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A novel recurrent chromosomal aberration involving chromosome 7 in childhood myelodysplastic syndrome
Cancer Genetics and Cytogenetics 201 (2010) 52e56
Short communication
A novel recurrent chromosomal aberration involving chromosome 7 in childhood myelodysplastic syndrome Libuse Lizcovaa,*, Zuzana Zemanovaa, Eva Malinovaa, Marie Jarosovab, Ester Mejstrikovac, Petr Smisekd, Dagmar Pospisilovae, Jan Staryd, Kyra Michalovaa a
Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 2, Prague, 128 08, Czech Republic b Department of Hemato-Oncology, University Hospital and Palacky University Olomouc, I.P.Pavlova 6, Olomouc, 779 00, Czech Republic c CLIP-Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine and University Hospital Motol, V uvalu 84, 150 06, Prague, Czech Republic d Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine and University Hospital Motol, V uvalu 84, 150 06, Prague, Czech Republic e Department of Paediatrics, University Hospital and Palacky University Olomouc, Czech Republic Received 6 November 2009; received in revised form 4 May 2010; accepted 4 May 2010
Abstract
Monosomy 7 and/or deletion of the long arm of chromosome 7 is a common cytogenetic aberration in children with myelodysplastic syndrome (MDS) and is associated with poor outcome. In this report, we present an unusual cytogenetic abnormality leading to loss of both the whole short and whole long arms of chromosome 7, which was found in the bone marrow cells of three pediatric patients with MDS. Using a combination of conventional and molecular cytogenetic methods, a tiny ‘‘dot-like’’ marker chromosome was found and described as der(7)del(7)(p11)del(7)(q11). Together with one previously published case, this chromosomal aberration represents a new rare recurrent karyotypic abnormality involving chromosome 7 in children with MDS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoetic stem cell disorders characterized by ineffective hematopoesis and increased risk of transformation to MDS-related leukemia. MDS is very uncommon in children and adolescents, accounting for less than 5% of all hematopoetic neoplasms, and is noted for great heterogeneity in presentation and clinical course [1]. In many cases, MDS is difficult to diagnose and to distinguish from other bone marrow (BM) failures, particularly in patients without increased blast count and hypocellular bone marrow. Thus, finding a recurrent chromosomal abnormality is one of the most important factors in confirming the diagnosis. Cytogenetic aberrations are detected in approximately 50e70% of children with primary MDS [2]. Monosomy 7 (7) and partial loss of the long arm of chromosome * Corresponding author. Tel.: þ420 224 962 431; fax: þ420 224 962 848. E-mail address: [email protected] (L. Lizcova). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.05.004
7 [del(7q)] are the most frequent ones, found in up to 40% of cases [3]. It was shown that karyotype changes typically indicate progression to advanced MDS. Children with monosomy 7 have significantly higher probability of progression than patients with other chromosomal abnormalities or normal karyotype [4]. It has been hypothesized that there is a tumor suppressor gene localized on 7q that contributes to the pathogenesis of the disease, but no putative gene has yet been identified [5]. Nevertheless, several structural chromosomal abnormalities, such as unbalanced translocations or interstitial deletions leading to loss of 7q, have been described in both children and adults with MDS. One of the uncommon mechanisms was shown to be loss of both the whole short and whole long arms of chromosome 7, with centromere retention described in few cases of adults with MDS [6e8]. To our knowledge, there is only one pediatric case published in the literature with this small ‘‘dot-like’’ chromosome consisting exclusively of chromosome 7 centromeric material [9]. In this report, we present three new cases of childhood MDS with this rare chromosomal aberration.
L. Lizcova et al. / Cancer Genetics and Cytogenetics 201 (2010) 52e56
2. Materials and methods 2.1. Conventional cytogenetics Bone marrow samples were cultured for 24 hours without stimulation. Chromosomal preparations were made by standard techniques using colcemide, hypotonic treatment, fixation in methanol/acetic acid, and G-banding. If available, at least 20 metaphases were analyzed. Chromosomal aberrations were described according to the International System for Human Cytogenetic Nomenclature [10]. 2.2. Fluorescence in situ hybridization (FISH) For the detection of monosomy 7 and/or loss of 7q, Vysis LSI D7S486 (7q31) SpectrumOrange/CEP 7 SpectrumGreen probe (Abbott Molecular, Des Plaines, IL), which hybridizes to the 7q31 region (orange signal) and to centromere (green signal), was used. All available metaphases and at least 200 interphase nuclei were analyzed. The cut-off level for positive value was determined on samples obtained from 10 healthy individuals and was established at 5%. Further FISH analyses [i.e. multicolor banding (mBAND) and subtelomeric FISH analyses] were carried out using the XCyte7 color kit (MetaSystems, Altlussheim, Germany) and Vysis ToTelVysion probe mix (Abbott Molecular), respectively. All FISH assays were performed according to the manufacturers’ recommendations. 2.3. Case 1 A 5-year-old boy was examined for anorexia, decrease of weight, polyuria, and polydipsia in February 2002 and subsequently further investigated for occurrence of lymphoblasts in peripheral blood (7%). Peripheral blood count showed 3.6109/L white blood cells (WBC), 103 g/L hemoglobin, and a platelet count of 175109/L. His bone marrow was hypercellular, with trilineage dysplasia and increased blast count (15e28%). The blasts were morphologically classified as M6 subtype. Flow cytometry demonstrated infiltration of bone marrow by an abnormal myeloid cell population (25%) with megakaryocytic differentiation (CD41, CD42b, and CD61) with aberrant expression of CD4, CD7, and CD56. Expression of CD235a (glycophorin A) was negative. Screening for the presence of PML/RARA, AML1/ETO, and CBFbeta/MYH11 fusion genes and FLT3/ITD was negative. The karyotype was designated by conventional cytogenetics as 46,XY,7,þmar[11]. The boy was diagnosed with MDS RAEBt (refractory anemia with excess blasts in transformation) and was referred for allogeneic bone marrow transplantation. A side diagnosis of diabetes insipidus without the need of replacement therapy was simultaneously assessed during the hospitalization. At the end of April 2002, he underwent bone marrow transplantation (BMT) from an HLAidentical brother without any complications and with complete allogenic hematopoesis since day þ49. Three years
53
after the BMT, an isolated BM relapse of myeloid malignancy was diagnosed during a routine check up. Conventional cytogenetics showed the identical chromosomal aberration as detected at the time of diagnosis. Expression of CD41, CD42be, and CD61 at relapse decreased immunophenotypically, newly aberrant expression of CD19 appeared in a proportion of blasts, and aberrant expression of CD7, CD56, and CD4 remained stable. Blasts were classified morphologically as M2 subtype. The patient started treatment according to BFM AML REZ 2001 HR protocol and was referred to a second sibling BMT. He received the transplant in August 2005, and at the time of this report, is in his second complete remission. 2.4. Case 2 A 4-year-old girl was diagnosed with MDS RC (refractory cytopenia) in June 2007 after experiencing 2 months of pancytopenia and macrocytic anemia. A complete blood count revealed 3.5109 WBC, 104 g/L hemoglobin, and a platelet count of 69109/L. Bone marrow examination showed hypocellular hematopoesis with dysplastic features, particularly in macrocytes. We did not identify an atypical population by flow cytometry; 1.4% of cells were CD34 positive. None of the fusion genes was found by reversetranscription polymerase chain reaction. Conventional cytogenetic analysis revealed two pathologic clones in a total of 21 metaphases evaluated: 45,XX,7[16]/46,XX, 7,þmar[3]/46,XX[2]. The girl started treatment with 6-mercaptopurine (50 mg/m2), and 3 months after diagnosis, she received a bone marrow transplant from an unrelated donor. She is still in her first complete remission. 2.5. Case 3 A 14-year-old boy was admitted to the hematologic department in February 2008 for a more detailed examination because of previously presented pancytopenia. Peripheral blood count showed 3.9109 WBC, 104 g/L hemoglobin, and a platelet count of 132109/L. Bone marrow aspiration and biopsy revealed hypocellular trilineage hematopoesis with dysplasia without increased blast count. No atypical population was found by flow cytometry; we identified only 0.2% of CD34þ precursors. No fusion genes were detected. A normal karyotype (46,XY[20]) was detected by conventional cytogenetics. The patient was diagnosed with MDS RC or, less possibly, moderately severe aplastic anemia in differential diagnosis. As a result, he was monitored on an ambulatory basis without specific treatment. One year from diagnosis, chromosomal abnormalities were detected in a routine bone marrow check up. The karyotype with two pathologic clones was described as 45,XY,7[8]/46,XY, 7,þmar[4]/46,XY[24]. There were no significant differences in other laboratory and clinical findings. At the time of this report, the patient is still in a good clinical course without therapy and is monitored only. In case treatment
54
L. Lizcova et al. / Cancer Genetics and Cytogenetics 201 (2010) 52e56
becomes necessary, he could receive a bone marrow transplant from an HLA-identical sister.
3. Results Cytogenetic analysis revealed chromosomal abnormalities at the time of diagnosis in cases 1 and 2. In patient 3, karyotypic changes were first detected one year from diagnosis (without previous treatment). Using conventional cytogenetics only, monosomy 7 together with a small ‘‘dotlike’’ marker chromosome was found in all three cases (Fig. 1). In case 1, the chromosomal aberration was detected in all analyzed metaphases. In two other cases, the marker chromosome was lost in part of the cells (i.e., monosomy 7 was the only aberration), and normal metaphases were seen, too. In all three patients, FISH with LSI 7q31 (SpectrumOrange)/CEP 7 (SpectrumGreen) DNA probe on metaphase cells with the small marker chromosome revealed one orange and one green signal on the normal chromosome 7 and another green signal hybridizing to a small marker chromosome, indicating that the marker chromosome was derived from the centromeric region of chromosome 7 (Fig. 1). Hybridization to interphase nuclei showed the following in individual patients: 7q31 deletion signal pattern (2G1O) and/or monosomy 7 (1G1O) and/or normal signal pattern (2G2O). Chromosomal breakpoints on the marker chromosome were determined by XCyte7 color kit (Fig. 1). As expected, FISH analyses of subtelomeric regions of chromosome 7 proved loss of these chromosomal parts on the marker chromosome. Altogether, the small marker chromosomes were composed exclusively of centromeric DNA and finally described in all three cases as der(7)del(7)(p11) del(7)(q11). The results of conventional cytogenetics and FISH analyses are summarized in Table 1.
4. Discussion Monosomy 7 or partial loss of 7q is associated with a variety of myeloid disorders, including childhood MDS.
Although the pathophysiologic relationship between this finding and childhood MDS is unclear, aberrations of chromosome 7 in these cases imply rather poor prognoses and are associated with high risk of transformation to acute leukemia [4]. At this time, hematopetic stem cell transplantation (HSCT) is the only curative therapy and is the treatment of choice for children with MDS and monosomy 7 [11]. As a result, assessment of karyotypic changes, especially those involving chromosome 7, is of great interest to researchers and is one of the most important factors at the time of diagnosis and during the course of the disease. We report three childhood MDS cases with monosomy 7 and an unusual marker chromosome that was subsequently shown to be composed solely of chromosome 7 centromeric region. Despite the fact that this minute chromosome was described in few cases of adult MDS [6e8], it had been published previously in just one pediatric case [9]. Moreover, except for one case, this marker chromosome in adults was part of a complex karyotype or was a result of karyotypic evolution during disease progression. In our patients (as well as in the only previous case), monosomy 7 with a small marker was the only cytogenetic aberration, and was detected at the time of diagnosis in two of them. In one case, the chromosomal abnormality was found one year from the diagnosis however, without any previous treatment. Thus, it obviously was not treatment related. In patients 2 and 3, two pathologic clones were detected: one with isolated monosomy 7 and a second one with monosomy 7, together with a small marker (derivative chromosome 7). Two different mechanisms leading to monosomy 7 are known: (1) mitotic nondisjunction or (2) via one- or multistep breakage-structural rearrangement followed by a progressive loss of the derivative chromosome 7. The second mechanism was reported particularly in patients who have had previous treatment or occupational exposure to potential carcinogens [8]. However, there is no evidence of such events in our patients’ histories. So, it is questionable whether monosomy 7 in these cases was caused by loss of the derivative 7 and represents clonal evolution, which seems more probable, or whether monosomy 7 and derivative 7 originated as two independent clones. On the other hand, in patient 1, as well as in the
Fig. 1. Results of conventional (A) and molecular cytogenetic analyses by FISH with LSI 7q31 SpectrumOrange/CEP 7 SpectrumGreen probe (B) and mBAND (C). Arrows indicate the marker chromosome.
L. Lizcova et al. / Cancer Genetics and Cytogenetics 201 (2010) 52e56
55
Table 1 Clinical and cytogenetic findings (FISH data are taken into account to characterize the marker chromosome) I-FISH Patient no.
Diagnosis
Age/sex
Date
Karyotype
CEP 7
LSI 7q31
% of nuclei
1
MDS RAEBt
5/M
2/2002
46,XY,der(7)del(7)(p11)del(7)(q11)[11]
4/2005 (relapse)
46,XY,der(7)del(7)(p11)del(7)(q11)[32]/46,XY[2]
þ/þ þ/þ þ/þ þ/þ þ/ þ/þ þ/þ þ/ þ/þ þ/ þ/þ þ/þ
þ/ þ/þ þ/ þ/þ þ/ þ/ þ/þ þ/ þ/þ þ/ þ/ þ/þ
79 21 67 23 53 5 42 2.5 97.5 15 7.5 77.5
2
MDS RC
4/F
6/2007
45,XX,7[16]/46,XX,der(7)del(7)(p11) del(7)(q11)[3]/46,XX[2]
3
MDS RC
14/M
2/2008
46,XY[20]
5/2009
45,XY,7[8]/46,XY,der(7)del(7)(p11) del(7)(q11)[4]/46,XY[24]
Abbreviations: þ/þ, probe signal present on both alleles; þ/, probe signal present on one allele only.
previously published pediatric case, only derivative 7 (without the presence of solely monosomy 7) was detected in all analyzed cells at the time of diagnosis and relapse. There were no specific morphologic or hematologic findings in our patients, consistent with an unusual chromosome 7 aberration. Nevertheless, in agreement with the study by Kardos et al. [4], in which children with refractory anemia and monosomy 7 had significantly higher levels of hemoglobin (median 106 g/L) than patients with normal karyotype (median 85 g/L), both of our patients with refractory cytopenia (cases 2 and 3) had Hb levels greater than 100g/L (104 g/L). Patient 1 suffered from diabetes insipidus, a rare complication described in patients with myeloid malignancies and monosomy 7 [12]. As HSCT is known to be the only curative therapy in patients with MDS and monosomy 7 [11], two of our patients (cases 1 and 2), as well as the previously reported child, underwent bone marrow transplantation and now they are living in first and second complete remission, respectively. Since the significance of small clones with monosomy 7 has to be determined [4], HSCT would be indicated in case of karyotypic evolution or clinical aggravation in patient 3. From a cytogenetic point of view, the pathologic clones (monosomy and derivative 7) in the patient were first detected in metaphase cells one year from diagnosis and were present above the cut-off limit in interphase nuclei, even though it was at a low percentage. This, in fact, reflects incipient karyotype evolution. The issue, whether to wait with HSCT for clinical aggravation or increase in pathological clone was proposed in the study of Kardos et al. [4], who concluded that HSCT early in the course of the disease and before progression was accompanied by significantly better post-transplantation survival in patients with MDS RC and monosomy 7. In conclusion, we present a novel rare recurrent cytogenetic aberration in children with MDS. Concerning the gene content, this finding reflects actual monosomy 7 as the centromeric chromosome 7 region was identified only
by hybridization analyses. This marker, due to its size, can easily be overlooked when examined by G-banding analysis only. That is the reason why all cases with an apparent monosomy 7 observed by conventional cytogenetics must be interpreted with caution and should be confirmed by molecular cytogenetic methods. In general, the exact characterization of the finding by all methods available is of utmost importance, especially in cases with a suspect recurrent aberration. Obviously, this is the first step in determining clinical significance of the specific genomic abnormality. Thus, whether this novel chromosomal abnormality represents only a new cytogenetic entity or a specific prognostic subgroup must be evaluated by conventional and molecular cytogenetic methods on a larger series of patients.
Acknowledgments This work was supported by grants from Czech Ministry of Education MSM 21620813, MSM LC535, and Czech Ministry of Health MZOVFN2005. References [1] Niemeyer CM, Baumann I. Myelodysplastic syndrome in children and adolescence. Semin Hematol 2008;45:60e70. [2] Elghetany MT. Myelodysplastic syndromes in children: a critical review of issues in the diagnosis and classification of 887 cases from 13 published series. Arch Pathol Lab Med 2007;131: 1110e6. [3] Hasle H, Alonzo TA, Auvrignon A, Behar C, Chang M, Creutzig U, et al. Monosomy 7 and deletion 7q in children and adolescents with acute myeloid leukemia: an international retrospective study. Blood 2007;109:4641e7. [4] Kardos G, Baumann I, Passmore SJ, Locatelli F, Hasle H, Schultz KR, et al. Refractory anemia in childhood: a retrospective analysis of 67 patients with particular reference to monosomy 7. Blood 2003;102:1997e2003. [5] Heerema NA, Nachman JB, Sather HN, La MK, Hutchinson R, Lange BJ, et al. Deletion of 7p or monosomy 7 in pediatric acute
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lymphoblastic leukemia is an adverse prognostic factor: a report from the Children’s Cancer Group. Leukemia 2004;18:939e47. [6] Baurmann H, Cherif D, Berger R. Interphase cytogenetics by fluorescent in situ hybridization (FISH) for characterization of monosomy-7-associated myeloid disorders. Leukemia 1993;7: 384e91. [7] Gibbons B, Lillington DM, Monard S, Young BD, Cheung KL, Lister TA, Kearney L. Fluorescence in situ hybridisation studies to characterise complete and partial monosomy 7 in myeloid disorders. Genes Chromosomes Cancer 1994;10:244e9. [8] Viguie´ F, Prigent Y, Ramond S, Baumelou E, Cadiou M, Dreyfus F, Zittoun R. Fluorescence in situ hybridization analysis of minute marker chromosomes in leukemia with monosomy 7. Leukemia 1995;9:1154e8.
[9] Zamecnikova A. Small marker chromosome and monosomy 7 in a pediatric patient with MDS. Cancer Genet Cytogenet 2008;180: 163e4. [10] Shaffer LG, Slovak ML, Campbell LJ, editors. ISCN: An International System for Human Cytogenetic Nomenclature. Basel: S. Karger, 2009. [11] Baumann I, Niemeyer CM, Bennett JM, Shannon K. Childhood myelodysplastic syndrome. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein SA, Thiele J, Vardim JW, editors. WHO Classification of Tumours of Heamapoietic and Lymphoid tissues. Lyon: IARC, 2008. pp. 104e7. [12] de la Chapelle A, Lahtinen R. Monosomy 7 predisposes to diabetes insipidus in leukaemia and myelodysplastic syndrome. Eur J Haematol 1987;39:404e11.
Short communication
A novel recurrent chromosomal aberration involving chromosome 7 in childhood myelodysplastic syndrome Libuse Lizcovaa,*, Zuzana Zemanovaa, Eva Malinovaa, Marie Jarosovab, Ester Mejstrikovac, Petr Smisekd, Dagmar Pospisilovae, Jan Staryd, Kyra Michalovaa a
Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 2, Prague, 128 08, Czech Republic b Department of Hemato-Oncology, University Hospital and Palacky University Olomouc, I.P.Pavlova 6, Olomouc, 779 00, Czech Republic c CLIP-Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine and University Hospital Motol, V uvalu 84, 150 06, Prague, Czech Republic d Department of Paediatric Haematology and Oncology, Charles University Prague, 2nd Faculty of Medicine and University Hospital Motol, V uvalu 84, 150 06, Prague, Czech Republic e Department of Paediatrics, University Hospital and Palacky University Olomouc, Czech Republic Received 6 November 2009; received in revised form 4 May 2010; accepted 4 May 2010
Abstract
Monosomy 7 and/or deletion of the long arm of chromosome 7 is a common cytogenetic aberration in children with myelodysplastic syndrome (MDS) and is associated with poor outcome. In this report, we present an unusual cytogenetic abnormality leading to loss of both the whole short and whole long arms of chromosome 7, which was found in the bone marrow cells of three pediatric patients with MDS. Using a combination of conventional and molecular cytogenetic methods, a tiny ‘‘dot-like’’ marker chromosome was found and described as der(7)del(7)(p11)del(7)(q11). Together with one previously published case, this chromosomal aberration represents a new rare recurrent karyotypic abnormality involving chromosome 7 in children with MDS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoetic stem cell disorders characterized by ineffective hematopoesis and increased risk of transformation to MDS-related leukemia. MDS is very uncommon in children and adolescents, accounting for less than 5% of all hematopoetic neoplasms, and is noted for great heterogeneity in presentation and clinical course [1]. In many cases, MDS is difficult to diagnose and to distinguish from other bone marrow (BM) failures, particularly in patients without increased blast count and hypocellular bone marrow. Thus, finding a recurrent chromosomal abnormality is one of the most important factors in confirming the diagnosis. Cytogenetic aberrations are detected in approximately 50e70% of children with primary MDS [2]. Monosomy 7 (7) and partial loss of the long arm of chromosome * Corresponding author. Tel.: þ420 224 962 431; fax: þ420 224 962 848. E-mail address: [email protected] (L. Lizcova). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.05.004
7 [del(7q)] are the most frequent ones, found in up to 40% of cases [3]. It was shown that karyotype changes typically indicate progression to advanced MDS. Children with monosomy 7 have significantly higher probability of progression than patients with other chromosomal abnormalities or normal karyotype [4]. It has been hypothesized that there is a tumor suppressor gene localized on 7q that contributes to the pathogenesis of the disease, but no putative gene has yet been identified [5]. Nevertheless, several structural chromosomal abnormalities, such as unbalanced translocations or interstitial deletions leading to loss of 7q, have been described in both children and adults with MDS. One of the uncommon mechanisms was shown to be loss of both the whole short and whole long arms of chromosome 7, with centromere retention described in few cases of adults with MDS [6e8]. To our knowledge, there is only one pediatric case published in the literature with this small ‘‘dot-like’’ chromosome consisting exclusively of chromosome 7 centromeric material [9]. In this report, we present three new cases of childhood MDS with this rare chromosomal aberration.
L. Lizcova et al. / Cancer Genetics and Cytogenetics 201 (2010) 52e56
2. Materials and methods 2.1. Conventional cytogenetics Bone marrow samples were cultured for 24 hours without stimulation. Chromosomal preparations were made by standard techniques using colcemide, hypotonic treatment, fixation in methanol/acetic acid, and G-banding. If available, at least 20 metaphases were analyzed. Chromosomal aberrations were described according to the International System for Human Cytogenetic Nomenclature [10]. 2.2. Fluorescence in situ hybridization (FISH) For the detection of monosomy 7 and/or loss of 7q, Vysis LSI D7S486 (7q31) SpectrumOrange/CEP 7 SpectrumGreen probe (Abbott Molecular, Des Plaines, IL), which hybridizes to the 7q31 region (orange signal) and to centromere (green signal), was used. All available metaphases and at least 200 interphase nuclei were analyzed. The cut-off level for positive value was determined on samples obtained from 10 healthy individuals and was established at 5%. Further FISH analyses [i.e. multicolor banding (mBAND) and subtelomeric FISH analyses] were carried out using the XCyte7 color kit (MetaSystems, Altlussheim, Germany) and Vysis ToTelVysion probe mix (Abbott Molecular), respectively. All FISH assays were performed according to the manufacturers’ recommendations. 2.3. Case 1 A 5-year-old boy was examined for anorexia, decrease of weight, polyuria, and polydipsia in February 2002 and subsequently further investigated for occurrence of lymphoblasts in peripheral blood (7%). Peripheral blood count showed 3.6109/L white blood cells (WBC), 103 g/L hemoglobin, and a platelet count of 175109/L. His bone marrow was hypercellular, with trilineage dysplasia and increased blast count (15e28%). The blasts were morphologically classified as M6 subtype. Flow cytometry demonstrated infiltration of bone marrow by an abnormal myeloid cell population (25%) with megakaryocytic differentiation (CD41, CD42b, and CD61) with aberrant expression of CD4, CD7, and CD56. Expression of CD235a (glycophorin A) was negative. Screening for the presence of PML/RARA, AML1/ETO, and CBFbeta/MYH11 fusion genes and FLT3/ITD was negative. The karyotype was designated by conventional cytogenetics as 46,XY,7,þmar[11]. The boy was diagnosed with MDS RAEBt (refractory anemia with excess blasts in transformation) and was referred for allogeneic bone marrow transplantation. A side diagnosis of diabetes insipidus without the need of replacement therapy was simultaneously assessed during the hospitalization. At the end of April 2002, he underwent bone marrow transplantation (BMT) from an HLAidentical brother without any complications and with complete allogenic hematopoesis since day þ49. Three years
53
after the BMT, an isolated BM relapse of myeloid malignancy was diagnosed during a routine check up. Conventional cytogenetics showed the identical chromosomal aberration as detected at the time of diagnosis. Expression of CD41, CD42be, and CD61 at relapse decreased immunophenotypically, newly aberrant expression of CD19 appeared in a proportion of blasts, and aberrant expression of CD7, CD56, and CD4 remained stable. Blasts were classified morphologically as M2 subtype. The patient started treatment according to BFM AML REZ 2001 HR protocol and was referred to a second sibling BMT. He received the transplant in August 2005, and at the time of this report, is in his second complete remission. 2.4. Case 2 A 4-year-old girl was diagnosed with MDS RC (refractory cytopenia) in June 2007 after experiencing 2 months of pancytopenia and macrocytic anemia. A complete blood count revealed 3.5109 WBC, 104 g/L hemoglobin, and a platelet count of 69109/L. Bone marrow examination showed hypocellular hematopoesis with dysplastic features, particularly in macrocytes. We did not identify an atypical population by flow cytometry; 1.4% of cells were CD34 positive. None of the fusion genes was found by reversetranscription polymerase chain reaction. Conventional cytogenetic analysis revealed two pathologic clones in a total of 21 metaphases evaluated: 45,XX,7[16]/46,XX, 7,þmar[3]/46,XX[2]. The girl started treatment with 6-mercaptopurine (50 mg/m2), and 3 months after diagnosis, she received a bone marrow transplant from an unrelated donor. She is still in her first complete remission. 2.5. Case 3 A 14-year-old boy was admitted to the hematologic department in February 2008 for a more detailed examination because of previously presented pancytopenia. Peripheral blood count showed 3.9109 WBC, 104 g/L hemoglobin, and a platelet count of 132109/L. Bone marrow aspiration and biopsy revealed hypocellular trilineage hematopoesis with dysplasia without increased blast count. No atypical population was found by flow cytometry; we identified only 0.2% of CD34þ precursors. No fusion genes were detected. A normal karyotype (46,XY[20]) was detected by conventional cytogenetics. The patient was diagnosed with MDS RC or, less possibly, moderately severe aplastic anemia in differential diagnosis. As a result, he was monitored on an ambulatory basis without specific treatment. One year from diagnosis, chromosomal abnormalities were detected in a routine bone marrow check up. The karyotype with two pathologic clones was described as 45,XY,7[8]/46,XY, 7,þmar[4]/46,XY[24]. There were no significant differences in other laboratory and clinical findings. At the time of this report, the patient is still in a good clinical course without therapy and is monitored only. In case treatment
54
L. Lizcova et al. / Cancer Genetics and Cytogenetics 201 (2010) 52e56
becomes necessary, he could receive a bone marrow transplant from an HLA-identical sister.
3. Results Cytogenetic analysis revealed chromosomal abnormalities at the time of diagnosis in cases 1 and 2. In patient 3, karyotypic changes were first detected one year from diagnosis (without previous treatment). Using conventional cytogenetics only, monosomy 7 together with a small ‘‘dotlike’’ marker chromosome was found in all three cases (Fig. 1). In case 1, the chromosomal aberration was detected in all analyzed metaphases. In two other cases, the marker chromosome was lost in part of the cells (i.e., monosomy 7 was the only aberration), and normal metaphases were seen, too. In all three patients, FISH with LSI 7q31 (SpectrumOrange)/CEP 7 (SpectrumGreen) DNA probe on metaphase cells with the small marker chromosome revealed one orange and one green signal on the normal chromosome 7 and another green signal hybridizing to a small marker chromosome, indicating that the marker chromosome was derived from the centromeric region of chromosome 7 (Fig. 1). Hybridization to interphase nuclei showed the following in individual patients: 7q31 deletion signal pattern (2G1O) and/or monosomy 7 (1G1O) and/or normal signal pattern (2G2O). Chromosomal breakpoints on the marker chromosome were determined by XCyte7 color kit (Fig. 1). As expected, FISH analyses of subtelomeric regions of chromosome 7 proved loss of these chromosomal parts on the marker chromosome. Altogether, the small marker chromosomes were composed exclusively of centromeric DNA and finally described in all three cases as der(7)del(7)(p11) del(7)(q11). The results of conventional cytogenetics and FISH analyses are summarized in Table 1.
4. Discussion Monosomy 7 or partial loss of 7q is associated with a variety of myeloid disorders, including childhood MDS.
Although the pathophysiologic relationship between this finding and childhood MDS is unclear, aberrations of chromosome 7 in these cases imply rather poor prognoses and are associated with high risk of transformation to acute leukemia [4]. At this time, hematopetic stem cell transplantation (HSCT) is the only curative therapy and is the treatment of choice for children with MDS and monosomy 7 [11]. As a result, assessment of karyotypic changes, especially those involving chromosome 7, is of great interest to researchers and is one of the most important factors at the time of diagnosis and during the course of the disease. We report three childhood MDS cases with monosomy 7 and an unusual marker chromosome that was subsequently shown to be composed solely of chromosome 7 centromeric region. Despite the fact that this minute chromosome was described in few cases of adult MDS [6e8], it had been published previously in just one pediatric case [9]. Moreover, except for one case, this marker chromosome in adults was part of a complex karyotype or was a result of karyotypic evolution during disease progression. In our patients (as well as in the only previous case), monosomy 7 with a small marker was the only cytogenetic aberration, and was detected at the time of diagnosis in two of them. In one case, the chromosomal abnormality was found one year from the diagnosis however, without any previous treatment. Thus, it obviously was not treatment related. In patients 2 and 3, two pathologic clones were detected: one with isolated monosomy 7 and a second one with monosomy 7, together with a small marker (derivative chromosome 7). Two different mechanisms leading to monosomy 7 are known: (1) mitotic nondisjunction or (2) via one- or multistep breakage-structural rearrangement followed by a progressive loss of the derivative chromosome 7. The second mechanism was reported particularly in patients who have had previous treatment or occupational exposure to potential carcinogens [8]. However, there is no evidence of such events in our patients’ histories. So, it is questionable whether monosomy 7 in these cases was caused by loss of the derivative 7 and represents clonal evolution, which seems more probable, or whether monosomy 7 and derivative 7 originated as two independent clones. On the other hand, in patient 1, as well as in the
Fig. 1. Results of conventional (A) and molecular cytogenetic analyses by FISH with LSI 7q31 SpectrumOrange/CEP 7 SpectrumGreen probe (B) and mBAND (C). Arrows indicate the marker chromosome.
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Table 1 Clinical and cytogenetic findings (FISH data are taken into account to characterize the marker chromosome) I-FISH Patient no.
Diagnosis
Age/sex
Date
Karyotype
CEP 7
LSI 7q31
% of nuclei
1
MDS RAEBt
5/M
2/2002
46,XY,der(7)del(7)(p11)del(7)(q11)[11]
4/2005 (relapse)
46,XY,der(7)del(7)(p11)del(7)(q11)[32]/46,XY[2]
þ/þ þ/þ þ/þ þ/þ þ/ þ/þ þ/þ þ/ þ/þ þ/ þ/þ þ/þ
þ/ þ/þ þ/ þ/þ þ/ þ/ þ/þ þ/ þ/þ þ/ þ/ þ/þ
79 21 67 23 53 5 42 2.5 97.5 15 7.5 77.5
2
MDS RC
4/F
6/2007
45,XX,7[16]/46,XX,der(7)del(7)(p11) del(7)(q11)[3]/46,XX[2]
3
MDS RC
14/M
2/2008
46,XY[20]
5/2009
45,XY,7[8]/46,XY,der(7)del(7)(p11) del(7)(q11)[4]/46,XY[24]
Abbreviations: þ/þ, probe signal present on both alleles; þ/, probe signal present on one allele only.
previously published pediatric case, only derivative 7 (without the presence of solely monosomy 7) was detected in all analyzed cells at the time of diagnosis and relapse. There were no specific morphologic or hematologic findings in our patients, consistent with an unusual chromosome 7 aberration. Nevertheless, in agreement with the study by Kardos et al. [4], in which children with refractory anemia and monosomy 7 had significantly higher levels of hemoglobin (median 106 g/L) than patients with normal karyotype (median 85 g/L), both of our patients with refractory cytopenia (cases 2 and 3) had Hb levels greater than 100g/L (104 g/L). Patient 1 suffered from diabetes insipidus, a rare complication described in patients with myeloid malignancies and monosomy 7 [12]. As HSCT is known to be the only curative therapy in patients with MDS and monosomy 7 [11], two of our patients (cases 1 and 2), as well as the previously reported child, underwent bone marrow transplantation and now they are living in first and second complete remission, respectively. Since the significance of small clones with monosomy 7 has to be determined [4], HSCT would be indicated in case of karyotypic evolution or clinical aggravation in patient 3. From a cytogenetic point of view, the pathologic clones (monosomy and derivative 7) in the patient were first detected in metaphase cells one year from diagnosis and were present above the cut-off limit in interphase nuclei, even though it was at a low percentage. This, in fact, reflects incipient karyotype evolution. The issue, whether to wait with HSCT for clinical aggravation or increase in pathological clone was proposed in the study of Kardos et al. [4], who concluded that HSCT early in the course of the disease and before progression was accompanied by significantly better post-transplantation survival in patients with MDS RC and monosomy 7. In conclusion, we present a novel rare recurrent cytogenetic aberration in children with MDS. Concerning the gene content, this finding reflects actual monosomy 7 as the centromeric chromosome 7 region was identified only
by hybridization analyses. This marker, due to its size, can easily be overlooked when examined by G-banding analysis only. That is the reason why all cases with an apparent monosomy 7 observed by conventional cytogenetics must be interpreted with caution and should be confirmed by molecular cytogenetic methods. In general, the exact characterization of the finding by all methods available is of utmost importance, especially in cases with a suspect recurrent aberration. Obviously, this is the first step in determining clinical significance of the specific genomic abnormality. Thus, whether this novel chromosomal abnormality represents only a new cytogenetic entity or a specific prognostic subgroup must be evaluated by conventional and molecular cytogenetic methods on a larger series of patients.
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