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Interphase cytogenetic analysis of clonality in peripheral blood cells from a patient with Down syndrome and acute megakaryoblastic leukemia
Cancer Genetics and Cytogenetics 148 (2004) 141–144
Short communication
Interphase cytogenetic analysis of clonality in peripheral blood cells from a patient with Down syndrome and acute megakaryoblastic leukemia Hong Changa,*, Dan Lia, Rakash Nayara, Charles Yeb, Wendy Laub, D. Robert Sutherlanda a
Department of Laboratory Hematology, Princess Margaret Hospital, University Health Network, University of Toronto, 610 University Avenue, 4-320, Toronto, Ontario M5G 2M9, Canada b Department of Hematopathology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Received 16 May 2003; received in revised form 12 June 2003; accepted 18 June 2003
Abstract
A combination of fluorescence-activated cell sorting and interphase fluorescence in situ hybridization (FISH) techniques was used to detect a clonal chromosomal marker in blasts, granulocytes, and T and B lymphocytes of the peripheral blood from a patient with Down syndrome and acute megakaryoblastic leukemia (AMKL) associated with trisomy 8 as a karyotypic abnormality. Immunophenotypic studies with flow cytometry showed two populations of leukemic blasts distinguished by their expression of the CD34 antigen. Interphase FISH studies revealed clonal trisomy 8 FISH signals in almost all blast cells, regardless of CD34 expression, as well as in a small subpopulation of granulocytes. Normal chromosome 8 signal patterns were detected in T and B cells and in a great majority of granulocytes. The present study provides evidence for the clonal involvement of leukemic blasts in AMKL of Down syndrome, indicating that a trisomy 8 abnormality may be a primary event in leukemogenesis. The transformation occurs in progenitor cells with limited myeloid differentiation and without involvement of lymphoid lineage cells. 쑖 2004 Elsevier Inc. All rights reserved.
1. Introduction Down syndrome (trisomy 21) is a chromosomal abnormality associated with increased risk of leukemia [1]. The majority of cases are acute megakaryoblastic leukemias (AMKL). Some studies have shown that the blasts in the myelodysplastic (MDS) AMKL of Down syndrome have in vitro properties of a multilineage precursor cell with potential for forming megakaryocytes, erythroid, and basophil/ mast cell progenitors [1,2], but not myeloid cells. It is not clear, however, whether different cell types (including granulocytes and T or B cells) are clonally interrelated in this particular type of leukemia of Down syndrome. Trisomy 8 is the most frequent chromosomal change associated with AMKL, occurring in ~20% of the cases [3–5]. In the present study, we evaluated the peripheral blood of a Down syndrome patient with AMKL who had trisomy 8. To try to delineate the clonal involvement, we sorted discrete leukocyte
* Corresponding author. Tel.: (416) 946-4635; fax: (416) 946-4616. E-mail address: [email protected] (H. Chang). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0165-4608(03)00273-5
subsets using fluorescence-activated cell sorting (FACS) and assessed each highly purified fraction using interphase fluorescence in situ hybridization (FISH) techniques.
2. Materials and methods 2.1. Patient A 2-year-old girl with Down syndrome presented with easy bruising. There was no prior history of transient leukemia in the neonatal period. Physical examination showed no organomegaly or lymphadenopathy. The complete blood counts showed hemoglobin 98 g/L, white blood cell count 5.3 × 109/L (10.9% granulocytes, 48.1% lymphocytes, 37% blast cells, and 3.9% monocytes), and platelets 41 × 109/L. The blasts exhibited undifferentiated morphology and basophilic cytoplasm. Bone marrow aspirates showed a dry tap, but biopsy revealed hypercellularity with significant fibrosis, clusters of dysplastic megakaryocytes, and reduction of both erythropoiesis and granulopoiesis. Cell-surface marker analysis with flow cytometry revealed blast cells expressing
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CD7, CD36, CD109, and CD11b and blast cells with weak expression of CD13, CD33, and CD34. Cytoplasmic marker analysis showed the blast cells to be unreactive with antibodies to MPO, CD3, and CD79a. Blasts were also negative for nuclear antigen TdT (all antibodies from Beckman Coulter, Miami, FL). Subsequent bone marrow biopsy analysis with immunohistochemical staining showed blasts weakly positive for CD41 and CD61. A diagnosis of AMKL associated with Down syndrome was made. Cytogenetic study on peripheral blood cells showed the following karyotype: 48,XX,⫹8,⫹21c[7]/47,XX,⫹21c[7]. 2.2. FACS Five milliliters of sodium-heparinized peripheral blood specimen was collected from the patient. Red blood cells were depleted by means of lysis with 1× ammonium chloride lysing buffer (Beckman Coulter) for 10 minutes at room temperature. Leukocytes were washed and resuspended in phosphate-buffered saline supplemented with 1% human serum albumin (PBS/HSA) (Bayer, Elkhart, IL). Cells were then counted and split into two fractions of 2 × 106 cells in
a volume of 50 µL. The first tube was stained with 20 µL CD45 PE-Cy5 (clone J33), 40 µL CD36 fluorescein isothiocyanate (FITC) (clone FA6-152), and 40 µL CD19 PE (clone J4.119). The second tube was stained with 20 µL of CD45 PE-Cy5, 40 µL of CD3 FITC (clone UCHT1), and 40 µL of CD34 PE (clone 581). Both tubes were incubated in the dark at 4⬚C for 30 minutes and then were washed twice and resuspended in 0.5 mL ice-cold PBS/HSA. Several fractions were sorted on an FACS Vantage cell sorter (BD Biosciences, San Jose, CA) equipped with an Enterprise laser (Coherent, Santa Clara, CA). As shown in Fig. 1, three major subsets of cells were identified based on CD45 expression and side scatter: region R1 contained the blast population, region R2 contained lymphocytes, and region R3 the granulocytes. Granulocytes were sorted directly from region R3. Because CD36 was expressed on virtually all blast cells (data not shown; CD36 is usually expressed on monocytes and megakaryocytes), CD36 was used to identify and sort the blasts from the primary blast region R1 as shown, using the sort gate R4. B cells within the lymphocyte gate were sorted based on CD19 expression
Fig. 1. FACS sorting and analysis of sorted fractions. Each peripheral blood sample was prepared and stained as described in the materials and methods section. Blasts from region R1 (A) were sorted for CD36 expression. Reanalysis of the sorted fraction is shown in region R4 (B). Lymphocytes from region R2 (A) were sorted into CD19⫹ and CD3⫹ fractions. Reanalyses of these fractions are shown in regions R5 (C) and R7 (F), respectively. Blasts from region R1 were also sorted for CD34 expression. Reanalysis of the CD34⫹ fraction is shown in region R6 (E). Granulocytes were directly sorted from region R3. Reanalysis of the sorted fraction is shown in region R3 (D).
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(region R5). From the second tube, blasts from region R1 were sorted for CD34 expression (region R6) and T cells from region R2 were sorted based on CD3 expression (region R7). The high purity of each sorted fraction was confirmed morphologically. 2.3. Interphase FISH Standard cytospin preparations were made from each of the described cell preparations and submitted for FISH analysis. Each slide was fixed in methanol–glacial acetic acid (3:1) and air-dried. Slides were pretreated in 2× standard saline citrate (SSC) at 37⬚C for 40 minutes, dehydrated in a series of three 2-minute incubations with 70, 90, and 100% ethanol, and then air-dried. To detect trisomy 8 anomalies, we used the commercial probe D8Z1 (Vysis, Downers Grove, IL), labeled in SpectrumGreen, which hybridizes to the α-satellite DNA in the centromere region of chromosome 8. Slides were then cover-slipped, sealed, denatured at 80⬚C for 4 minutes, and allowed to hybridize for 16–20 hours at 42⬚C. After hybridization, the slides were washed in 0.4% SSC at 72⬚C for 2 minutes and then transferred to 1× phosphate buffer detergent at room temperature for 1 minute. After this step, the antifade was added, and cells were viewed with a fluorescent microscope equipped with a double band-pass filter for FITC and Texas Red. A total of 300 consecutive interphase nuclei were counted for the presence of three abnormal signals for chromosome 8 (Fig. 2). Based on normal peripheral blood leukocytes as baseline controls, the cutoff for false positives was 5%.
3. Results and discussion Five subset cell populations (T cell, B cell, granulocyte, total blast, and CD34⫹ blast populations) were isolated from the peripheral blood of the patient by means of FACS. Among blast cell populations, 20% were CD34⫹. Each of the five subsets was evaluated for the presence of trisomy 8 as a clonal marker with FISH studies. As shown in Table 1, neither T-cell (CD3⫹) nor B-cell (CD19⫹) populations carried trisomy 8. Trisomy 8 was observed in 94% of CD34⫹ and 96% of CD34⫺ blast populations, respectively; it was also detected in a minor population of granulocytes (8%). Down syndrome AMKL is frequently associated with trisomy 8. This chromosomal abnormality occurs also in patients with myelodysplasia (MDS) without Down syndrome; however, in Down syndrome, there is evidence that trisomy 8 is strongly associated with AMKL, and the marrow is characterized by an abnormal and selective proliferation of dysplastic megakaryocytes [6]. The cure rate with chemotherapy is higher in Down syndrome patients, compared with a low rate in other groups [7,8]. Several clonal studies in non–Down syndrome patients with trisomy 8 have indicated that this abnormality is involved in the myeloid and monocyte series, and possibly an earlier stem
Fig. 2. Trisomy 8 in sorted cell populations as detected with FISH. (A) Lymphocytes (T cells) with normal two signals for CEP 8 probe. (B) Blast cells with three signals for CEP 8, indicating trisomy 8. (C) Granulocytes with two or three signals for CEP 8.
cell [9,10]. In contrast, Zipursky et al. [11] reported a series of patients of Down syndrome with MDS and trisomy 8. From in vitro colony assays for bone marrow cells and subsequent FISH analysis, they found no evidence of this chromosomal abnormality in myeloid and lymphoid cells, suggesting that the progenitor cells in MDS of Down syndrome have the potential of forming cells of megakaryocytic and erythroid lineages. Ma et al. [12] evaluated bone marrow cells from a Down syndrome patient with AMKL and trisomy 8 with FISH studies and then with Wright–Giemsa
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Table 1 Frequency of FISH signals of the chromosome 8-specific probe in sorted cell populations Signal frequency (% of 300 cells) Immunophenotypic expression and cell type ⫹
CD3 (T cells) CD19⫹ (B cells) CD34⫹ blast cells CD36⫹ (total blast cells) Granulocytes
1 signal
2 signals
3 signals
4 signalsa
6.7 6.3 0.7 0.3 1.0
92.2 92.0 3.3 3.0 91.0
1.3 1.7 94.0 95.7 8.0
— — 2.0 2.0 —
5% cutoff rate. a M-dashes indicate no cells with four signals observed.
staining for microscopic identification of cell lineages; they showed that trisomy 8 was confined to the blast population, not detected in myeloid cells, erythroid blasts, or lymphocytes. Furthermore, trisomy 8 was present in a proportion of blasts only (61%), therefore likely representing a secondary genetic change in leukemogenesis. In this study, we conducted a FISH-based clonality analysis of peripheral blood cells from a Down syndrome patient with AMKL and trisomy 8. Five discrete subsets of leukocytes were purified through FACS and analyzed with interphase FISH with a centromeric chromosome 8 probe. We showed that almost all blast cells (regardless of CD34 antigen expression) contained trisomy 8, suggesting that this chromosomal abnormality may be involved in the primary leukemogenesis. Trisomy 8 was not detectable in T or B lymphocytes of our patient. In contrast to other reports, in the present study trisomy 8 was detected in a small population of mature myeloid cells. Although the level of detection was only slightly above the cutoff false positive value, the trisomy 8 is unlikely to be due to the presence of contaminating blast cells in the sorted granulocyte, because all the positive cells were segmented neutrophils. This observation should be confirmed in more case studies. Nevertheless, our results provide evidence that in AMKL of Down syndrome, T or B lymphocytes are not clonally involved, leukemic transformation may occur in progenitor cells with only limited myeloid differentiation. Acknowledgment The authors thank Dr. A. Zipursky for helpful suggestions and M. Chan for preparing the manuscript. References [1] Zipursky A, Brown E, Christensen H, Sutherland R, Doyle J. Leukemia and/or myeloproliferative syndrome in neonates with Down’s syndrome. Semin Perinatol 1997;21:97–101.
[2] Suda T, Suda J, Miura Y, Hayashi Y, Eguchi M, Tadokoro K, Saito M. Clonal analysis of basophil differentiation in bone marrow cultures from a Down’s syndrome patient with megakaryoblastic leukemia. Blood 1985;66:1278–83. [3] Broomhead AF, Kwan YL, Zell NA, Lam-Po-Tang PR, O’Gorman Hughes D. Acute leukemia characterized by trisomy 8 in Down’s syndrome. Pathology 1985;17:111–4. [4] Hecht F, Hecht BK, Morgan R, Sandberg AA, Link MP. Chromosome clues to acute leukemia in Down’s syndrome. Cancer Genet Cytogenet 1986;21:93–8. [5] Hayashi Y, Eguchi M, Syugita K, Nakazawa S, Sato T, Kojima S, Bessho F, Konishi S, Inaba T, Hanada R, Yamamoto K. Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down’s syndrome. Blood 1988;72:15–23. [6] Zipursky A, Thorner P, De Harven E, Christensen H, Doyle J. Myelodysplasia and acute megakaryoblastic leukemia in Dow’s syndrome. Leuk Res 1994;18:163–71. [7] Ravindranath Y, Abella E, Krischer J, Wiley J, Inoue S, Harris M, Chauvenet A, Alvarado CS, Dubowy R, Ritchey AK, Land V, Steuber CP, Weinstein H. Acute myeloid leukemia (AML) in Down’s syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML study 8498. Blood 1992;80:2210–4. [8] Lange B. The management of neoplastic disorders of hematopoiesis in children with Down’s syndrome. Br J Haematol 2000;110:512–24. [9] Parlier V, Tiainen M, Beris P, Miescher PA, Knuutila S, Jotterand Bellomo M. Trisomy 8 detection in granulomonocytic erythrocytic and megakaryocytic lineages by chromosomal in situ suppression hybridization in a case of refractory anemia with ringed sideroblasts complicating the course of paroxysmal nocturnal hemoglobinuria. Br J Haematol 1992;81:296–304. [10] Anastasi J, Feng J, Le Beau MM, Larson RA, Rowley JD, Vardiman JW. Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: Lineage, response to growth factor therapy, and clone expansion. Blood 1993;81:1580–5. [11] Zipursky A, Wang H, Brown EJ, Squire J. Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome. Blood 1994;84:2278–82. [12] Ma SK, Lee ACW, Wan TSK, Lam CK, Chan LC. Trisomy 8 as a secondary genetic change in acute megakaryoblastic leukemia associated with Down’s syndrome. Leukemia 1999;13:491–2.
Short communication
Interphase cytogenetic analysis of clonality in peripheral blood cells from a patient with Down syndrome and acute megakaryoblastic leukemia Hong Changa,*, Dan Lia, Rakash Nayara, Charles Yeb, Wendy Laub, D. Robert Sutherlanda a
Department of Laboratory Hematology, Princess Margaret Hospital, University Health Network, University of Toronto, 610 University Avenue, 4-320, Toronto, Ontario M5G 2M9, Canada b Department of Hematopathology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Received 16 May 2003; received in revised form 12 June 2003; accepted 18 June 2003
Abstract
A combination of fluorescence-activated cell sorting and interphase fluorescence in situ hybridization (FISH) techniques was used to detect a clonal chromosomal marker in blasts, granulocytes, and T and B lymphocytes of the peripheral blood from a patient with Down syndrome and acute megakaryoblastic leukemia (AMKL) associated with trisomy 8 as a karyotypic abnormality. Immunophenotypic studies with flow cytometry showed two populations of leukemic blasts distinguished by their expression of the CD34 antigen. Interphase FISH studies revealed clonal trisomy 8 FISH signals in almost all blast cells, regardless of CD34 expression, as well as in a small subpopulation of granulocytes. Normal chromosome 8 signal patterns were detected in T and B cells and in a great majority of granulocytes. The present study provides evidence for the clonal involvement of leukemic blasts in AMKL of Down syndrome, indicating that a trisomy 8 abnormality may be a primary event in leukemogenesis. The transformation occurs in progenitor cells with limited myeloid differentiation and without involvement of lymphoid lineage cells. 쑖 2004 Elsevier Inc. All rights reserved.
1. Introduction Down syndrome (trisomy 21) is a chromosomal abnormality associated with increased risk of leukemia [1]. The majority of cases are acute megakaryoblastic leukemias (AMKL). Some studies have shown that the blasts in the myelodysplastic (MDS) AMKL of Down syndrome have in vitro properties of a multilineage precursor cell with potential for forming megakaryocytes, erythroid, and basophil/ mast cell progenitors [1,2], but not myeloid cells. It is not clear, however, whether different cell types (including granulocytes and T or B cells) are clonally interrelated in this particular type of leukemia of Down syndrome. Trisomy 8 is the most frequent chromosomal change associated with AMKL, occurring in ~20% of the cases [3–5]. In the present study, we evaluated the peripheral blood of a Down syndrome patient with AMKL who had trisomy 8. To try to delineate the clonal involvement, we sorted discrete leukocyte
* Corresponding author. Tel.: (416) 946-4635; fax: (416) 946-4616. E-mail address: [email protected] (H. Chang). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0165-4608(03)00273-5
subsets using fluorescence-activated cell sorting (FACS) and assessed each highly purified fraction using interphase fluorescence in situ hybridization (FISH) techniques.
2. Materials and methods 2.1. Patient A 2-year-old girl with Down syndrome presented with easy bruising. There was no prior history of transient leukemia in the neonatal period. Physical examination showed no organomegaly or lymphadenopathy. The complete blood counts showed hemoglobin 98 g/L, white blood cell count 5.3 × 109/L (10.9% granulocytes, 48.1% lymphocytes, 37% blast cells, and 3.9% monocytes), and platelets 41 × 109/L. The blasts exhibited undifferentiated morphology and basophilic cytoplasm. Bone marrow aspirates showed a dry tap, but biopsy revealed hypercellularity with significant fibrosis, clusters of dysplastic megakaryocytes, and reduction of both erythropoiesis and granulopoiesis. Cell-surface marker analysis with flow cytometry revealed blast cells expressing
142
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CD7, CD36, CD109, and CD11b and blast cells with weak expression of CD13, CD33, and CD34. Cytoplasmic marker analysis showed the blast cells to be unreactive with antibodies to MPO, CD3, and CD79a. Blasts were also negative for nuclear antigen TdT (all antibodies from Beckman Coulter, Miami, FL). Subsequent bone marrow biopsy analysis with immunohistochemical staining showed blasts weakly positive for CD41 and CD61. A diagnosis of AMKL associated with Down syndrome was made. Cytogenetic study on peripheral blood cells showed the following karyotype: 48,XX,⫹8,⫹21c[7]/47,XX,⫹21c[7]. 2.2. FACS Five milliliters of sodium-heparinized peripheral blood specimen was collected from the patient. Red blood cells were depleted by means of lysis with 1× ammonium chloride lysing buffer (Beckman Coulter) for 10 minutes at room temperature. Leukocytes were washed and resuspended in phosphate-buffered saline supplemented with 1% human serum albumin (PBS/HSA) (Bayer, Elkhart, IL). Cells were then counted and split into two fractions of 2 × 106 cells in
a volume of 50 µL. The first tube was stained with 20 µL CD45 PE-Cy5 (clone J33), 40 µL CD36 fluorescein isothiocyanate (FITC) (clone FA6-152), and 40 µL CD19 PE (clone J4.119). The second tube was stained with 20 µL of CD45 PE-Cy5, 40 µL of CD3 FITC (clone UCHT1), and 40 µL of CD34 PE (clone 581). Both tubes were incubated in the dark at 4⬚C for 30 minutes and then were washed twice and resuspended in 0.5 mL ice-cold PBS/HSA. Several fractions were sorted on an FACS Vantage cell sorter (BD Biosciences, San Jose, CA) equipped with an Enterprise laser (Coherent, Santa Clara, CA). As shown in Fig. 1, three major subsets of cells were identified based on CD45 expression and side scatter: region R1 contained the blast population, region R2 contained lymphocytes, and region R3 the granulocytes. Granulocytes were sorted directly from region R3. Because CD36 was expressed on virtually all blast cells (data not shown; CD36 is usually expressed on monocytes and megakaryocytes), CD36 was used to identify and sort the blasts from the primary blast region R1 as shown, using the sort gate R4. B cells within the lymphocyte gate were sorted based on CD19 expression
Fig. 1. FACS sorting and analysis of sorted fractions. Each peripheral blood sample was prepared and stained as described in the materials and methods section. Blasts from region R1 (A) were sorted for CD36 expression. Reanalysis of the sorted fraction is shown in region R4 (B). Lymphocytes from region R2 (A) were sorted into CD19⫹ and CD3⫹ fractions. Reanalyses of these fractions are shown in regions R5 (C) and R7 (F), respectively. Blasts from region R1 were also sorted for CD34 expression. Reanalysis of the CD34⫹ fraction is shown in region R6 (E). Granulocytes were directly sorted from region R3. Reanalysis of the sorted fraction is shown in region R3 (D).
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(region R5). From the second tube, blasts from region R1 were sorted for CD34 expression (region R6) and T cells from region R2 were sorted based on CD3 expression (region R7). The high purity of each sorted fraction was confirmed morphologically. 2.3. Interphase FISH Standard cytospin preparations were made from each of the described cell preparations and submitted for FISH analysis. Each slide was fixed in methanol–glacial acetic acid (3:1) and air-dried. Slides were pretreated in 2× standard saline citrate (SSC) at 37⬚C for 40 minutes, dehydrated in a series of three 2-minute incubations with 70, 90, and 100% ethanol, and then air-dried. To detect trisomy 8 anomalies, we used the commercial probe D8Z1 (Vysis, Downers Grove, IL), labeled in SpectrumGreen, which hybridizes to the α-satellite DNA in the centromere region of chromosome 8. Slides were then cover-slipped, sealed, denatured at 80⬚C for 4 minutes, and allowed to hybridize for 16–20 hours at 42⬚C. After hybridization, the slides were washed in 0.4% SSC at 72⬚C for 2 minutes and then transferred to 1× phosphate buffer detergent at room temperature for 1 minute. After this step, the antifade was added, and cells were viewed with a fluorescent microscope equipped with a double band-pass filter for FITC and Texas Red. A total of 300 consecutive interphase nuclei were counted for the presence of three abnormal signals for chromosome 8 (Fig. 2). Based on normal peripheral blood leukocytes as baseline controls, the cutoff for false positives was 5%.
3. Results and discussion Five subset cell populations (T cell, B cell, granulocyte, total blast, and CD34⫹ blast populations) were isolated from the peripheral blood of the patient by means of FACS. Among blast cell populations, 20% were CD34⫹. Each of the five subsets was evaluated for the presence of trisomy 8 as a clonal marker with FISH studies. As shown in Table 1, neither T-cell (CD3⫹) nor B-cell (CD19⫹) populations carried trisomy 8. Trisomy 8 was observed in 94% of CD34⫹ and 96% of CD34⫺ blast populations, respectively; it was also detected in a minor population of granulocytes (8%). Down syndrome AMKL is frequently associated with trisomy 8. This chromosomal abnormality occurs also in patients with myelodysplasia (MDS) without Down syndrome; however, in Down syndrome, there is evidence that trisomy 8 is strongly associated with AMKL, and the marrow is characterized by an abnormal and selective proliferation of dysplastic megakaryocytes [6]. The cure rate with chemotherapy is higher in Down syndrome patients, compared with a low rate in other groups [7,8]. Several clonal studies in non–Down syndrome patients with trisomy 8 have indicated that this abnormality is involved in the myeloid and monocyte series, and possibly an earlier stem
Fig. 2. Trisomy 8 in sorted cell populations as detected with FISH. (A) Lymphocytes (T cells) with normal two signals for CEP 8 probe. (B) Blast cells with three signals for CEP 8, indicating trisomy 8. (C) Granulocytes with two or three signals for CEP 8.
cell [9,10]. In contrast, Zipursky et al. [11] reported a series of patients of Down syndrome with MDS and trisomy 8. From in vitro colony assays for bone marrow cells and subsequent FISH analysis, they found no evidence of this chromosomal abnormality in myeloid and lymphoid cells, suggesting that the progenitor cells in MDS of Down syndrome have the potential of forming cells of megakaryocytic and erythroid lineages. Ma et al. [12] evaluated bone marrow cells from a Down syndrome patient with AMKL and trisomy 8 with FISH studies and then with Wright–Giemsa
144
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Table 1 Frequency of FISH signals of the chromosome 8-specific probe in sorted cell populations Signal frequency (% of 300 cells) Immunophenotypic expression and cell type ⫹
CD3 (T cells) CD19⫹ (B cells) CD34⫹ blast cells CD36⫹ (total blast cells) Granulocytes
1 signal
2 signals
3 signals
4 signalsa
6.7 6.3 0.7 0.3 1.0
92.2 92.0 3.3 3.0 91.0
1.3 1.7 94.0 95.7 8.0
— — 2.0 2.0 —
5% cutoff rate. a M-dashes indicate no cells with four signals observed.
staining for microscopic identification of cell lineages; they showed that trisomy 8 was confined to the blast population, not detected in myeloid cells, erythroid blasts, or lymphocytes. Furthermore, trisomy 8 was present in a proportion of blasts only (61%), therefore likely representing a secondary genetic change in leukemogenesis. In this study, we conducted a FISH-based clonality analysis of peripheral blood cells from a Down syndrome patient with AMKL and trisomy 8. Five discrete subsets of leukocytes were purified through FACS and analyzed with interphase FISH with a centromeric chromosome 8 probe. We showed that almost all blast cells (regardless of CD34 antigen expression) contained trisomy 8, suggesting that this chromosomal abnormality may be involved in the primary leukemogenesis. Trisomy 8 was not detectable in T or B lymphocytes of our patient. In contrast to other reports, in the present study trisomy 8 was detected in a small population of mature myeloid cells. Although the level of detection was only slightly above the cutoff false positive value, the trisomy 8 is unlikely to be due to the presence of contaminating blast cells in the sorted granulocyte, because all the positive cells were segmented neutrophils. This observation should be confirmed in more case studies. Nevertheless, our results provide evidence that in AMKL of Down syndrome, T or B lymphocytes are not clonally involved, leukemic transformation may occur in progenitor cells with only limited myeloid differentiation. Acknowledgment The authors thank Dr. A. Zipursky for helpful suggestions and M. Chan for preparing the manuscript. References [1] Zipursky A, Brown E, Christensen H, Sutherland R, Doyle J. Leukemia and/or myeloproliferative syndrome in neonates with Down’s syndrome. Semin Perinatol 1997;21:97–101.
[2] Suda T, Suda J, Miura Y, Hayashi Y, Eguchi M, Tadokoro K, Saito M. Clonal analysis of basophil differentiation in bone marrow cultures from a Down’s syndrome patient with megakaryoblastic leukemia. Blood 1985;66:1278–83. [3] Broomhead AF, Kwan YL, Zell NA, Lam-Po-Tang PR, O’Gorman Hughes D. Acute leukemia characterized by trisomy 8 in Down’s syndrome. Pathology 1985;17:111–4. [4] Hecht F, Hecht BK, Morgan R, Sandberg AA, Link MP. Chromosome clues to acute leukemia in Down’s syndrome. Cancer Genet Cytogenet 1986;21:93–8. [5] Hayashi Y, Eguchi M, Syugita K, Nakazawa S, Sato T, Kojima S, Bessho F, Konishi S, Inaba T, Hanada R, Yamamoto K. Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down’s syndrome. Blood 1988;72:15–23. [6] Zipursky A, Thorner P, De Harven E, Christensen H, Doyle J. Myelodysplasia and acute megakaryoblastic leukemia in Dow’s syndrome. Leuk Res 1994;18:163–71. [7] Ravindranath Y, Abella E, Krischer J, Wiley J, Inoue S, Harris M, Chauvenet A, Alvarado CS, Dubowy R, Ritchey AK, Land V, Steuber CP, Weinstein H. Acute myeloid leukemia (AML) in Down’s syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML study 8498. Blood 1992;80:2210–4. [8] Lange B. The management of neoplastic disorders of hematopoiesis in children with Down’s syndrome. Br J Haematol 2000;110:512–24. [9] Parlier V, Tiainen M, Beris P, Miescher PA, Knuutila S, Jotterand Bellomo M. Trisomy 8 detection in granulomonocytic erythrocytic and megakaryocytic lineages by chromosomal in situ suppression hybridization in a case of refractory anemia with ringed sideroblasts complicating the course of paroxysmal nocturnal hemoglobinuria. Br J Haematol 1992;81:296–304. [10] Anastasi J, Feng J, Le Beau MM, Larson RA, Rowley JD, Vardiman JW. Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: Lineage, response to growth factor therapy, and clone expansion. Blood 1993;81:1580–5. [11] Zipursky A, Wang H, Brown EJ, Squire J. Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome. Blood 1994;84:2278–82. [12] Ma SK, Lee ACW, Wan TSK, Lam CK, Chan LC. Trisomy 8 as a secondary genetic change in acute megakaryoblastic leukemia associated with Down’s syndrome. Leukemia 1999;13:491–2.