autologous peripheral blood stem cell transplantation; R-DHAP ¼ rituximab, dexametazone, cytosar, cisplatin; CTX ¼ cyclophosphamide;
MTX ¼ methotrexate.
Amplification of AML1 in t-MDS/t-AML
MK Andersen et al
198
Leukemia

translocated to chromosome band 21q22 consisted of small
interspersed layers of material from 9p and 21q, including the
4–5 copies of AML1 inserted between 9p24 and 21q22
(Figure 1b).
Patient II disclosed four or five apparently normal copies of
AML1 on a derivative of chromosome 18 made up of small
layers of material from chromosomes 18, 9, 14, and 21 (Figure
1c and d). In some cells, material from chromosome 9 could not
be identified on the der(18). The extra copies of AML1 were
interspersed between material from chromosome 14 (Figure 1d).
Patient III disclosed four apparently normal copies of AML1
on different chromosome arms: One copy was on the normal
chromosome 21, another was located on a der(19) to which a
segment of 21q was translocated, and two copies were located
on a der(21) to which material from chromosome 19 was
inserted between two segments of 21q each containing a copy
of AML1 (Figure 1e and f). In addition, five copies of AML1 were
detected in a large proportion of interphase cells.
A mutation of TP53 was detected in all three patients. Patient
A had a 734G-A base change resulting in a G245D amino-
acid substitution, patient B had a IVS5-2 A-C base change
resulting in disruption of a splice site, and patient C had a
731G-T base change resulting in a G245V amino-acid
substitution (Table 1). Similar to what has been reported in
childhood ALL,
19
point mutations of AML1 were not detected in
any of the three patients.
The three patients with amplification or duplication of AML1
observed in the present study share striking similarities; all three
patients were older, had previously received therapy with
alkylating agents, had very complex karyotypes, and presented
mutations of TP53.
The mechanisms underlying gene amplification in cancer are
far from solved. DNA breakage followed by breakage-fusion
bridge formation is one suggested mechanism,
20
and experi-
mental studies have demonstrated that mutational inactivation
of TP53 is associated with gene amplification and aneuploi-
dy.
21,22
The results of our previous study
18
and of the present
study support and important role of TP53 mutations in gene
amplification and duplication as 10 out of 11 patients with
amplification or duplication of MLL or AML1 presented TP53
mutations. The excess of patients with amplification or duplica-
tion of MLL or AML1 following therapy with alkylating agents
could relate to a double effect of these drugs, that is, DNA
breakage and induction of mutation of TP53.
So far only few studies have evaluated the genetic con-
sequences of amplification or duplication of the MLL and AML1
genes. In 31 patients with MDS or AML and gain of 11q23,
Poppe et al
23
demonstrated by real-time quantitative RT-PCR
increased expression of the unrearranged MLL gene in parallel
with an increasing number of MLL copies. The overexpression of
MLL apparently resulted in a gain of function, as it was
associated with increased expression of HOXA9, a downstream
target of MLL.
23
Similarly, Mikhail et al
24
detected enhanced
AML1 expression in nine patients with ALL and extra copies of
AML1. Five of these patients had only a nonconstitutional
trisomy 21 and four patients had tandem copies of AML1 on a
der(21). Experimentally, overexpression of the unrearranged
AML1 gene has been shown to have oncogenic potential.
25
Surprisingly, in the study by Mikhail et al,
24
also nine out of the
remaining 32 patients without amplification of AML1 showed
overexpression of the gene suggesting that other mechanisms
than gene amplification may be involved in overexpression of
AML1.
The often rather large size of the amplified regions on 21q has
raised the question whether other genes in the proximity of
AML1 could be of importance. Recently, amplification of 21q
was shown to result in overexpression of other genes than AML1
located to 21q such as the APP, the ETS2, and the ERG genes.
26
The present study strongly confirms and extends a close
association between mutation of TP53 and chromosomal
instability underlying amplification of chromosome bands
11q23 and 21q22. It is of interest that mutations of TP53 and
complex karyotypes are also very common in many solid
tumors.
Acknowledgements
We are indebted to Inge-Lise Frost Andersen and to Pia Bech for
excellent technical assistance in performing the FISH analyses and
the mutation analyses of TP53 and AML1, respectively.
Figure 1 FISH images of metaphase cells from three patients with
amplification or duplication of AML1. (a) Patient I presenting tandemly
repeated AML1 signals located on a dic(9;21) shown by a probe
spanning AML1 (red) and TEL (green). (b) The dic(9;21) from patient I is
made up of interchanging layers of material from chromosomes 9 and
21 as demonstrated by WCP probes for chromosomes 9 (green) and
21(red). (c) Patient II presenting tandemly repeated AML1 signals (red)
located on a der(18). (d) In patient II part of the der(18) is made up of
interchanging layers of chromosomes 14 and 21 as shown by WCP 14
(red) and WCP 21 (green) probes and a centromere-specific probe of
chromosome 18 (also green). (e) M-FISH karyotype from patient III
showing unbalanced translocations resulting in a der(19) and a
der(21). (f) Patient III presenting single AML1 signals (red) on the
normal chromosome 21 and on the der(19), and two AML1 signals on
the der(21).
Amplification of AML1 in t-MDS/t-AML
MK Andersen et al
199
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