Download Expression analyses identify MLL as a prominent target of 11q23

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NEOPLASIA
Expression analyses identify MLL as a prominent target of 11q23 amplification
and support an etiologic role for MLL gain of function in myeloid malignancies
Bruce Poppe, Jo Vandesompele, Claudia Schoch, Charlotta Lindvall, Krzysztof Mrózek, Clara D. Bloomfield, H. Berna Beverloo,
Lucienne Michaux, Nicole Dastugue, Christian Herens, Nurten Yigit, Anne De Paepe, Anne Hagemeijer, and Frank Speleman
MLL amplification was recently recognized as a recurrent aberration in acute
myeloid leukemia (AML) and myelodysplastic syndrome (MDS), associated with
adverse prognosis and karyotype complexity. Here we present detailed results
of fluorescence in situ hybridization
(FISH) and expression analyses of MLL
and 5 selected 11q candidate oncogenes
(CBL, DDX6, ETS1, FLI1, and PLZF) in 31
patient samples and one cell line with
11q23 gain. FISH analyses revealed that
the 11q23 amplicon invariably encom-
passed MLL, DDX6, ETS1, and FLI1,
whereas expression analyses identified
MLL and DDX6 as the most differentially
expressed genes among samples with
and without 11q23 copy gain or amplification. In MLL-amplified samples, a significant transcriptional up-regulation of
MEIS1, PROML1, ADAM10, NKG2D, and
ITPA was noted. Further analyses, designed to elucidate a possible role of the
11q overexpressed genes (MLL, DDX6,
FLI1, and ETS1) in unselected MDS and
AML samples, revealed a significant up-
regulation of MLL in MDS. Our findings
confirm the MLL gene as a prominent
target of 11q23 amplification and provide
further evidence for an etiologic role for
MLL gain of function in myeloid malignancies. In addition, our results indicate that
the transcriptional program associated
with MLL rearrangements and MLL overexpression displays significant similarities. (Blood. 2004;103:229-235)
© 2004 by The American Society of Hematology
Introduction
Genomic amplification is a frequently encountered acquired genetic aberration in malignant proliferations and typically leads to
inappropriate (over)expression of one or more oncogenes located
within the amplicon (for a review, see Schwab1). Cytogenetically,
genomic amplification is most frequently apparent from the
presence of homogeneously staining regions and double minute
chromosomes. In contrast to solid tumors, genomic amplification is
rarely detected in hematologic malignancies. Genes that have
drawn particular interest in acute myeloid leukemia (AML) and
myelodysplastic syndromes (MDSs), with respect to genomic
overrepresentation, are MYC, ETV6, MLL, and RUNX1 (AML1,
CBFA2) and ribosomal RNA genes.2-9 For any of these genes,
however, an oncogenic role of copy number gain or amplification
in myeloid malignancies has not been demonstrated so far.
Recently, 11q23 overrepresentation was described as a new
cytogenetic entity in myeloid malignancies. In cases with 11q23
amplification, the MLL gene was consistently shown to be amplified,2,5,7,10-14 although double minute chromosomes containing
more distally located sequences have been described occasionally.15,16 Using fluorescence in situ hybridization (FISH) with MLL
flanking probes, 2 distinct patterns were identified: MLL amplification on homogeneously staining regions or double minutes
and MLL low-copy gain due to the retention of MLL copies on
extra or derivative chromosomes 11. In view of the consistent
overrepresentation of MLL in the reported leukemias with 11q23
gain or amplification and given the putative gain of function of
the gene as a result of fusion with various partner genes, this
oncogene was assumed to be a prime target that drives the 11q23
amplicon formation.
Thus far, the role of MLL (and flanking candidate oncogenes) in
the various structural defects leading to 11q23 gain or amplification
has remained largely unexplored. To better characterize the consequences of 11q23 overrepresentation, we studied the expression
patterns of several oncogenes located within the amplified region.
Besides MLL, we analyzed the expression of CBL, DDX6, ETS1,
FLI1, and PLZF, which were selected from a 20-Mb genomic
interval encompassing MLL. Genes that were highly overexpressed
in 11q23-amplified malignancies were then studied in a series of
unselected patients with AML and MDS with diploid 11q23 status
to evaluate their possible contribution to the leukemic phenotype.
From the Center for Medical Genetics, University Hospital Ghent, Belgium;
Laboratory for Leukemia Diagnostics, University Hospital Grosshadern,
Ludwig-Maximilians-University of Munich, München, Germany; Department of
Molecular Medicine, Karolinska Hospital and Institute, Stockholm, Sweden;
Division of Hematology and Oncology, Comprehensive Cancer Center, Arthur
G. James Cancer Hospital and Richard J. Solove Research Institute, Ohio
State University, Columbus; Departments of Clinical Genetics/Cell Biology and
Genetics, Erasmus MC, Rotterdam, The Netherlands; Department of
Hematology and Center for Human Genetics, Cliniques Universitaires SaintLuc, Brussels, Belgium; Department of Human Genetics, CHU Sart Tilman,
Liège, Belgium; Laboratoire d’Hématologie, CHU Toulouse, France; Centre for
Human Genetics, University of Leuven, Belgium.
grant no. G.0310.01, VEO grant 011V1302, and by grant P30 CA16058 from
the National Cancer Institute, Bethesda, MD, and the Coleman Leukemia
Research Fund, St Paul, MN. B.P. is a research fellow of the Fund for Scientific
Research of Flanders. N.Y. is supported by the Fund for Scientific Research of
Flanders (FWO-Vlaanderen). L.M. is partially supported by a grant from the
Salus Sanguinis Foundation. H.B.B. is partially supported by a grant from the
Association for International Cancer Research (no. 99-111).
Reprints: Frank Speleman, Centre for Medical Genetics, University Hospital
Ghent, De Pintelaan 185, 9000 Ghent, Belgium; e-mail: franki.speleman@
ugent.be.
Submitted June 30, 2003; accepted August 22, 2003. Prepublished online as
Blood First Edition Paper, August 28, 2003; DOI 10.1182/blood-2003-06-2163.
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.
Supported by the Fund for Scientific Research of Flanders (FWO-Vlaanderen)
© 2004 by The American Society of Hematology
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
229
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230
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
POPPE et al
Because our current understanding of leukemic transformation
mediated by MLL fusion genes supports a role for MLL gain of
function and because MLL rearranged leukemias were recently
shown to be characterized by specific gene expression profiles,17-20
we explored possible transcriptional similarities in MLL rearranged
and MLL overexpressing samples. To this purpose we investigated
the expression of selected genes, overexpressed in MLL rearranged
leukemias (MEIS1, FLT3, PROML1, ADAM10, CCNA1, LMO2,
NKG2D, and ITPA),17-19 in relation to the genomic overrepresentation and overexpression of MLL.
Patients, materials, and methods
Patient samples
Samples from patients with MDS or AML and MLL copy gain or
amplification were collected retrospectively from 9 genetic centers. Samples
were considered eligible for inclusion based on the presence of 3 or more
copies of the MLL gene, demonstrated by FISH. Samples displaying MLL
rearrangement, as demonstrated either by FISH or Southern blot, were
excluded from the analyses. Table 1 summarizes the characteristics of the
patients. The initial sample set, used to identify 11q23 overexpressed genes,
included normal bone marrow (n ⫽ 9), AML samples with normal karyotypes (n ⫽ 8), and samples with increased MLL copy number (groups A, B,
and C; see “Results,” n ⫽ 31). This sample set was also used to analyze the
expression levels of the genes, overexpressed in MLL rearranged leukemia.
Further quantification of 11q up-regulated genes was performed in a series
of unselected patients, diagnosed with AML (n ⫽ 11) or MDS (n ⫽ 11), and
normal bone marrow samples (n ⫽ 14). Patient characteristics from this
separate patient cohort are given in Table 2.
Conventional and molecular cytogenetics
Unstimulated bone marrow cells were cultured and harvested according to
standard procedures. Karyotypic analysis was performed on G-, R- or
Q-banded metaphases in all cases; karyotypes of patients no. 1 to 6, 8 to 10,
14, 15, 17, 18, 20, 21, 24, and 28 to 30 were further characterized using
spectral karyotyping or multiplex FISH (Table 1). The karyotypes were
described according to the 1995 recommendations of the International
System for Human Cytogenetic Nomenclature (ISCN).21 The following
probes were selected for FISH: the LSI MLL dual-color probe (Vysis,
Downers Grove, IL) and gene-specific 11q probes: RP11-770K18 (DDX6),
RP11-835G21 (FLI1), and RP11-1007G5 (ETS1). To evaluate the extent of
the amplicon and to facilitate the interpretation of the 11q23 copy number,
an ATM gene-specific probe at 11q22.3 (RP11-241D13) was included in the
hybridizations. Clone isolation, labeling, and FISH were performed as
previously described,7 using biotin-16-dUTP and digoxigenin-11-dUTP
(Roche Diagnostics Belgium, Vilvoorde, Belgium) as haptens, or according
to the manufacturer’s recommendations. The identity of the genomic clones
was validated by polymerase chain reaction (PCR), using the primers
designed for real-time reverse transcription–PCR (RT-PCR); in addition,
these clones were tested on normal controls. As each hybridization was
performed with 2 differentially labeled probes, the evaluation of the
hybridization pattern and signal intensity served as a reciprocal internal
control. Hybridization signals in each sample were counted by 3 independent operators and were analyzed and interpreted at the interphase and
metaphase levels. The average number of hybridization signals observed by
the 3 different operators in 100 aneuploid interphase nuclei was calculated
to assign each sample to the different subgroups (“Results”).
RNA isolation, cDNA synthesis, and quantitative real-time PCR
RNA was isolated using the RNeasy Midi Kit (Qiagen, Hilden, Germany)
or the Trizol reagent (Invitrogen, Merelbeke, Belgium) according to the
manufacturer’s instructions. DNase treatment, cDNA synthesis, primer
design, and SYBR Green I RT-PCR were performed as previously
described.22 Reactions were performed on an ABI Prism 5700 Sequence
Detector (Applied Biosystems, Foster City, CA). Real-time RT-PCR data
analysis and expression normalization were performed using multiple
internal control genes, as described elsewhere.23 Positional candidate 11q23
genes were selected from a 20-Mb genomic interval encompassing MLL,
based on the human annotated genome sequence, available in the public
domain (http://genome.ucsc.edu/ and http://www.ensembl.org/). Selected
genes were CBL, DDX6, ETS1, FLI1, and PLZF. HOXA9 expression
analysis was performed to evaluate the functionality of the MLL peptide in
MLL overexpressing samples. Expression analysis of a number of mixedlineage leukemia-specific genes (MEIS1, FLT3, PROML1, ADAM10,
CCNA1, LMO2, NKG2D, and ITPA) was performed to assess possible
similarities between MLL rearranged and MLL overexpressing leukemias.17-19 Primer sequences are available from the Real-Time PCR Primer
and Probe Database, RTPRimerDB, Web site at: http://realtimeprimerdatabase.ht.st (RTPrimerDB ID: 682-696).24
Data analysis
Statistical analysis was performed using SPSS software version 11.0 (SPSS,
Chicago, IL). The nonparametric Mann-Whitney U test was used to
evaluate the significance of difference in mean expression levels between
the patient subgroups. Correlation analyses were performed using the
Spearman rank correlation coefficient. All described P values are 2-sided.
Results
Patient characteristics
Clinical characteristics of the 31 patients are summarized in Table 1.
Median age at diagnosis was 65.0 years (range, 26-84 years); the
male-to-female ratio was 1.6:1.0. All patients were diagnosed with
myeloid malignancies (AML and MDS), most frequently AML M2
(n ⫽ 13) followed by AML M1 (n ⫽ 7); only 5 patients displayed the
typical AML M4 or AML M5 associated with MLL gene rearrangements (patients no. 7, 15, 17, 20, and 27). Cytogenetic and FISH
findings of the complex karyotypes were previously reported for 6
patients (Table 1). Twenty-five additional patients included in this study
showed similar complex patterns of chromosomal rearrangements,
including the high frequency of aberrations involving chromosome 5
(⫺5 or unbalanced rearrangements resulting in loss of 5q). Of the 31
patients included in this study, 4 had received prior chemotherapy or
radiotherapy. In 4 of the AML patients a previous phase of MDS was
documented or suspected clinically or morphologically.
Banding, MLL gene locus FISH, and multiplex-FISH confirmed
the previously reported complex aberrations in the UoC-M1 cell line.25
Characterization of the 11q23 amplicon and definition of
patient subgroups
FISH characterization of the 11q23 amplicon using gene-specific
probes for MLL, DDX6, FLI1, and ETS1 (covering approximately
10 Mb) revealed that in all cases these 4 genes were invariably
implicated in both low copy number 11q23 gains and in high-level
amplifications. Across the various samples, the 11q23 copy number
varied considerably from 3 copies, due to unbalanced rearrangements, through significantly increased but countable copy numbers
(5-10 MLL copies) up to a hardly countable number of fluorescent
signals in samples containing homogeneously staining regions or
double minute chromosomes. Based on the number of MLL copies
and the results from routine cytogenetic analysis, 3 different patient
groups were defined. Group A (n ⫽ 16) included samples with 3 to
5 copies of the MLL gene (low copy gain) due to retention of MLL
on abnormal chromosomes with extra segments from 11q; group B
(n ⫽ 11) comprised samples with MLL amplification (5-10 MLL
copies), not evidenced by banding analyses; and group C (n ⫽ 4)
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BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
MLL OVEREXPRESSION IN 11q23-AMPLIFIED AML AND MDS
231
Table 1. Clinical characteristics and cytogenetic findings in patients with MLL amplification
Case
no.
Age,
y
Sex
Diagnosis*
Group†
Type
Karyotype‡
1
68
M
MDS/RAEB
A
—
47,XY,der(1)t(1;20)(p34;q?),der(2)(:2p?1232q11.2::17::7q3137qter),der(7)(:2q11.232q37::7p22
Ref§
7 (30)
37q22),der(11)(qter3q?::p?3qter),⫹der(11)del(11)(p12)del(11)(q13q23),der(17)t(17;20),⫹der
(17),⫺20
2
63
M
AML M0
C
—
45,XY,der(2)t(2;18)(p13;p11.?3),del(5)(q13q33),der(7)t(7;11),dup(11)(pter3q24::q?243q?),der
7 (26)
(11;12)(q10:q10),der(13)t(7:13)(q?;q21),⫹der(13)t(7;13),⫺16,der(17)t(2;17)(p13;q21),der(17)
t(7;17)(?;q24),der(18)t(18;20)(q?;p11.2),der(20)t(7;20)
3
76
M
AML M2
C
—
46,XY,dic(1;19)(19pter319qter::1p3631p13::19p12319q13.1::1p3631qter),der(5)t(5;17)(q13;
7 (35)
q11.2⬃q22),dup(11)(pter3q24::q?243q?),der(17)r(5;17),⫺19,i(22)(q10),⫹i(22)
4
75
F
AML M2
C
CT,
46,XX,del(3)(p12p21),del(5)(q24q34),i(9)(p10),del(11)(q14),⫹1⬃23dmin
—
45,XY,dic(5;18)(q13;p11.2),r(11)(p15q25),⫹dicr(11;11)(p13q25;p13q25),⫺13,ins(16;11)(p13;?),
—
RT
5
76
M
AML M2
B
PM?
der(17)t(13;17)(q?;q25)
6
64
F
AML M2
A
—
49,XX,⫹X,der(4)t(4;18)(q24;q?),⫹der(4)t(4;18)(q24;q?),del(5)(q13q31),⫺7,⫹8,⫹10,der(12)t(4;
—
12)(q24;p13),der(16)t(11;16)(?;q21),der(18)del(18)(p11)del(18)(q11)
7
61
M
AML M4
A
—
46,XY,der(11)t(11;11)(p15;q11)
—
8
63
M
AML M1
B
—
48,XY,der(4)t(4;21)(q35;q?),⫹5,del(5)(q13q33),der(6)(6pter36q11::17?317?::21?321?::6q11
—
9
58
F
MDS/RAEB
A
RT
47⬃48,X,t(X;5)(q13;q13),del(5)(q15q31),⫹6,der(11)dup(11)(q13q23)t(11;21)(q25;q11),⫹r(11)x3,
36qter),der(8)t(8;21)(p23;q?),⫹der(11),⫹r(11),⫺17,der(21)t(17;21)(?;q11.2)
—
⫺18,⫺21
10
72
M
AML M1
A
—
47⬃49,XY,⫺2,dic(5;17)(q11;p11),der(7)t(2;7)(q11;q11)t(2;12)(q37;q12),⫹8,⫹r(11)x3,der(12)t(2;12)
—
(?;q12),⫹13
11
71
M
AML
A
RE
49,XY,der(2)t(2;16)(p25;?),⫺5,r(5),⫹der(8)t(8;15)(q13;q25),⫹10,der(11)t(11;12),⫹der(11)x3,⫺12,
—
⫹13,der(15)t(8;15)(q?;q25),der(16)t(12;16)(?;q24),⫺17,⫺18
12
66
F
MDS/RAEB
A
—
40,XX,⫺2,⫺3,⫺5,der(6;16)dic(6;16)(q16;p13)t(2;16)(?;q24),r(7)(p12q11.2),i(11)(q10),der(12)t(12;21) —
13
67
M
AML M2
B
—
45,XY,del(5)(q13q31),der(11)t(11;11)(q23;q?),⫺15
—
14
65
F
AML M2
A
—
43,X,⫺X,der(5)t(5;7)(p11;q32)del(5)(q13q31),der(7)t(5;7)(p11;q32),qdp(11)(q23q25),⫺17,⫺18
—
15
61
M
AML M4
B
CT
46,XY,del(5)(q13q31),der(22)ins(22;11)(q11;q?q?)
—
16
84
F
AML M2
A
—
44,XX,der(5)t(5;17)(q11;q11),der(11)t(11;22)(q23;q11)dup(11;22)(q22q23;q11q13),⫹der(11)dup
—
17
57
F
AML M4
B
—
48,XX,⫹der(11),⫹der(11)
—
18
43
M
AML M2
B
PM?
44,XY,del(5)(q13q33),dic(6;17)(p23;p13),der(11),⫺18
—
19
64
M
AML M1
A
PM
46,XY,der(17)t(11;17)(q13;p13)
—
20
26
F
AML M4
A
—
46,XX,del(5)(q13q33),der(11)t(11;17)(p15;q21),⫹r(11)(p11q25),⫺17
—
21
63
M
AML M2
A
—
42,XY,der(5)del(5)(p15)del(5)(q13),⫺7,der(12)t(12;16)(p11;p11),ins(12;5)(p13;q?q?),⫺16,⫺18,
—
(p12;q?)t(3;21)(p22;?),⫺13,der(15)t(3;15)(q21;p10),der(17)t(13;17)(q11;p12),der(21;22)(q10;q10)
(11)(q22q25)t(11;22)(q23;q11)dup(11;22)(q22q23;q11q13),dic(12;15)(p11;p11),⫺17,⫺22
der(22;22)(p10;p10)
22
82
M
AML M1
A
—
44,XY,del(5)(q31),der(5)t(5;17)(q13;q?),trp(11)(q14q25),⫺17,der(17)t(5;17)(q?;q25),⫺8
—
23
46
F
MDS/
B
CT
44,XX,⫺5,der(7)t(5;7)(p11;p11)t(5;7)(q13;q22)t(5;7)(q23;q34),⫺11,der(18)t(11;18)(?;p11.2)
—
24
46
F
AML M2
B
—
42,X,⫺X,⫺3,der(5)t(5;17)(q11;q11)ins(5;11)(p13;?),⫺9,del(11)(p11.2),r(11),⫹der(11)dup(11)
—
RAEB-T
(q?q?)t(11;21)(q?;q?)t(3;21)(q11;q?),⫺17,⫺21
25
80
M
AML M0
B
—
42⬃43,XY,⫺3,der(5)t(5;19)(q11.2;?),del(6)(p22),der(9)t(6;9)(p22;q34),der(11;15)(q10;q10),der(12)
—
t(12;19)(p11;?),⫺17,⫺19,der(20)t(11;20)(q2?3;q13.3)
26
74
F
AML M1
B
—
49,X,t(X;1)(p11;p11),t(1;22)(q31;q12),der(3)t(3;11)(q12;q21⬃23),del(5)(q21q31),der(6)(17pter3
—
17p1?::15q2?13q11::14q323q11::6p2336qter),der(7)t(6;7)(p23;q22),⫹der(8)t(3;8)(q21;q24),
⫹r(11),⫺14,⫹15,ider(15)(q10)t(11;15)(q13;q?)x2,der(17)t(15;17)(q2?1;p1?),⫹22
27
58
M
AML M5A
C
—
45,XY,ins(4;11)(q2?;q?q?),del(5)(q13q33),dup(11)(q?q?),der(12)t(11;12)(q?;p11.2)hsr(11)(q23),⫺16
—
28
80
M
AML M1
A
—
44,X,⫺Y,del(5)(q13q33),der(5)t(5;11)(q35;q13),⫺7,der(21)(21pter321q2?1::7?::21q?::7?::21q?
10 (3)
29
48
M
AML M2
B
PM
45⬃46,XY,der(5)t(5;17)(q11;q11),r(11;11)(p15q25;11q?),⫺17,⫹mar
10 (15)
30
68
F
AML M2
A
—
45,XX,inv(4)(p15.3q23),inv(5)(p15.3q11.1),der(5)del(5)(q13q35)t(5;13)(q13;q21),der(13)t(5;13)
10 (24)
31
77
M
AML
A
—
46,XY,t(1;21)(q31;q21),der(7)t(7;11)(p22;q23)dup(11)(q23q24),dup(11)(q23q24),⫹dup(11)(q23q24)
UoC-M1
—
—
—
—
—
43X,⫺Y,add(5)(q13),dic(5;9)(p15;p13),⫺7,⫹der(9)t(9;19)(q11;q11),dic(9;?;16;?)(9qter⫺9p1?3::
::7?::21q?::7?)
(q35;q21),der(16)t(10;16)(q22;p13),⫺18,der(19)t(11;19)(q13;p13.3)
24
?::16p11⫺16q22::?),⫺11,⫹16,dic(16;21)(q11;p12),der(17)t(7;17)(p14;p12),⫺19,⫺19,⫹mar1,
⫹mar2,⫹mar3
RAEB indicates refractory anemia with excess blasts; CT, prior chemotherapy; RT, prior radiotherapy; PM: prior MDS; RE, relapse; RAEB-T, refractory anemia with excess
blasts in transformation; and —, not applicable.
*Disease phenotype according to the French-American-British classification.
†Group as defined in “Results.”
‡Dominant clonal aberration or composite karyotype. Karyotypes of patients no. 1-6, 8-10, 14, 15, 17, 18, 20, 21, 24 and 28-30 were characterized using spectral
karyotyping or multiplex-FISH.
§References reporting previously published cases; case numbers identifying the individual patient samples in the respective reports are indicated in parentheses.
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POPPE et al
Table 2. Clinical characteristics and cytogenetic findings of patients
included in the cohort to determine the significance of differential
MLL, DOX6, FLI1, and ETS1 expression in normal marrow, MDS,
and AML
Case
no.
Age,
y
Sex
1
64
M
MDS/RAEB
45⬃46, XY, ⫺5, ⫺7, ⫺14, ⫺16, ⫺19, ⫺20,
2
69
F
MDS/RAEB-T
46,XX
3
61
M
MDS/RAEB
46,XY
4
69
M
MDS/RA
46,XY
5
70
F
MDS/RAEB
46,XX
6
78
M
MDS/RA
46,XY
7
48
M
MDS/RAEB
46,XY
8
76
F
MDS/RAEB-T
46,XX,der(3)t(3;?)(q21;?),t(9;22)(q34;q11.2)
9
55
F
MDS/RA
46,XX
10
36
M
MDS/RA
46,XY
11
38
F
MDS/RA
46,XX
12
71
F
AML M2
46,XX
13
78
F
sAML
46,XX
14
51
M
AML M1
47,XY,⫹13
15
78
F
AML M0
46,X,add(X)(p2?2),del(20)(q11.2)
16
76
F
AML M2
47,XX,i(8q),⫹der(8)t(1;8)(q12;p23)
17
79
M
AML M4
48⬃50,XY,del(5)(q31q33),⫺12, ⫺13, ⫺14,
Diagnosis*
Karyotype†
⫺21, ⫹6⬃7mar
⫺16, ⫺18, ⫺20, ⫺21, ⫹9⬃11mar
18
68
M
AML M5
46,XY
19
45
F
AML M1
46,XX
20
45
F
AML M4
46,XX,add(16)(q23-24)
21
64
M
AML M1
46,XY
22
66
F
AML M2
46,XX
RA indicates refractory anemia; sAML, secondary AML.
*Disease phenotype according to the French-American-British classification.
†Dominant clonal aberration.
contained samples with a high number of MLL signals that could
not be reliably counted, resulting from cytogenetically detectable
homogeneously staining regions or double minute chromosomes
(Table 1). These 3 arbitrarily defined groups, associated with an
increased 11q23 copy number, were further used to determine the
significance of the expression levels of the different oncogenes
studied in relation to the 11q23 amplification status.
Figure 1. Correlation of oncogene overexpression to 11q23 copy number
status. The geometric averages of the normalized expression of MLL, DDX6, FLI1,
PLZF, CBL, and ETS1 in the different patient groups (“Results”); group A (f), group B
(u), and group C (䡺). P values indicate the significance of the difference in observed
expression in 2 by 2 analyses of the different patient groups, as identified by the
Mann-Whitney test. Significant P values are indicated in bold.
the functionality of the MLL transcript in MLL-amplified cases.
Assuming the MLL protein is functional, increased MLL expression should result in increased transcription of one or more of its
target genes, as anticipated from its physiologic role.26 To this
purpose, expression of HOXA9, a well-known MLL target gene,
was analyzed. HOXA9 was significantly up-regulated in samples
with high-level MLL amplification (Figure 2), underscoring the fact
that the MLL peptide encoded by the amplified allele maintained its
property of supporting homeotic transcription.
Similarities in MLL-overexpressing and
MLL-rearranged leukemias
Because recent reports identified a number of genes that were
specifically up-regulated in leukemias with an MLL rearrangement,17-19 we investigated a possible relationship between increased MLL expression and expression of a number of these mixed
lineage leukemia-associated genes: MEIS1, FLT3, PROML1,
ADAM10, CCNA1, LMO2, NKG2D, and ITPA. Five of these 8
Expression analysis of MLL, DDX6, CBL, ETS1, PLZF, and FLI1
in relation to the 11q23 status
Expression levels of 6 selected 11q23 oncogenes (MLL, DDX6,
CBL, ETS1, PLZF, and FLI1) were compared to the genomic 11q23
status, according to the 3 different subgroups, as just defined. In a
first analysis, we compared samples with MLL low copy number
gains (group A) versus samples containing high-level MLL amplification (groups B and C combined as a single subset). This revealed
a highly significant transcriptional up-regulation of MLL, DDX6,
FLI1, and CBL (P ⫽ .00021, P ⫽ .00053, P ⫽ .0072, and
P ⫽ .0063, respectively) in cases with increased 11q23 copy
number. Subsequently, the expression patterns for all 6 oncogenes
were studied in the 3 separate patient groups (Figure 1). In these
analyses, highly significant expression differences were identified
consistently only for MLL and DDX6, when considering all 2-by-2
comparisons of groups A, B, and C. The MLL overrepresenting cell
line UoC-M1,25 containing 4 MLL copies, revealed an increased
MLL expression, comparable to group B samples.
MLL overexpression results in MLL gain of function
Limited patient material precluded functional tests at the protein
level and, therefore, we adopted an indirect approach for studying
Figure 2. Correlation of expression of HOXA9 and of selected mixed-lineage
leukemia-specific genes with 11q23 copy number status. The geometric averages of the normalized expression of the different genes in the different patient
groups (“Results”); group A (f), group B ( ), and group C (䡺). P values indicate the
significance of the difference in observed expression in 2-by-2 analyses of the
different patient groups, as identified by the Mann-Whitney test. Significant P values
are indicated in bold.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
genes, PROML1, ADAM10, NKG2D, ITPA, and MEIS1, were
strongly up-regulated, specifically in the samples displaying highlevel 11q23 amplification (Figure 2). In addition, considering all
group A, B, and C samples, the expression of these 5 genes was
significantly correlated to MLL expression (Spearman ␳ and P
values: PROML1: 0.52, P ⫽ .003; ADAM10: 0.68, P ⫽ .0003;
NKG2D: 0.51, P ⫽ .004; ITPA: 0.74, P ⫽ .0003; MEIS1: 0.37,
P ⫽ .040).
Expression analysis of MLL, DDX6, ETS1, and FLI1 in
unselected AML and MDS samples
Because the expression analysis from 11q23-amplified leukemias
could point to a role for gain of function of MLL, DDX6, and to a
lesser extent of FLI1 and ETS1, in myeloid malignancies in general
(ie, irrespective of the 11q23 copy number), we studied the
expression levels of these genes in a separate series of normal
marrow (n ⫽ 14), MDS (n ⫽ 11), and AML (n ⫽ 11) samples,
without 11q23 rearrangements. Significantly increased MLL expression in malignant samples was noted (P ⫽ .028), whereas the
expression of DDX6, FLI1, and ETS1 was similar in normal and
malignant bone marrow. Increased MLL expression was especially
noted in MDS samples (P ⫽ .00068), whereas the level of MLL
expression in normal marrow and AML samples did not differ
significantly (Figure 3).
Discussion
The introduction of FISH with MLL-specific probes in routine
diagnostic analyses and the application of multicolor investigations
for dissection of complex karyotypic changes in AML and MDS
have led to the identification of a new patient subgroup with 11q23
overrepresentation or amplification, consistently associated with an
increased MLL copy number. The molecular consequences of these
particular 11q23 imbalances have remained largely unexplored
thus far. Here, we investigated 31 AML and MDS samples with
known MLL copy number gain or MLL high-level amplification.
First, 11q23 gain and amplification patterns were assessed in
further detail using FISH with probes for selected oncogenes, that
is, MLL, DDX6, FLI1, and ETS1, covering a considerable genomic
distance within 11q23. Three distinct patterns of gain or amplification were discerned: group A (n ⫽ 16) showed low copy gain of
unrearranged MLL due to the presence of additional chromosome
11 material on derivative, marker, or ring chromosomes; group B
(n ⫽ 11) was characterized by MLL amplification, unnoticed on
Figure 3. Expression of MLL and HOXA9 in unselected patient samples.
Geometric average of the MLL (䡺) and HOXA9 (u) expression in normal marrow
(n ⫽ 14), MDS (n ⫽ 11), and AML (n ⫽ 11) samples, with normal 11q23 copy number
in the absence of an MLL gene rearrangement. P values indicate the significance of
the difference in observed expression in 2-by-2 analyses of the different patient
groups, as identified by the Mann-Whitney test.
MLL OVEREXPRESSION IN 11q23-AMPLIFIED AML AND MDS
233
karyotypic evaluation, whereas group C (n ⫽ 4) contained samples
with a high level of MLL amplification, cytogenetically characterized by the presence of homogeneously staining regions or double
minute chromosomes.
A major goal of this study was to investigate the consequences
of 11q23 gain and amplification on the expression level of MLL, the
presumed prime target of the amplicon, and 5 other candidate genes
located within the implicated 11q23 segment, DDX6, FLI1, ETS1,
CBL, and PLZF. The highly differential expression levels of DDX6
and MLL, in relation to the 11q23 status, suggest that both MLL and
DDX6 may drive the amplification process. Support for the
presence of functional MLL protein, albeit indirect, and for a gain
of function mechanism for MLL in cases with MLL amplification
was demonstrated by significant correlation between MLL and
HOXA9 expression, because HOXA9 is a well-known downstream
(positively up-regulated) target of MLL. MLL gain of function is
also the mechanism thought to mediate leukemic transformation in
MLL-rearranged leukemias. Recently, these leukemias were shown
to be characterized by a specific gene expression profile.17-20 Here,
we showed that some of the mixed-lineage leukemia-associated
genes are significantly overexpressed in MLL-overexpressing
samples, thus suggesting that the transcriptional consequences of
MLL rearrangement and overexpression display noteworthy similarities. Of note, in a recent report several genes were identified that
are up-regulated in B- as well as T-lineage MLL-rearranged
ALLs.18 Intriguingly, we found a significant up-regulation in
MLL-amplified samples for genes that are overexpressed in both Tand B-lineage MLL-rearranged leukemias (such as MEIS1, HOXA9,
and ADAM10), whereas this correlation was absent for myeloid
genes that are specifically overexpressed in MLL-rearranged BALLs, such as FLT3 and CCNA1. Although the current number of
patients and genes included in this study remains limited, these data
seem to underscore the activation of similar genes in MLLrearranged and -overexpressing leukemias. Because the identification of a specific transcriptional program associated with MLL
rearrangement is expected to facilitate the identification of novel
targets for therapeutic intervention,27 these targets might also prove
to be relevant in MLL-overexpressing samples. This is of particular
importance in view of the poor prognosis often associated with
MLL-rearranged leukemias and the extremely poor survival of
patients with AML/MDS with MLL amplification. These clinical
and expression similarities for MLL-rearranged and MLLoverexpressing leukemias might be explained by a common
transcriptional program underlying the resistance to conventional
chemotherapy, which could account for the adverse clinical course
typifying these leukemias. Further studies requiring larger numbers
of homogeneously treated patients are required to determine the
relationship between MLL expression and prognosis.
Further support for a specific role of MLL comes from our
preliminary analysis of expression levels of both MLL and the other
selected 11q23 genes in unselected AML and MDS samples. From
these analyses, a role for gain of function for DDX6, FLI1, and
ETS1 in myeloid malignancies is unlikely because neither of these
genes displays significant expression differences between normal
marrow, MDS, and AML samples. In contrast, MLL was shown to
be significantly up-regulated in MDS (P ⫽ .00068). The patterns of
expression and correlation profiles of MLL and HOXA9 are in
keeping with a fundamental role for both genes in leukemic
transformation. Evidently, because these data are derived from
relatively small and heterogeneous sample sets, the presented data
need confirmation from larger studies. However, these results could
point to an important role for MLL gain of function in MDS, or at
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
234
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
POPPE et al
least in a subset of MDS patients. Consequently, MLL overexpression might prove to be an interesting prognostic indicator or
even a target for therapeutic intervention in MDS. Additionally,
the presented results encourage further research aimed at
analyzing the transforming potential of MLL gain of function, as
a consequence of increased MLL expression, but also as a result
of activating mutations.
The observed increased expression of DDX6 in 11q23 amplified
leukemias is also of possible biologic interest. DDX6 belongs to a
family of RNA helicase genes (DEAD box proteins), which are
believed to be involved in a number of developmental processes.
DDX6 as well as other DEAD box protein family members have
been shown to be involved in malignant transformation, particularly as a result of increased expression. DDX6 was originally
cloned from a B-cell lymphoma cell line (RC-K8), in which it is
overexpressed due to juxtaposition to the IGH enhancer and has
been suggested to be implicated in colorectal carcinogenesis.28,29
DDX1 is often coamplified in MYCN-amplified tumors, most
notably in neuroblastoma.30 Yet another DEAD box protein
family member, DDX10, is involved in MDS and AML,
particularly in therapy-related cases, by fusion to NUP98 as a
consequence of the inv11(p15q22).31 Similarly to DDX1, DDX6
could play an essential role in 11q23-amplified leukemias and
further studies concerning the role of this gene in this patient
subset should be performed.
The presence of a patient group with multiple 11q23 copies
without cytogenetic evidence for amplification (group B) is intriguing. In a subset of patients this is in keeping with the presence of
individual MLL signals on multiple der11 chromosomes. In other
patients, however, multiple clustered signals were apparent. Given
the extremely rapid disease progression in these patients, these
samples might represent an intermediate stage toward high-level
amplifications observed in group C. A similar mechanism has been
suggested for MYCN amplification in neuroblastoma.32
Although our analyses have focused on the role of MLL and
other adjacent 11q oncogenes in 11q23 overrepresentation, there is
some evidence for a more distally located amplicon. In 2 separate
reports, Crossen et al15 and Sait et al16 have identified 2 patients
with double minute chromosomes containing ETS1, but not MLL.
Arguably, 11q amplification comprises 2 different critical regions:
the more predominant targeting MLL and a more rare type
involving more distally located genes. Because ETS1 was amplified in the 2 described patients,15,16 this gene might seem an
attractive candidate oncogene involved in these 11q23 amplifications. On the other hand, in our series, ETS1 was only overexpressed in 3 of the 4 samples characterized by high-level ETS1
amplification. Therefore, we consider it unlikely that ETS1 is the
primary target of these more distally localized amplifications.
In conclusion, our analyses, which addressed the functional
consequences of oncogene amplification in AML and MDS, have
revealed that genomic amplification in these malignancies behaves
in a manner comparable to that in solid tumors and other
hematologic malignancies with an obvious correlation between
copy number increases and increased gene expression. MLL and
DDX6 showed a highly differential expression between samples
with and without 11q23 gain and therefore were retained as key
targets of 11q23 amplification. MLL overexpression was shown to
result in an MLL gain of function because it was associated with
increased expression of one of its physiologic downstream targets,
HOXA9. In addition, transcriptional similarities between MLL
amplified and MLL rearranged leukemias were identified and a
possible role for MLL overexpression as an early event in
malignant transformation was described.
Acknowledgment
This text presents research results of the Belgian program of
Interuniversity Poles of Attraction initiated by the Belgian State,
Prime Minister’s Office, Science Policy Programming. Scientific
responsibility is assumed by the authors.
References
1. Schwab M. Oncogene amplification in solid tumors. Semin Cancer Biol. 1999;9:319-325.
2. Andersen MK, Christiansen DH, Kirchhoff M,
Pedersen-Bjergaard J. Duplication or amplification of chromosome band 11q23, including the
unrearranged MLL gene, is a recurrent abnormality in therapy-related MDS and AML, and is
closely related to mutation of the TP53 gene and
to previous therapy with alkylating agents. Genes
Chromosomes Cancer. 2001;31:33-41.
3. Andreasson P, Johansson B, Billström R, Garwicz S, Mitelman F, Höglund M. Fluorescence in
situ hybridization analyses of hematologic malignancies reveal frequent cytogenetically unrecognized 12p rearrangements. Leukemia. 1998;12:
390-400.
4. Fonatsch C, Nowotny H, Pittermann-Höcker E, et
al. Amplification of ribosomal RNA genes in acute
myeloid leukemia. Genes Chromosomes Cancer.
2001;32:11-17.
5. Michaux L, Wlodarska I, Stul M, et al. MLL amplification in myeloid leukemias: a study of 14 cases
with multiple copies of 11q23. Genes Chromosomes Cancer. 2000;29:40-47.
6. Streubel B, Valent P, Lechner K, Fonatsch C. Amplification of the AML1(CBFA2) gene on ring chromosomes in a patient with acute myeloid leukemia and a constitutional ring chromosome 21.
Cancer Genet Cytogenet. 2001;124:42-46.
7. Van Limbergen H, Poppe B, Michaux L, et al.
Identification of cytogenetic subclasses and re-
curring chromosomal aberrations in AML and
MDS with complex karyotypes using M-FISH.
Genes Chromosomes Cancer. 2002;33:60-72.
8. Nowell P, Finan J, Dalla Favera R, et al. Association of amplified oncogene c-myc with an abnormally banded chromosome 8 in a human leukaemia cell line. Nature. 1983;306:494-497.
9. Fugazza G, Bruzzone R, Puppo L, Patrone F,
Sessarego M. Amplified c-MYC sequences localized by fluorescence in-situ hybridization on
double minute chromosomes in acute myeloid
leukemias. Leuk Res. 1997;21:703-709.
10. Mrózek K, Heinonen K, Theil KS, Bloomfield CD.
Spectral karyotyping in patients with acute myeloid leukemia and a complex karyotype shows
hidden aberrations, including recurrent overrepresentation of 21q, 11q, and 22q. Genes Chromosomes Cancer. 2002;34:137-153.
11. Schoch C, Haferlach T, Bursch S, et al. Loss of
genetic material is more common than gain in
acute myeloid leukemia with complex aberrant
karyotype: a detailed analysis of 125 cases using
conventional chromosome analysis and fluorescence in situ hybridization including 24-color
FISH. Genes Chromosomes Cancer. 2002;
35:20-29.
12. Avet-Loiseau H, Godon C, Li JY, et al. Amplification of the 11q23 region in acute myeloid leukemia. Genes Chromosomes Cancer. 1999;26:166170.
13. Cuthbert G, Thompson K, McCullough S, et al.
MLL amplification in acute leukaemia: a United
Kingdom Cancer Cytogenetics Group (UKCCG)
study. Leukemia. 2000;14:1885-1891.
14. Streubel B, Valent P, Jager U, et al. Amplification
of the MLL gene on double minutes, a homogeneously staining region, and ring chromosomes in
five patients with acute myeloid leukemia or myelodysplastic syndrome. Genes Chromosomes
Cancer. 2000;27:380-386.
15. Crossen PE, Morrison MJ, Rodley P, Cochrane J,
Morris CM. Identification of amplified genes in a
patient with acute myeloid leukemia and double
minute chromosomes. Cancer Genet Cytogenet.
1999;113:126-133.
16. Sait SNJ, Qadir MU, Conroy JM, Matsui S-I,
Nowak NJ, Baer MR. Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndrome: identification of new amplification regions by fluorescence in situ hybridization
and spectral karyotyping. Genes Chromosomes
Cancer. 2002;34:42-47.
17. Armstrong SA, Staunton JE, Silverman LB, et al.
MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002;30:41-47.
18. Ferrando AA, Armstrong SA, Neuberg DS, et al.
Gene expression signatures in MLL-rearranged
T-lineage and B-precursor acute leukemias:
dominance of HOX dysregulation. Blood. 2003;
102:262-268.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 1 JANUARY 2004 䡠 VOLUME 103, NUMBER 1
19. Rozovskaia T, Ravid-Amir O, Tillib S, et al. Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements.
Proc Natl Acad Sci U S A. 2003;100:7853-7858.
24.
20. Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia
by gene expression profiling. Cancer Cell. 2002;
1:133-143.
25.
21. Mitelman F, ed. ISCN 1995. An International System for Human Cytogenetic Nomenclature.
Basel, Switzerland: Karger; 1995.
26.
22. Vandesompele J, De Paepe A, Speleman F.
Elimination of primer-dimer artifacts and genomic
coamplification using a two-step SYBR green I
real-time RT-PCR. Anal Biochem. 2002;303:
95-98.
23. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RTPCR data by geometric averaging of multiple in-
27.
28.
MLL OVEREXPRESSION IN 11q23-AMPLIFIED AML AND MDS
ternal control genes. Genome Biol. 2002;3:
research 34.1-34.11.
Pattyn F, Speleman F, De Paepe A, Vandesompele J. RTPrimerDB: the real-time PCR primer
and probe database. Nucleic Acids Res. 2003;31:
122-123.
Allen RJ, Smith SD, Moldwin RL, et al. Establishment and characterization of a megakaryoblast
cell line with amplification of MLL. Leukemia.
1998;12:1119-1127.
Yu BD, Hess JL, Horning SE, Brown GAJ, Korsmeyer SJ. Altered Hox expression and segmental
identity in Mll-mutant mice. Nature. 1995;378:
505-508.
Armstrong SA, Kung AL, Mabon ME, et al. Inhibition of FLT3 in MLL: validation of a therapeutic
target identified by gene expression based classification. Cancer Cell. 2003;3:173-183.
Akao Y, Seto M, Yamamoto K, et al. The RCK
gene associated with t(11;14) translocation is distinct from the MLL/ALL-1 gene with t(4;11) and
235
t(11;19) translocations. Cancer Res. 1992;52:
6083-6087.
29. Nakagawa Y, Morikawa H, Hirata I, et al. Overexpression of rck/p54, a DEAD box protein, in human colorectal tumours. Br J Cancer. 1999;80:
914-917.
30. Squire JA, Thorner PS, Weitzman S, et al. Coamplification of MYCN and a DEAD box gene
(DDX1) in primary neuroblastoma. Oncogene.
1995;10:1417-1422.
31. Arai Y, Hosoda F, Kobayashi H, et al. The
inv(11)(p15q22) chromosome translocation of de
novo and therapy-related myeloid malignancies
results in fusion of the nucleoporin gene, NUP98,
with the putative RNA helicase gene, DDX10.
Blood. 1997;89:3936-3944.
32. Corvi R, Savelyeva L, Schwab M. Duplication of
N-MYC at its resident site 2p24 may be a mechanism of activation alternative to amplification in
human neuroblastoma cells. Cancer Res. 1995;
55:3471-3474.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2004 103: 229-235
doi:10.1182/blood-2003-06-2163 originally published online
August 28, 2003
Expression analyses identify MLL as a prominent target of 11q23
amplification and support an etiologic role for MLL gain of function in
myeloid malignancies
Bruce Poppe, Jo Vandesompele, Claudia Schoch, Charlotta Lindvall, Krzysztof Mrózek, Clara D.
Bloomfield, H. Berna Beverloo, Lucienne Michaux, Nicole Dastugue, Christian Herens, Nurten Yigit,
Anne De Paepe, Anne Hagemeijer and Frank Speleman
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