Cancer Genetics and Cytogenetics 200 (2010) 167e169

Short communication

Complex karyotype defined by molecular cytogenetic FISH and M-FISH

in an infant with acute megakaryoblastic leukemia and neurofibromatosis
Terezinha de Jesus Marques-Sallesa,b, Hasmik Mkrtchyanc, Edinalva Pereira Leitea,
Eliane Maria Soares-Venturaa, Maria Tereza Cartaxo Muniza, Elizangela Ferreira Silvaa,
Thomas Liehrc,*, Maria Luiza Macedo Silvad, Neide Santosb
a
Pediatric Oncohematology Center, Hospital Oswaldo Cruz, University of Pernambuco, Recife, PE, Brazil
b
Genetic Department, Federal University of Pernambuco, Recife, PE Brazil
c
Jena University Hospital, Institute of Human Genetics and Anthropology, D-07740 Jena, Germany
d
Cytogenetic Department, National Center for Bone Marrow Transplant (CEMO-INCA), National Cancer Institute, Rio de Janeiro, RJ, Brazil
Received 29 October 2009; received in revised form 19 February 2010; accepted 3 March 2010

Abstract Acute myeloid leukemia in childhood is a heterogeneous group of diseases, and different epidemi-
ologic factors are involved in the etiopathogenesis. Genetic syndromes are one of the predisposing
factors of acute myeloid leukemia (AML), including Down syndrome, Bloom syndrome, and neuro-
fibromatosis. Acute megakaryoblastic leukemia (AMKL) is the main subtype in Down syndrome
infants, and acquired chromosomal anomalies are closely related to the physiopathology of the
illness. The main chromosomal anomalies in AMKL are structural, such as t(1;22); however,
complex karyotypes are also common. Here we describe the case of an infant with neurofibroma-
tosis developing AMKL with a complex karyotype including 5q and 17q deletions, TP53 deletion,
and an unusual unbalanced chromosomal translocation t(11;19)(q13;p13), leading to three copies of
the MLL gene. Ó 2010 Published by Elsevier Inc.

1. Introduction including neoplasia of the central and peripheral nervous

system, especially optic nerve glioma and neurofibrosarco-
Acute myeloid leukemia (AML) accounts for ~20% of
ma. Childhood myeloid leukemia with monosomy 7 or
the cases of acute leukemia in children [1]. Genetic disor-
juvenile myelomonocytic leukemia have been the main
ders, including Down syndrome, Bloom syndrome, and
hematological syndromeeassociated diseases reported in
neurofibromatosis type 1 (NF1), among others, are consid-
NF1 [2,5]. In malignant hematological disease of patients
ered predisposing factors for the development of an AML
with NF1, further mutations in the NF1 gene have been
[2,3]. Acute megakaryoblastic leukemia (AMKL) is a form
detected in complex karyotypes, as well as mutations in
of AML (FrencheAmericaneBritish subtype AML-M7). It
the tumor suppressor gene TP53 [6,7].
has a bimodal distribution, with a peak in the adult phase
In this report we describe a new and rare association of
and another peak in children of !3 years of age, showing
NF1 and AMKL, the latter presenting complex karyotypic
an incidence that varies from 3.1% to 14.6% in this child-
changes including three copies of MLL and a deletion of
hood group [4].
TP53.
Neurofibromatosis type 1 has an autosomal dominant
pattern of inheritance; 50% of patients inherit the condition
from one parent and the remainder acquire it as a new 2. Materials and methods
mutation. The neurofibromin 1 gene (NF1) is located on
17q11.2; it has 51 exons and is 350 kb in length. The clin- 2.1. Case history
ical features of NF1 are multiple hyperpigmentary spots on
An 11-month-old mulatto girl was admitted for diagnosis
the skin and subcutaneous tumors. Neurofibromatosis type
and treatment at the Oncohematology Pediatric Center
1 is associated with a high prevalence of diverse tumors,
(CEONHPE), Recife, Brazil, in May 2006. On admission
* Corresponding author. Tel.: þ49-3641-935533; fax: þ49-3641- the child presented with diarrhea and sepsis. Physical exam-
935582. ination revealed apathy, café-au-lait spots, pedunculate
E-mail address: i8lith@mti.uni-jena.de (T. Liehr). facial tumor near the right ear, poor general state of health,
0165-4608/$ e see front matter Ó 2010 Published by Elsevier Inc.
doi:10.1016/j.cancergencyto.2010.03.003
168 T. de Jesus Marques-Salles et al. / Cancer Genetics and Cytogenetics 200 (2010) 167e169
serious malnutrition (weight 5.2 kg), generalized edemas,
cutaneous and mucous bleeding, and enlarged liver and
spleen. She had a history of parental consanguinity. The
hemoglobin level was 6.4 g/dL, the white blood cell count
was 74  109/L with 80% blasts, the platelet count was
20  109/L, and the lactate dehydrogenase level was
2.739 UI. The morphologic evaluation revealed hypocellular
bone marrow infiltrated by 83% undifferentiated blast
cells, some with cytoplasmatic extensions. Sudan Black
cytochemical reactions were negative. The blast cells
expressed CD45, CD34, CD7, CD33, CD61, and CD13,
compatible with a diagnosis of AML-M7. The child was
treated according to the AML BFM-98 protocol. After
induction of remission, she presented serious agranulocy-
tosis (480 leukocytes/mm3), diarrhea, and indications of
sepsis. She was treated with antibiotics therapy, but she
died, 21 days after the diagnosis.
2.2. Conventional cytogenetic banding analysis
Bone marrow cells obtained at diagnosis were cultivated
for 24 hours without mitogenic stimulation and processed
as previously described [8]; GTG banding was done ac-
cording to standard protocol [9]. Chromosomes were iden-
tified and described in accordance with ISCN 2009 [10].
2.3. Molecular cytogenetic analysis
Fluorescence in situ hybridization (FISH) was performed
using the Vysis LSI MLL dual-color, break-apart rearrange-
ment and LSI p53 (SpectrumOrange) probes (Abbott Molec-
ular, Des Plaines, IL; Wiesbaden-Delkenheim, Germany),
according to the manufacturer’s instructions. Multicolor
FISH (M-FISH) using 24 whole-chromosome painting
probes was performed as previously described [11,12].
Fig. 1. G-banding analysis indicates karyotype as 46,XX,del(5)(q31),add(10)(q26),del(10)(q22),der(19)t(11;19)(q13;p13).
3. Results
The GTG-banding revealed the karyotype as 46,XX,del(5)
(q31),add(10)(q26),del(10)(q22),der(19)t(11;19)(q13;p13)[13]/
46,XX[7] (Fig. 1). To further delineate the karyotypic
changes and to confirm the alterations identified with
GTG-banding, a more detailed investigation using M-FISH
revealed the karyotype as follows: 46,XX,der(5)t(5;10),der
(10)t(1;10),del(10)(q?),del(17)(p?),der(19)t(11;19) (Fig. 2A).
As expected, the LSI MLL dual-color, break-apart rearrange-
ment probe showed 3 hybridization signals in 196 of 200
interphase nuclei; however, the LSI p53 probe showed only
one signal in 30% of interphase cells analyzed (Fig. 2B).
4. Discussion
Acute megakaryoblastic leukemia is a biological hetero-
geneous form of AML and at least two entities are recog-
nized, one that occurs in children with Down syndrome
and another in the absence of Down syndrome. The cytoge-
netic profile in AMKL is well characterized by a karyotype
with numerical abnormalities, t(1;22) rearrangement, and
other structural changes [13].
Notably, the complex karyotype in our case presented
only translocations and deletions, but no numerical
changes. The chromosomes involved in the observed abnor-
malities are frequently altered in AMKL. The deletion of
the TP53 gene in 17p13.2 and the translocation
t(11;19)(q13;p13) seen in this patient are also suggested
to be important alterations in terms of disease development
or progression, and chromosome 19 is known as hotspot of
rearrangements in AMKL.
De novo mutation of TP53 occurs in !10% of AML
cases; it is often associated with a cytogenetically visible
 T. de Jesus Marques-Salles et al. / Cancer Genetics and Cytogenetics 200 (2010) 167e169
Fig. 2. (A) Fluorescence in situ hybridization (FISH) using the probe LSI p53 together with a whole chromosome painting (wcp) probe showed a deletion in
the TP53 region on one chromosome in 30% of the cells. (B) With multicolor-FISH, the complex karyotype was confirmed and refined as
46,XX,der(5)t(5;10),der(10)t(1;10),del(10)(q?),del(17)(p?),der(19)t(11;19). Arrows indicate derivative chromosomes.
deletion of the chromosome band 17p13, complex karyo-
types, and poor prognosis. Many cases were reported to
present also a deletion in 5q, suggesting coordination
between TP53 function and loss of a putative tumor
suppressor gene at 5q [7]. The TP53 deletion is described
in congenital diseases with AML, but the mechanism is
unknown. Rücker et al. [14] identified recurrent cryptic
deletion in 17q11.2, where the NF1 gene is located, and
suggested that in AML the main target of the deletion in
chromosome 17 is NF1, and not TP53.
To our knowledge, the present case represents the first
reported association of NF1 and infant AMKL. Molecular
cytogenetic studies revealed both TP53 and 5q deletion,
in addition to an unbalanced t(11;19)(q13;p13), leading to
3 copies of MLL, but rearrangements within the MLL gene
were absent.
Acknowledgments
This work was supported in part by the Support Founda-
tion for Science and Technology of Pernambuco State
(Fundação de Amparo à Ciência e Tecnologia do Estado
de Pernambuco [FACEPE]), Coordination for Improvement
of Higher EducationeGerman Academic Exchange Service
(Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
SuperioreDeutscher Akademischer Austausch Dienst
[CAPES/DAAD]) (D/07/09624), and Monika Kutzner-
Stiftung. The authors are grateful to Children and Life
Program for the helpful support in the childhood cancer
network care.
References
[1] Viana MB, Cunha KCCMS, Ramos G, Murao M. Acute myeloid
leukemia in childhood: 15-year experience in a single institution
[In Portuguese]. J Pediatr (Rio J) 2003;79:489e96.
[2] Maris JM, Wiersma SR, Mahgoub N, Thompson P, Geyer RJ,
Hurwitz CG, et al. Monosomy 7 myelodysplastic syndrome and other
169
second malignant neoplasms in children with neurofibromatosis
type 1. Cancer 1997;79:1438e46.
[3] Korf BR. Malignancy in neurofibromatosis type 1. Oncologist 2000;
5:477e85.
[4] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,
Gralnick HR, et al. Proposed revised criteria for the classification
of acute myeloid leukemia: a report of the FrencheAmericane
British Cooperative group. Ann Intern Med 1985;103:620e5.
[5] DeBella K, Szudek J, Friedman JM. Use of the National Institutes of
Health criteria for diagnosis of neurofibromatosis 1 in children.
Pediatrics 2000;105:608e14.
[6] Suela J, Largo C, Ferreira B, Alvarez S, Robledo M, González-
Neira A, et al. Neurofibromatosis 1, and not TP53, seems to be the
main target of chromosome 17 deletions in de novo acute myeloid
leukemia. J Clin Oncol 2007;25:1151e2.
[7] Lai JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M,
Lepelley P, et al. Myelodysplastic syndromes and acute myeloid
leukemia with 17p deletion: an entity characterized by specific dys-
granulopoı̈esis and high incidence of P53 mutations. Leukemia
1995;9:370e81.
[8] Macedo Silva ML, Raimondi SC, Abdelhay E, Gross M,
Mkrtchyan H, de Figueiredo AF, et al. Banding and molecular cyto-
genetic studies detected a CBFB-MYH11 fusion gene that appeared as
abnormal chromosomes 1 and 16 in a baby with acute myeloid
leukemia FAB M4-Eo. Cancer Genet Cytogenet 2008;182:56e60.
[9] Verma RS, Babu A. Human chromosomes: a manual of basic
techniques. 2nd ed. New York: Pergamon Press, 1995.
[10] Shaffer LG, Slovak ML, Campbell LJ, editors. ISCN 2009: an
international system for human cytogenetic nomenclature. Basel:
S. Karger, 2009. 2009.
[11] Liehr T, Claussen U. Multicolor-FISH approaches for the character-
ization of human chromosomes in clinical genetics and tumor cytoge-
netics. Curr Genomics 2002;3:213e35.
[12] Liehr T, Starke H, Weise A, Lehrer H, Claussen U. Multicolor FISH
probe sets and their applications. Histol Histopathol 2004;19:
229e37.
[13] Dastugue N, Lafage-Pochitaloff M, Pagès MP, Radford I, Bastard C,
Talmant P, et al. Groupe Français d0 Hematologie Cellulaire. Cytoge-
netic profile of childhood and adult megakaryoblastic leukemia (M7):
a study of the Groupe Français de Cytogénétique Hématologique
(GFCH). Blood 2002;100:618e26.
[14] Rücker FG, Bullinger L, Schwaenen C, Lipka DB, Wessendorf S,
Fröhling S, et al. Disclosure of candidate genes in acute myeloid
leukemia with complex karyotype using microarray-based molecular
characterization. J Clin Oncol 2006;24:3887e94.