Complex_karyotype_defined_by_molecular_c.pdf

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 epidemiologic 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 neurofibromatosis. 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 neurofibromatosis 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 Acute myeloid leukemia (AML) accounts for ~20% of the cases of acute leukemia in children [1]. Genetic disorders, including Down syndrome, Bloom syndrome, and neurofibromatosis type 1 (NF1), among others, are considered predisposing factors for the development of an AML [2,3]. Acute megakaryoblastic leukemia (AMKL) is a form of AML (FrencheAmericaneBritish subtype AML-M7). It has a bimodal distribution, with a peak in the adult phase and another peak in children of !3 years of age, showing an incidence that varies from 3.1% to 14.6% in this childhood group [4]. 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 mutation. The neurofibromin 1 gene (NF1) is located on 17q11.2; it has 51 exons and is 350 kb in length. The clinical features of NF1 are multiple hyperpigmentary spots on the skin and subcutaneous tumors. Neurofibromatosis type 1 is associated with a high prevalence of diverse tumors, * Corresponding author. Tel.: þ49-3641-935533; fax: þ49-3641935582. E-mail address: [email protected] (T. Liehr). 0165-4608/$ e see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.cancergencyto.2010.03.003

including neoplasia of the central and peripheral nervous system, especially optic nerve glioma and neurofibrosarcoma. Childhood myeloid leukemia with monosomy 7 or juvenile myelomonocytic leukemia have been the main hematological syndromeeassociated diseases reported in NF1 [2,5]. In malignant hematological disease of patients with NF1, further mutations in the NF1 gene have been detected in complex karyotypes, as well as mutations in the tumor suppressor gene TP53 [6,7]. In this report we describe a new and rare association of NF1 and AMKL, the latter presenting complex karyotypic changes including three copies of MLL and a deletion of TP53.

2. Materials and methods 2.1. Case history An 11-month-old mulatto girl was admitted for diagnosis and treatment at the Oncohematology Pediatric Center (CEONHPE), Recife, Brazil, in May 2006. On admission the child presented with diarrhea and sepsis. Physical examination revealed apathy, cafe´-au-lait spots, pedunculate facial tumor near the right ear, poor general state of health,

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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 agranulocytosis (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 according to standard protocol [9]. Chromosomes were identified 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 rearrangement and LSI p53 (SpectrumOrange) probes (Abbott Molecular, 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].

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 rearrangement 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 heterogeneous form of AML and at least two entities are recognized, one that occurs in children with Down syndrome and another in the absence of Down syndrome. The cytogenetic 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 abnormalities 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

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).

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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 karyotypes, 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. Ru¨cker 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.

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Acknowledgments This work was supported in part by the Support Foundation for Science and Technology of Pernambuco State (Fundac¸a˜o de Amparo a` Cieˆncia e Tecnologia do Estado de Pernambuco [FACEPE]), Coordination for Improvement of Higher EducationeGerman Academic Exchange Service (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel SuperioreDeutscher Akademischer Austausch Dienst [CAPES/DAAD]) (D/07/09624), and Monika KutznerStiftung. The authors are grateful to Children and Life Program for the helpful support in the childhood cancer network care.

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