Cancer Genetics and Cytogenetics 170 (2006) 171e174

Short communication

High-level amplification of the RUNX1 gene in two cases

of childhood acute lymphoblastic leukemia
Zaida Garcı́a-Casadoa, José Cerveraa, Amparo Verdeguerb, Maria Tassoc, Ana Valenciaa,
Juan C. Pajueloa, Armando V. Mena-Durana, Eva Barragánd, Margarita Blanesa,
Pascual Boluferd, Miguel A. Sanza,*
a
Hematology Service, University Hospital La Fe, Avenida Campanar, 21, 46009 Valencia, Spain
b
Unit of Pediatric Oncology, University Hospital La Fe, Valencia, Spain
c
Division of Pediatric Oncology, Alicante University Hospital, Carretera San Vicente del Raspeig s/n, 03690 San Vicente del Raspeig, Alicante, Spain
d
Molecular Biology Laboratory (Department of Medical Biopathology), University Hospital La Fe, Valencia, Spain
Received 8 March 2006; received in revised form 10 May 2006; accepted 23 May 2006

Abstract The RUNX1 (alias AML1) gene is involved in several patterns of chromosomal translocations and
rearrangements associated with human acute leukemia. Often, multiple signals for AML1 have been
observed in childhood acute lymphoblastic leukemia (ALL) due to frequent polysomy of chromo-
some 21 in this leukemia. Additionally, high-level amplification of AML1, in the absence of polys-
omy of chromosome 21, has been reported in childhood ALL. We report two new cases of
childhood ALL, without a ETV6/RUNX1 (alias TEL/AML1) rearrangement, showing high-level am-
plification of the AML1 gene detected by fluorescence in situ hybridization and comparative geno-
mic hybridization analysis. The first case was an 11-year-old girl with 7e12 signals for AML1 in
nearly 84% of the cells, and the loss of a TEL allele. In the second patient, a 6-year-old girl, mul-
tiple copies of the AML1 gene were also observed in 99% of the cells, although no deletion of TEL
was found. The similarity in the clinicobiologic features of all the cases with this abnormality points
to an emerging molecular cytogenetic subgroup of B-cell precursor ALL and suggests a possible
dosage effect of AML1 in the pathogenesis of leukemia. Ó 2006 Elsevier Inc. All rights reserved.

1. Introduction cytogenetic subgroup in ALL and suggesting a possible

The RUNX1 gene (alias AML1, the form used here) is lo-
cated on 21q22 and encodes a DNA-binding protein that
contains a runt homology domain. This gene is involved
in several patterns of chromosomal translocations and rear-
rangements associated with human acute leukemia. The
most common structural abnormality is t(12;21)(p13;q22),
present in ~25% of childhood B-cell acute lymphoblastic
leukemia (B-ALL) [1e4], and resulting in the ETV6/
RUNX1 (alias TEL/AML1, the form used here) fusion gene
[5]. But the AML1 gene is also involved in translocation
t(8;21) in acute myeloid leukemia (AML) and in transloca-
tion t(3;21) in myelodysplasia and in the blast phase of
chronic myeloid leukemia (CML). Recently, amplification
of the 21q22 region including AML1 has been reported in
childhood ALL, pointing to an emerging molecular
* Corresponding author. Tel.: and fax: þ34-6-386 8757.
E-mail address: msanz@uv.es (M.A. Sanz).
0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2006.05.011
dosage effect of AML1 in the pathogenesis of leukemia
[6e12].
We report here two cases of childhood ALL that pre-
sented high-level amplification of the AML1 gene detected
by fluorescence in situ hybridization (FISH) and compara-
tive genomic hybridization (CGH).
2. Case report
2.1. Case 1
An 11-year-old girl was admitted to the hospital because
of asthenia for 10e15 days, accompanied by fever in the
last few days. Her grandfather had died of hepatocellular
carcinoma. On physical examination she was pale, with iso-
lated petechiae and cutaneous cafe-au-lait stains on her face
and right flank. She also had a hard 6-cm hepatomegaly and
splenomegaly. Routine blood analysis showed hemoglobin
at 79 g/L, platelets 15
109/L, white blood cell (WBC)
count 1.49
109/L (absolute neutrophil count ANC
172 Z. Garcı́a-Casado et al. / Cancer Genetics and Cytogenetics 170 (2006) 171e174
9
0.11
10 /L, blasts 12%), lactate dehydrogenase LDH
798 U/L, aspartate aminotransferase AST 150 U/L, alanine
aminotransferase ALT 117 U/L, and no alteration in coag-
ulation tests. Bone marrow (BM) aspirate revealed the pres-
ence of 96% small blast cells with very scanty agranular
cytoplasm and irregular nucleus without evident nucleoli.
Cerebrospinal fluid obtained by lumbar spine puncture
showed no infiltration of central nervous system. She was
diagnosed with ALL of high risk because of her age. Immu-
nophenotyping yielded a classification for this leukemia as
common B-ALL. She started chemotherapy following the
SHOP-99 protocol (prednisone, vincristine, daunorubicin,
L-asparaginase, and intrathecal therapy) and achieved com-
plete remission. Seven months later she remained in com-
plete remission.
2.2. Case 2
A 7-year-old girl presented with a 3-week history of as-
thenia and she had turned pale in the last week. Twenty-five
days before admission, she had an upper right lobe pneu-
monia cured with antibiotics. She had a family history of
colon cancer affecting her grandfather. On physical exami-
nation she was pale; she was well nourished and had no
signs of hemorrhage. Lymph nodes were palpable in sub-
maxillar and inguinal regions. Routine analysis showed: he-
moglobin at 31 g/L, platelets 19
109/L, WBC count 6.8

109/L. BM aspirate showed the presence of 100% infil-
tration by small and medium size blast cells. Immunophe-
notyping yielded a classification for this leukemia as
common ALL. She started standard-risk chemotherapy fol-
lowing the SHOP-99 protocol. When a BM aspiration on
day 14 after initiation of treatment showed persistence of
20% blast cells, she was upgraded to high-risk ALL and
methotrexate was added to her treatment. She achieved
complete remission; 19 months later she was alive and dis-
ease free.
3. Materials and methods
3.1. Cytogenetics and FISH analysis
Cytogenetic analysis was performed on 24-h unstimu-
lated BM cell cultures. Chromosomes were treated and
stained with trypsineGiemsa banding (GTG-banding).
Poor quality of the metaphases in both cases prevented kar-
yotype description. FISH screening was performed on fixed
BM cell suspensions for prognostically significant abnor-
malities (i.e., TEL/AML1 and BCR/ABL fusion) and rear-
rangements of the MLL gene. The dual-color probe kits
LSI TEL/AML1 ES and LSI BCR/ABL ES and the LSI
MLL locus probe (Vysis, Downers Grove, IL) were used.
3.2. Comparative genomic hybridization
CGH was performed according to Kallioniemi et al. [13]
with some modifications. The normal (reference) DNA and
tumor (test) DNA were labeled by nick translation with
SpectrumRed and SpectrumGreen dUTP respectively (Vy-
sis). The size of the nick-translated fragments, tested with
agarose electrophoresis, ranged from 300 to 3,000 bp.
Briefly, 1 mg of each labeled DNA was hybridized to nor-
mal metaphase slides in the presence of Cot-1 DNA (Vysis)
for 72 h at 37 C. After hybridization, stringency washes
were performed and chromosomes were counterstained
with 40 ,6-diamidino-2-phenylindole (DAPI). Image acquisi-
tion, processing, and evaluation were done with a Nikon
Eclipse E600 epifluorescence microscope equipped with
a charge-coupled device camera and ISIS image analysis
software (MetaSystems, Altlussheim, Germany). Three-
color images (with green for test DNA, red for reference
DNA, and blue for the DAPI counterstaining) were ac-
quired from >10 metaphase spreads. The threshold values
for detection of genomic imbalances were determined as
0.80 for losses and 1.20 for gains.
3.3. Cell cycle analysis
Quantitative measurement of cellular DNA content was
performed with flow cytometry using a Coulter DNA
reagents kit (Beckman Coulter, Fullerton, CA) in a FACS-
Calibur cytometer (BD-Becton Dickinson Benelux, Erem-
bodegem, Belgium).
4. Results
Both patients were negative for TEL/AML1 fusion and
for BCR/ABL fusion, tested by either FISH or PCR, and
for rearrangements of the MLL gene (tested by FISH). Nev-
ertheless, FISH analysis at presentation using TEL/AML1
dual-color probe revealed multiple copies of AML1 in both
cases. In the interphase FISH analysis of patient 1, nearly
84% of the analyzed nuclei showed 7-12 copy signals of
AML1, with one TEL signal absent (Fig. 1A). Patient 2 also
presented extra copies of AML1 in 99% of the nuclei, but
only the two normal signals for TEL were seen (Fig. 1B).
The DNA index was calculated by measuring the DNA con-
tent of cells with propidium iodide; both samples had an in-
dex of 1.0 (indicating that they were diploid). The CGH
data showed a high-level amplification of the 21q22 region,
confirming the results obtained by FISH study (Fig. 1C).
5. Discussion
Polysomy of chromosome 21 is a frequent finding in he-
matologic malignant diseases, mainly in ALL, leading to an
increased number of copies of the AML1 gene; however,
amplification of AML1 has also been reported in patients
without this polysomy. Niini et al. [6] first reported the
high-level amplification of AML1 in children with ALL in
2000. They presented a FISH analysis of AML1 in a series
of 112 children with B-cell ALL, of whom 40 showed more
than two signals of AML1, but only 2 of the 40 cases
 Z. Garcı́a-Casado et al. / Cancer Genetics and Cytogenetics 170 (2006) 171e174
Fig. 1. (A,B) FISH results on interphase nuclei with a specific TEL/AML1 dual-color DNA probe (AML1 in red and TEL in green). (A) Patient 1 presented
multiple copy signals of AML1 and one TEL signal was missing (indicated by arrow). (B) Patient 2 likewise showed extra copies of AML1, but two normal
signals for TEL (arrows). (C) Partial GTG-banding karyotype and CGH profile of chromosomes 21 from both patients, showing the amplification threshold
exceeded for the 21q22 region.
presented multiple AML1 signals tandemly located on a de-
rivative chromosome 21. Other reports have described sim-
ilar abnormalities in ALL (extra copies of the AML1 gene
on derivative chromosomes 21, or marker chromosomes),
as well as cases with normal karyotype; more than 40 cases
have been described in the literature, with an estimated
incidence of 1.5% [10e12].
From January 1999 to December 2005, samples from
110 consecutive pediatric patients with ALL were evalu-
ated at our center. Two of these patients presented amplifi-
cation of AML1, with an incidence of 1.8%. Here, we
describe two cases of childhood B-cell precursor ALL,
without a TEL/AML1 rearrangement, presenting AML1 am-
plification detected by FISH and CGH analysis and, in one
case, with a TEL allele deletion. The loss of one allele TEL
has been reported previously and does not seems to confer
differences in the outcome of the patients [14,15]. Unfortu-
nately, the poor quality of the metaphases in both our cases
did not allow the karyotype analysis. Although the DNA in-
dex suggests a normal diploid pattern, we cannot rule out
the presence of some extra chromosomes (e.g., a duplicated
21 chromosome), due to the limited sensitivity of flow
cytometry.
The similarity in the clinicobiologic features of all cases
with AML1 amplification suggests that this abnormality
characterizes an emerging molecular cytogenetic subgroup
of B-cell precursor ALL, potentially different from cases
with t(12;21) and other recurrent chromosomal abnormali-
ties. This amplification is less frequently found in adult
ALL, and likewise is less common in myeloid diseases
(in which AML1 is involved in other rearrangements).
The additional copies of the AML1 gene would result in
the dysregulation of the normal gene product, contributing
to the pathogenesis of ALL. In fact, the AML1 gene codifies
for a transcription factor that is crucial for regulating the
173
expression of multiple genes involved in hematopoiesis.
Nevertheless, the mechanism underlying the physical ef-
fects of this abnormality in leukemogenesis warrant further
investigation.
References
[1] Borkhardt A, Cazzaniga G, Viehmann S, Valsecchi MG, Ludwig WD,
Burci L, Mangioni S, Schrappe M, Riehm H, Lampert F, Basso G,
Masera G, Harbott J, Biondi A. Associazione Italiana Ematologia On-
cologia Pediatrica and the BerlineFrankfurteMunster Study Group.
Incidence and clinical relevance of TEL/AML1 fusion genes in chil-
dren with acute lymphoblastic leukemia enrolled in the German and
Italian multicenter therapy trials. Blood 1997;90:571e7.
[2] Liang DC, Chou TB, Chen JS, Shurtleff SA, Rubnitz JE,
Downing JR, Pui CH, Shih LY. High incidence of TEL/AML1 fusion
resulting from a cryptic t(12;21) in childhood B-lineage acute lym-
phoblastic leukemia in Taiwan. Leukemia 1996;10:991e3.
[3] Romana SP, Poirel H, Leconiat M, Flexor MA, Mauchauffe M,
Jonveaux P, Macintyre EA, Berger R, Bernard OA. High frequency
of t(12;21) in childhood B-lineage acute lymphoblastic leukemia.
Blood 1995;86:4263e9.
[4] Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC,
Hancock ML, Chan GC, Pui CH, Grosveld G, Downing JR. TE-
L/AML1 fusion resulting from a cryptic t(12;21) is the most common
genetic lesion in pediatric ALL and defines a subgroup of patients
with an excellent prognosis. Leukemia 1995;9:1985e9.
[5] Romana SP, Mauchauffe M, Le Coniat M, Chumakov I, Le
Paslier D, Berger R, Bernard OA. The t(12;21) of acute lympho-
blastic leukemia results in a TEL-AML1 gene fusion. Blood
1995;85:3662e70.
[6] Niini T, Kanerva J, Vettenranta K, Saarinen-Pihkala UM, Knuutila S.
AML1 gene amplification: a novel finding in childhood acute lympho-
blastic leukemia. Haematologica 2000;85:362e6.
[7] Morel F, Herry A, Le Bris MJ, Douet-Guilbert N, Le Calvez G,
Marion V, Berthou C, De Braekeeler M. AML1 amplification in a case
of childhood acute lymphoblastic leukemia. Cancer Genet Cytogenet
2002;137:142e5.
[8] Alvarez Y, Coll MD, Bastida P, Ortega JJ, Caballin MR. AML1 am-
plification in a child with acute lymphoblastic leukemia. Cancer
Genet Cytogenet 2003;140:58e61.
174 Z. Garcı́a-Casado et al. / Cancer Genetics and Cytogenetics 170 (2006) 171e174
[9] Harewood L, Robinson H, Harris R, Al-Obaidi MJ, Jalali GR,
Martineau M, Moorman AV, Sumption N, Richards S, Mitchell C,
Harrison CJ. Amplification of AML1 on a duplicated chromosome
21 in acute lymphoblastic leukemia: a study of 20 cases. Leukemia
2003;17:547e53.
[10] Soulier J, Trakhtenbrot L, Najfeld V, Lipton JM, Mathew S, Avet-
Loiseau H, De Braekeleer M, Salem S, Baruchel A, Raimondi SC,
Raynaud SD. Amplification of band q22 of chromosome 21, includ-
ing AML1, in older children with acute lymphoblastic leukemia: an
emerging molecular cytogenetic subgroup. Leukemia 2003;17:
1679e82.
[11] Robinson HM, Broadfield ZJ, Cheung KL, Harewood L, Harris RL,
Jalali GR, Martineau M, Moorman AV, Taylor KE, Richards S,
Mitchell C, Harrison CJ. Amplification of AML1 in acute lympho-
blastic leukemia is associated with a poor outcome. Leukemia 2003;
17:2249e50.
[12] Ferro MT, Hernaez R, Sordo MT, Garcia-Sagredo JM, Garcia-
Miguel P, Fernandez Guijarro M, Lopez J, Villalon C, Vallcorba I,
Cabello P, San Roman C. Chromosome 21 tandem repetition and
AML1 (RUNX1) gene amplification. Cancer Genet Cytogenet 2004;
149:11e6.
[13] Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW,
Waldman F, Pinkel D. Comparative genomic hybridization for molec-
ular cytogenetic analysis of solid tumors. Science 1992;258:818e21.
[14] Cave H, Cacheux V, Raynaud S, Brunie G, Bakkus M, Cochaux P,
Preudhomme C, Lai JL, Vilmer E, Grandchamp B. ETV6 is the target
of chromosome 12p deletions in t(12;21) childhood acute lympho-
cytic leukemia. Leukemia 1997;11:1459e64.
[15] Kempski H, Chalker J, Chessells J, Sturt N, Brickell P, Webb J,
Clink JM, Reeves B. An investigation of the t(12;21) rearrangement
in children with B-precursor acute lymphoblastic leukaemia using cy-
togenetic and molecular methods. Br J Haematol 1999;105:684e9.