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Cytogenetic Triclonality in T-Cell Acute Lymphoblastic Leukemia
Cytogenetic Triclonality in T-Cell Acute Lymphoblastic Leukemia: A Conventional and Molecular Cytogenetic Study K. F. Wong, C. C. So, and L. P. Siu
ABSTRACT: Cytogenetically-unrelated clones are infrequently seen in hematologic malignancies, and are particularly uncommon in acute lymphoblastic leukemia. We report a case of T-cell acute lymphoblastic leukemia with L2 morphology which demonstrated three cytogenetically distinct clones: 46,XY, t(2;9)(p21;q34)/46,XY,del(6)(q21q23)/47,XX,18. Interphase cytogenetic analysis by fluorescence in situ hybridization (FISH) confirmed the presence of trisomy 8 in a significant proportion of lymphoblasts, while reverse transcription-polymerase chain reaction (RT-PCR) did not show the presence of BCR/ABL fusion. This is the first report describing the occurrence of cytogenetic triclonality in de novo T-cell acute lymphoblastic leukemia. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Approximately two thirds of all acute lymphoblastic leukemias (ALL) have recognizable nonrandom cytogenetic abnormalities. Most cases show consistently-occurring structural abnormalities such as t(1;19)(q23;p13), del(6) (q15-21) and t(9;22)(q34;q11); however, additional karyotypic changes are not uncommon [1]. These secondary changes occur almost invariably in the background of the primary abnormalities, suggesting evolution of subclones from the original single clone during disease progression. On the other hand, karyotypic disparity is a rare occurrence in ALL at the time of diagnosis [2–4]. We report a case of Ph-negative T-cell ALL showing three distinct and unrelated clones on cytogenetic analysis. CASE REPORT AND CYTOGENETICS An 11-year-old boy presented with weight loss and neck and shoulder pain for 2 months. Clinical examination showed hepatomegaly and generalized lymphadenopathy. There were also multiple erythematous skin rashes over the chest and limbs. Chest X-ray revealed a mediastinal mass. Peripheral blood count showed: hemoglobin 11.3 g/dL, platelets 166 3 109/L, and leukocytes 136 3 109/L, with 32% neutrophils, 7% lymphocytes, 7% monocytes, 1%
eosinophils, and 53% lymphoblasts. The lymphoblasts were medium to large in size, and had irregular to deeply clefted nuclei, small to distinct nucleoli, and scanty to fair amount of basophilic, sometimes vacuolated, cytoplasm. Bone marrow examination showed a hypercellular marrow with adequate megakaryocytes, normal granulopoiesis, and depressed erythropoiesis. Lymphoblasts accounted for 70% of the marrow nucleated cells. They were cytochemically inert, being negative for myeloperoxidase, Sudan Black B and esterases. Immunophenotypic study showed that they expressed CD2, CD3, CD7, and TdT but were negative for B-cell, myeloid, and megakaryocytic markers. A diagnosis of T-cell ALL was made. Cytogenetic analysis was performed by overnight fluorodeoxyuridine synchronized culture of the marrow cells. Metaphase chromosomes were banded by trypsin/Giemsa and karyotyped according to the ISCN [5]. Three abnormal and unrelated clones were found with 46,XY,t(2;9)(p21;q34)[11]/ 46,idem,dup(1)(p22p36)[3]/46,XY,del(6)(q21q23)[2]/47, XY,18[8] (Fig. 1), in addition to a normal clone of 46, XY[5]. He was treated with systemic chemotherapy which included prednisolone, vincristine, daunorubicin, and L-asparaginase.
MOLECULAR CYTOGENETIC STUDIES From the Department of Pathology, Queen Elizabeth Hospital, Hong Kong, S. A. R. China. Address reprint requests to: Dr. K. F. Wong, Department of Pathology, Queen Elizabeth Hospital, 30 Gascoigne Road, Kowloon, Hong Kong, S. A. R. China. Received September 17, 1998; accepted April 23, 1999. Cancer Genet Cytogenet 116:77–80 (2000) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Fluorescence In Situ Hybridization (FISH) Fluorescence in situ hybridization was performed on cells from the cytogenetic preparation stored in methanol:glacial acetic acid (3:1). Control cells were obtained from normal individuals. Fluorescence in situ hybridization detection of chromosome 8 was carried out using digoxi-
0165-4608/00/$–see front matter PII S0165-4608(99)00096-5
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Figure 1 G-banded karyotype showing (A) 46,XY,t(2;9)(p21;q34), (B) 46,XY,del(6)(q21q23), and (C) 47,XY,18. genin-labeled chromosome 8 a-satellite DNA probe (Boehringer Mannheim, Germany). Briefly, the slides were denatured in 70% formamide and 2 3 standard sodium citrate (SSC) at 708C. They were then dehydrated sequentially in 70%, 90%, and 100% cold ethanol. The probes were applied onto the slides, and the slides were incubated overnight in a moist chamber at 378C. After washing in 2 3 SSC, the hybridized probes were detected by using rhodamine-conjugated sheep anti-digoxigenin antibody (Boehringer Mannheim). Cells were scored under ultraviolet light; 8.5% of the lymphoblasts showed three hybrid-
ization signals, while the false-positive rate (three signals) was 2%. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Because the breakpoint of the chromosome 9 in the t(2;9) translocation was located at 9q34, a masked t(9;22) (q34;q11) translocation had to be excluded. Reserve transcription-polymerase chain reaction using primer sets specific for both the major and minor breakpoint cluster regions on the BCR gene was performed on the leukemic
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Triclonal ALL
Figure 1 Continued.
lymphoblasts to detect the presence of BCR/ABL fusion transcripts as previously described [6]. No BCR/ABL fusion transcript could be demonstrated.
DISCUSSION Hematologic malignancies have often been considered a monoclonal disorder, that is, neoplastic cells are derived from a single mutated cell. This is supported by the frequent demonstration of a single abnormal clone in most leukemias and myelodysplastic syndromes on conventional cytogenetic analysis. Karyotypic divergence is an uncommon finding in hematologic malignancies at the time of diagnosis, but is more commonly seen in patients with a prior history of cytotoxic therapy [2–4, 7, 8]. In most instances, cytogenetic evolution occurs with additional chromosomal abnormalities found in a background of related karyotypes. Nevertheless, cytogenetically unrelated clones can sometimes be found in hematologic malignancies, and have been reported to occur more frequently in AML of monocytic lineage [2, 4, 7]. Karytoypic disparity is, however, rarely seen in ALL, with a reported incidence of only 0–0.4% [3, 4] and is often not seen even in large reported series on cytogenetic findings of ALL [9, 10]. In the reported cases, karyotypic diversity occurs mainly in the form of cytogenetic biclonality. In this report, we describe a case of ALL with three distinct and unrelated clones on conventional cytogenetic analysis. To the best of our knowledge, this is the first report to describe such an occurrence in de novo T-cell ALL. Because ALL patients with t(9;22)(q34;q11) often develop other chromosomal abnormalities (trisomy 8 being
one of them) during disease progression [1], the occurrence of trisomy 8 and rearrangement involving 9q34 in our patient raises the possibility of a masked t(9;22), with the trisomy 8 representing the cytogenetic evolution of the primary clone. However, molecular study with RT-PCR did not show any BCR/ABL fusion transcript in our patient. Although karyotypic disparity is rare in ALL on conventional cytogenetic analysis, genetic diversity appears to be common at the molecular level. By using PCR for the genes encoding the immunoglobulin and complementarity-determining region 3, biclonality could be demonstrated in more than one fifth of the cases of B-cell ALL [11, 12]. It is therefore possible that the cytogenetically unrelated clones are actually subclones with underlying common but submicroscopic karyotypic aberration, and the different chromosomal abnormalities may just represent clonal evolution of the ancestral clone.
REFERENCES 1. Heim S, Mitelman F (1986): Secondary chromosome aberrations in the acute leukemias. Cancer Genet Cytogenet 22:331– 338. 2. Furuya T, Morgan R, Sandberg AA (1992): Cytogenetic biclonality in malignant hematologic disorders. Cancer Genet Cytogenet 63:25–28. 3. Heim S, Mitelman F (1989): Cytogenetically unrelated clones in hematological neoplasms. Leukemia 3:6–8. 4. Kobayashi H, Kaneko Y, Maseki N, Sakurai M (1990): Karyotypically unrelated clones in acute leukemias and myelodysplastic syndromes. Cancer Genet Cytogenet 47:171–178. 5. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel.
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6. So CC, Wong KF, Chung JSL, Kwong YL (1999): BCR/ABL translocation in adult acute lymphoblastic leukaemia—comparison of conventional and interphase cytogenetic studies. Cancer Genet Cytogenet 110:19–22.
10. Raimondi SC, Behm FG, Roberson PK, Pui CH, Rivera GK, Murphy SB, Williams DL (1988): Cytogenetics of childhood T-cell leukemia. Blood 72:1560–1566.
7. Wong KF, Kwong YL, Tang KC (1995): Biclonal acute monoblastic leukemia showing del(7q) and concomitant trisomies 9 and 22. Cancer Genet Cytogenet 82:70–72.
11. Scrideli, CA, Simoes AL, Defavery R, Bernardes JE, Duarte MH, Tone LG (1997): Childhood B lineage acute lymphoblastic leukemia clonality study by polymerase chain reaction. J Pediatr Hematol Oncol 19:516–522.
8. Wong KF, So CC (1997): Biclonal B-cell chronic lymphocytic leukemia with inv(14). Cancer Genet Cytogenet 94:135–137. 9. Katz JA, Taylor LD, Carroll A, Elder FFB, Mahoney DH (1991): Cytogenetic features of childhood acute lymphoblastic leukemia: a concordance study on a Pediatric Oncology Group study. Cancer Genet Cytogenet 55:249–256.
12. Beishuizen A, Hahlen K, Hagemeijer A, Verhoeven MA, Hooijkaas H, Adriaansen HJ, Wolvers-Tettero IL, van Wering ER, van Dongen J (1991): Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursors B-cell origin. Leukemia 5:657–667.
ABSTRACT: Cytogenetically-unrelated clones are infrequently seen in hematologic malignancies, and are particularly uncommon in acute lymphoblastic leukemia. We report a case of T-cell acute lymphoblastic leukemia with L2 morphology which demonstrated three cytogenetically distinct clones: 46,XY, t(2;9)(p21;q34)/46,XY,del(6)(q21q23)/47,XX,18. Interphase cytogenetic analysis by fluorescence in situ hybridization (FISH) confirmed the presence of trisomy 8 in a significant proportion of lymphoblasts, while reverse transcription-polymerase chain reaction (RT-PCR) did not show the presence of BCR/ABL fusion. This is the first report describing the occurrence of cytogenetic triclonality in de novo T-cell acute lymphoblastic leukemia. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Approximately two thirds of all acute lymphoblastic leukemias (ALL) have recognizable nonrandom cytogenetic abnormalities. Most cases show consistently-occurring structural abnormalities such as t(1;19)(q23;p13), del(6) (q15-21) and t(9;22)(q34;q11); however, additional karyotypic changes are not uncommon [1]. These secondary changes occur almost invariably in the background of the primary abnormalities, suggesting evolution of subclones from the original single clone during disease progression. On the other hand, karyotypic disparity is a rare occurrence in ALL at the time of diagnosis [2–4]. We report a case of Ph-negative T-cell ALL showing three distinct and unrelated clones on cytogenetic analysis. CASE REPORT AND CYTOGENETICS An 11-year-old boy presented with weight loss and neck and shoulder pain for 2 months. Clinical examination showed hepatomegaly and generalized lymphadenopathy. There were also multiple erythematous skin rashes over the chest and limbs. Chest X-ray revealed a mediastinal mass. Peripheral blood count showed: hemoglobin 11.3 g/dL, platelets 166 3 109/L, and leukocytes 136 3 109/L, with 32% neutrophils, 7% lymphocytes, 7% monocytes, 1%
eosinophils, and 53% lymphoblasts. The lymphoblasts were medium to large in size, and had irregular to deeply clefted nuclei, small to distinct nucleoli, and scanty to fair amount of basophilic, sometimes vacuolated, cytoplasm. Bone marrow examination showed a hypercellular marrow with adequate megakaryocytes, normal granulopoiesis, and depressed erythropoiesis. Lymphoblasts accounted for 70% of the marrow nucleated cells. They were cytochemically inert, being negative for myeloperoxidase, Sudan Black B and esterases. Immunophenotypic study showed that they expressed CD2, CD3, CD7, and TdT but were negative for B-cell, myeloid, and megakaryocytic markers. A diagnosis of T-cell ALL was made. Cytogenetic analysis was performed by overnight fluorodeoxyuridine synchronized culture of the marrow cells. Metaphase chromosomes were banded by trypsin/Giemsa and karyotyped according to the ISCN [5]. Three abnormal and unrelated clones were found with 46,XY,t(2;9)(p21;q34)[11]/ 46,idem,dup(1)(p22p36)[3]/46,XY,del(6)(q21q23)[2]/47, XY,18[8] (Fig. 1), in addition to a normal clone of 46, XY[5]. He was treated with systemic chemotherapy which included prednisolone, vincristine, daunorubicin, and L-asparaginase.
MOLECULAR CYTOGENETIC STUDIES From the Department of Pathology, Queen Elizabeth Hospital, Hong Kong, S. A. R. China. Address reprint requests to: Dr. K. F. Wong, Department of Pathology, Queen Elizabeth Hospital, 30 Gascoigne Road, Kowloon, Hong Kong, S. A. R. China. Received September 17, 1998; accepted April 23, 1999. Cancer Genet Cytogenet 116:77–80 (2000) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Fluorescence In Situ Hybridization (FISH) Fluorescence in situ hybridization was performed on cells from the cytogenetic preparation stored in methanol:glacial acetic acid (3:1). Control cells were obtained from normal individuals. Fluorescence in situ hybridization detection of chromosome 8 was carried out using digoxi-
0165-4608/00/$–see front matter PII S0165-4608(99)00096-5
78
K. F. Wong et al.
Figure 1 G-banded karyotype showing (A) 46,XY,t(2;9)(p21;q34), (B) 46,XY,del(6)(q21q23), and (C) 47,XY,18. genin-labeled chromosome 8 a-satellite DNA probe (Boehringer Mannheim, Germany). Briefly, the slides were denatured in 70% formamide and 2 3 standard sodium citrate (SSC) at 708C. They were then dehydrated sequentially in 70%, 90%, and 100% cold ethanol. The probes were applied onto the slides, and the slides were incubated overnight in a moist chamber at 378C. After washing in 2 3 SSC, the hybridized probes were detected by using rhodamine-conjugated sheep anti-digoxigenin antibody (Boehringer Mannheim). Cells were scored under ultraviolet light; 8.5% of the lymphoblasts showed three hybrid-
ization signals, while the false-positive rate (three signals) was 2%. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Because the breakpoint of the chromosome 9 in the t(2;9) translocation was located at 9q34, a masked t(9;22) (q34;q11) translocation had to be excluded. Reserve transcription-polymerase chain reaction using primer sets specific for both the major and minor breakpoint cluster regions on the BCR gene was performed on the leukemic
79
Triclonal ALL
Figure 1 Continued.
lymphoblasts to detect the presence of BCR/ABL fusion transcripts as previously described [6]. No BCR/ABL fusion transcript could be demonstrated.
DISCUSSION Hematologic malignancies have often been considered a monoclonal disorder, that is, neoplastic cells are derived from a single mutated cell. This is supported by the frequent demonstration of a single abnormal clone in most leukemias and myelodysplastic syndromes on conventional cytogenetic analysis. Karyotypic divergence is an uncommon finding in hematologic malignancies at the time of diagnosis, but is more commonly seen in patients with a prior history of cytotoxic therapy [2–4, 7, 8]. In most instances, cytogenetic evolution occurs with additional chromosomal abnormalities found in a background of related karyotypes. Nevertheless, cytogenetically unrelated clones can sometimes be found in hematologic malignancies, and have been reported to occur more frequently in AML of monocytic lineage [2, 4, 7]. Karytoypic disparity is, however, rarely seen in ALL, with a reported incidence of only 0–0.4% [3, 4] and is often not seen even in large reported series on cytogenetic findings of ALL [9, 10]. In the reported cases, karyotypic diversity occurs mainly in the form of cytogenetic biclonality. In this report, we describe a case of ALL with three distinct and unrelated clones on conventional cytogenetic analysis. To the best of our knowledge, this is the first report to describe such an occurrence in de novo T-cell ALL. Because ALL patients with t(9;22)(q34;q11) often develop other chromosomal abnormalities (trisomy 8 being
one of them) during disease progression [1], the occurrence of trisomy 8 and rearrangement involving 9q34 in our patient raises the possibility of a masked t(9;22), with the trisomy 8 representing the cytogenetic evolution of the primary clone. However, molecular study with RT-PCR did not show any BCR/ABL fusion transcript in our patient. Although karyotypic disparity is rare in ALL on conventional cytogenetic analysis, genetic diversity appears to be common at the molecular level. By using PCR for the genes encoding the immunoglobulin and complementarity-determining region 3, biclonality could be demonstrated in more than one fifth of the cases of B-cell ALL [11, 12]. It is therefore possible that the cytogenetically unrelated clones are actually subclones with underlying common but submicroscopic karyotypic aberration, and the different chromosomal abnormalities may just represent clonal evolution of the ancestral clone.
REFERENCES 1. Heim S, Mitelman F (1986): Secondary chromosome aberrations in the acute leukemias. Cancer Genet Cytogenet 22:331– 338. 2. Furuya T, Morgan R, Sandberg AA (1992): Cytogenetic biclonality in malignant hematologic disorders. Cancer Genet Cytogenet 63:25–28. 3. Heim S, Mitelman F (1989): Cytogenetically unrelated clones in hematological neoplasms. Leukemia 3:6–8. 4. Kobayashi H, Kaneko Y, Maseki N, Sakurai M (1990): Karyotypically unrelated clones in acute leukemias and myelodysplastic syndromes. Cancer Genet Cytogenet 47:171–178. 5. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel.
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K. F. Wong et al.
6. So CC, Wong KF, Chung JSL, Kwong YL (1999): BCR/ABL translocation in adult acute lymphoblastic leukaemia—comparison of conventional and interphase cytogenetic studies. Cancer Genet Cytogenet 110:19–22.
10. Raimondi SC, Behm FG, Roberson PK, Pui CH, Rivera GK, Murphy SB, Williams DL (1988): Cytogenetics of childhood T-cell leukemia. Blood 72:1560–1566.
7. Wong KF, Kwong YL, Tang KC (1995): Biclonal acute monoblastic leukemia showing del(7q) and concomitant trisomies 9 and 22. Cancer Genet Cytogenet 82:70–72.
11. Scrideli, CA, Simoes AL, Defavery R, Bernardes JE, Duarte MH, Tone LG (1997): Childhood B lineage acute lymphoblastic leukemia clonality study by polymerase chain reaction. J Pediatr Hematol Oncol 19:516–522.
8. Wong KF, So CC (1997): Biclonal B-cell chronic lymphocytic leukemia with inv(14). Cancer Genet Cytogenet 94:135–137. 9. Katz JA, Taylor LD, Carroll A, Elder FFB, Mahoney DH (1991): Cytogenetic features of childhood acute lymphoblastic leukemia: a concordance study on a Pediatric Oncology Group study. Cancer Genet Cytogenet 55:249–256.
12. Beishuizen A, Hahlen K, Hagemeijer A, Verhoeven MA, Hooijkaas H, Adriaansen HJ, Wolvers-Tettero IL, van Wering ER, van Dongen J (1991): Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursors B-cell origin. Leukemia 5:657–667.