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Three isodicentric Philadelphia chromosomes in acute phase of chronic myeloid leukemia: A case report
Three Isodicentric Philadelphia Chromosomes in Acute Phase of Chronic Myeloid Leukemia: A Case Report G. Kovacs, A. Georgii, and K. Mainzer
ABSTRACT: This report describes a case of blast crisis in chronic myeloid leukemia with the occurrence of isodicentric Ph chromosomes. We briefly review observations on isoPh chromosomes in chronic myeloid leukemia. INTRODUCTION Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in the leukemic cells of about 90% of patients with this disease. The most c o m m o n additional chromosome aberration at the time of blast transformation is the appearance of a second Ph chromosome. A n isochromosome 17q and trisomies 8 and 19 are also often present. However, an isodicentric Ph chromosome is a very rare karyotypic change in the acute phase of CML. Whang-Peng et ah first reported five cases of CML with dicentric Ph chromosomes [1]. Prigogina et al. described a dicentric Ph chromosome detected at the time of acceleration in the blood and bone marrow, as well as in the spleen, of a patient suffering from CML [2]. Moreover, iso- and isodicentric Ph chromosomes have been observed in the chronic and the accelerated phases of three CML patients [3, 4]. In this article, we describe a case of CML with the appearance of one, two, or three isodicentric Ph chromosomes in the acute phase of the disease.
MATERIALS AND METHODS Case Report A 37-year-old male patient was referred for investigation of elevated white cell and platelet counts (discovered accidentally during investigation for a pyloric ulcer) to the Altona Hospital, Hamburg, in October 1978. No l y m p h nodes were palpable, and both the liver and spleen were normal in size. Hematologic investigation at the time of admission showed a white cell count of 160 × 109/L, a red cell count of 4.75 × 1012/L, platelets of 564 × 109/L, and hemoglobin of 14.4 g/dl. The white cell
From the Laboratoryof Cytogenetics(G.K.),the Institute of Pathology (A.G.),Medical School Hanover, Hanover, and the Departmentof Medicine,Altona Hospital, (K.M.)Hamburg,F.R.G. Address requests for reprints to Dr. G. Kovacs, Laboratory of Cytogenetics, Institute of Pathology, Medical School Hanover, P.O. Box 61 01 40, D-3000 Hanover 61, F.R.G. Received October 12, 1984; accepted February 28, 1985.
29 © 1986 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet20:29-33 (1986) 0165-4608/86/$03.50
30
o. Kovacs et al. differential count was 36% segmented neutrophils, 33% bands, 14% metamyelocytes, 11% myelocytes, 6% promyelocytes, 1% blasts, 5% lymphocytes, and 3% normoblasts. The neutrophil alkaline phosphatase score was negative. Bone marrow aspirate examination, as well as the marrow trephine biopsy, showed hypercellularity due to increased granulopoiesis and megakaryopoiesis. There was a moderate diffused increase in marrow fibrosis. The hematologic and trephine findings corresponded with a diagnosis of CML. Myleran therapy was initiated, and when the peripheral blood count returned to normal, maintenance chemotherapy was resumed, using intermittent courses of Myleran. The repeat marrow trephine biopsy findings in May 1980 corresponded to those observed in 1978. In August 1983, the patient was readmitted to the hospital because of bronchopneumonia with a greatly elevated white cell count of 311 x 109/L. Differential count showed 66% myeloblasts, 1% promyelocyte, 2% myelocytes, 8% metamyelocytes, 9% bands, 13% segmented neutrophils, and 1% eosinophils. Marrow trephine biopsy showed a hypercellular marrow with moderate diffuse fibrosis, confirming the acute blastic phase of CML. Small, discrete cervical, axillary, and inguinal lymph nodes were palpable, and both liver and spleen were enlarged 4 cm below the costal margin. A diagnosis was made of CML in blast crisis. Treatment with vincristine and prednisone was without effect, and the patient died 5 days after admission. Postmortem findings revealed changes in the sternum, femur, and vertebra that corresponded to an acute transformation of CML with complete overgrowth of the bone marrow by myeloblasts. Enlarged lymph nodes in axillary, cervical, and inguinal regions, splenomegaly (1055 g), hepatomegaly (3120 g), and enlarged kidneys (each 200 g) were found with leukemic infiltration. Both lungs showed confluent bronchopneumonia, with leukemic infiltration in the parenchyma and on the visceral pleura.
Cytogenetic Studies Chromosome analyses were carried out on bone marrow aspirates using the direct method and on peripheral blood cultures after 24 hr of incubation. Heparinized blood and bone marrow samples were centrifuged, and white blood cells were washed twice in a medium without fetal calf serum. The cells then were processed for direct preparation or cultured in RPMI 1640 medium supplemented with 15% fetal calf serum for 24 hr. Colchicine was added only during the hypotonic treatment at a final concentration of 0.1 p.g/ml. After fixing, air-dried preparations were made, and GTG- and GBG-bandings were performed. RESULTS AND DISCUSSION Eighty-one metaphases from unstimulated blood cultures were analyzed using GTGbanding; 38 metaphases from bone marrow were also analyzed using this method. The karyotyping of bone marrow, as well as of peripheral blood cells, revealed a simple clonal evolution following a linear pathway (Table 1). The first step was a classical translocation between chromosomes #9 and #22. In the second step, subsequent Ph chromosomes underwent abnormal replication, resulting in the first isodicentric Ph chromosome, which further replicated abnormally, resulting in the second and third isochromosomes 2 2 q - . The banding pattern of isodicentric Ph chromosomes was identical in all cells analyzed (Fig. 1A). When the metaphases were stained with CBG-technique, it was obvious that these isochromosomes were dicentric (Fig. 1B). The translocated segment on chromosome #9 was not further
31
Isodicentric Ph Chromosomes in CML
Table 1
Chromosome aberrations in bone marrow and peripheral blood cells analyzed with G-banding No. of cells
Stemline Sideline 1 Sideline 2 Sideline 3
Karyotypes
Bone marrow
Blood
47,XY,9q +, - 22, + 2idicPh 46,XY,t(9;22) 46,XY,9q+,-22,+idicPh 48,XY,9q+,-22,+3idicPh
22 3 5 8
43 9 11 18
increased, and no additional chromosome material was detected on any other chromosomes. Two isodicentric Ph chromosomes were found in 55% of metaphases; thus, the stemline karyotype was 47,XY,9q + , - 2 2 , + 2idicPh. Isodicentric Ph chromosomes present in this case are apparently identical to those found by Whang-Peng et al. [1]. These authors postulated that the isodicentric Ph chromosome represents two Ph chromosomes "joined at the satellite region of the short arms by either fusion or translocation." According to this hypothesis, the Ph c h r o m o s o m e must be first duplicated, triplicated, or futher replicated, and must then undergo centric fusion or translocation, resulting in iso- or isodicentric Ph chromosomes. A Ph c h r o m o s o m e with isodicentric Ph chromosome(s) appearing in the same cell must also occur. This p a t h w a y of the genesis of isochromosomes does not seem to be possible w h e n v i e w e d against the clonal evolution in our case: there was no evidence for the appearance of both a Ph c h r o m o s o m e and an isodicentric Ph chromosome in the same cells. Instead, it is possible that an isoPh chromosome arises through " m i s d i v i s i o n " of the centromere (the centromere divides crosswise) or through homologous adjacent symmetrical exchange at the centromeric region of a Ph c h r o m o s o m e [5, 6]. The second and third isochromosomes could be p r o d u c e d through n o n d i s j u n c t i o n or selective endoreduplication. In the stemline karyotype of the case presented here, there were four copies of the rearranged region of chromosome #22. However, in 22% of the cells analyzed, the karyotype revealed six copies of this region. This extreme increase in the amount of the Ph chromosome occurs very rarely in CML; only a few cases have been reported with more than two Ph chromosomes. In one of five cases described by Whang-Peng et al., two dicentric Ph chromosomes occurred in 12% of the cells [1]. Furthermore, Stoll and Oberling [7] reported a case with three Ph chromosomes, and a h y p o t r i p l o i d stemline with four Ph chromosomes was described in an erythroleukemic acute phase of CML [8]. To our knowledge, the present case of CML is the first to reveal three isodicentric Ph chromosomes, and in this way, six copies of the rearranged sequence of chromosome #22. Recently, it has been demonstrated by recombinant DNA technology that the Ph translocation is reciprocal [9]. The h u m a n c-abl oncogene has been found consistently on chromosome #22, even in c o m p l e x Ph translocations [10]. Furthermore, the presence of a specific chromosomal breakpoint on chromosome #22 w i t h i n a limited region has been described [11]. It appears that the translocation of the c-abl oncogene in its transcriptionally active site on c h r o m o s o m e #22 is more important in the promotion, as well as in the progression, of the leukemic process than the rearrangement on chromosome #9. Two or more copies of the abnormal chromosome # 9 have been very rarely reported; however, the d u p l i c a t i o n of the Ph chromosome is the more c o m m o n additional chromosome aberration in the acute phase of CML.
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Isodicentric Ph Chromosomes in CML
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ge 121/21-1). We would like to thank Miss Anja Stiirmer for excellent technical assistance.
REFERENCES 1. Whang-Peng J, Knutsen TA, Lee EC (1973): Brief communication: Dicentric Ph I chromosome. J Natl Cancer Inst 51:2009-2012. 2. Prigogina EL, Fleischman EW, Volkova MA, Frenkel MA (1978): Chromosome abnormalities and clinical and morphologic manifestations of chronic myeloid leukemia. Hum Genet 41:143-156. 3. Mitelman F, Levan G, Nilsson PG, Brandt L (1976): Non-random karyotypic evolution in chronic myeloid leukemia. Int J Cancer 18:24-30. 4. Hagemeijer A, Stenfert Kroeze WF, Abels J (1980): Cytogenetic follow-up of patients with nonlymphocytic leukemia. I. Philadelphia chromosome positive chronic myeloid leukemia. Cancer Genet Cytogenet 2:317-326. 5. Darlington CD (1939): Misdivision and the genetics of the centromere. J Genet 37:341364. 6. Oshimura M, Kakati S, Sandberg AA (1977): Possible mechanisms for the genesis of common chromosome abnormalities, including isochromosomes and the Philadelphia chromosome. Cancer Res 37:3501-3507. 7. Stoll C, Oberling F (1979): Non-random clonal evolution in 45 cases of chronic myeloid leukemia. Leuk Res 3:61-66. 8. Hjorth M, Mark J, Tibblin E (1980): A hypotriploid stemline with 4 Ph 1 chromosomes in erythroleukemic blast crisis of a CML-patient with a long survival time. Hereditas 93:333336. 9. Groffen J, Heisterkamp N, Stephenson JR, Geurts van Kessel A, de Klein A, Grosveld G, Bootsma D (1983): c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia. J Exp Med 158:9-15. 10. de Klein A, Geurts van Kessel A, Grosveld G, Bartram CR, Hagemeijer A, Bootsma D, Spurr NK, Heisterkamp N, Groffen J, Stephenson JR (1982): A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300:765767. 11. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93-99.
ABSTRACT: This report describes a case of blast crisis in chronic myeloid leukemia with the occurrence of isodicentric Ph chromosomes. We briefly review observations on isoPh chromosomes in chronic myeloid leukemia. INTRODUCTION Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in the leukemic cells of about 90% of patients with this disease. The most c o m m o n additional chromosome aberration at the time of blast transformation is the appearance of a second Ph chromosome. A n isochromosome 17q and trisomies 8 and 19 are also often present. However, an isodicentric Ph chromosome is a very rare karyotypic change in the acute phase of CML. Whang-Peng et ah first reported five cases of CML with dicentric Ph chromosomes [1]. Prigogina et al. described a dicentric Ph chromosome detected at the time of acceleration in the blood and bone marrow, as well as in the spleen, of a patient suffering from CML [2]. Moreover, iso- and isodicentric Ph chromosomes have been observed in the chronic and the accelerated phases of three CML patients [3, 4]. In this article, we describe a case of CML with the appearance of one, two, or three isodicentric Ph chromosomes in the acute phase of the disease.
MATERIALS AND METHODS Case Report A 37-year-old male patient was referred for investigation of elevated white cell and platelet counts (discovered accidentally during investigation for a pyloric ulcer) to the Altona Hospital, Hamburg, in October 1978. No l y m p h nodes were palpable, and both the liver and spleen were normal in size. Hematologic investigation at the time of admission showed a white cell count of 160 × 109/L, a red cell count of 4.75 × 1012/L, platelets of 564 × 109/L, and hemoglobin of 14.4 g/dl. The white cell
From the Laboratoryof Cytogenetics(G.K.),the Institute of Pathology (A.G.),Medical School Hanover, Hanover, and the Departmentof Medicine,Altona Hospital, (K.M.)Hamburg,F.R.G. Address requests for reprints to Dr. G. Kovacs, Laboratory of Cytogenetics, Institute of Pathology, Medical School Hanover, P.O. Box 61 01 40, D-3000 Hanover 61, F.R.G. Received October 12, 1984; accepted February 28, 1985.
29 © 1986 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet20:29-33 (1986) 0165-4608/86/$03.50
30
o. Kovacs et al. differential count was 36% segmented neutrophils, 33% bands, 14% metamyelocytes, 11% myelocytes, 6% promyelocytes, 1% blasts, 5% lymphocytes, and 3% normoblasts. The neutrophil alkaline phosphatase score was negative. Bone marrow aspirate examination, as well as the marrow trephine biopsy, showed hypercellularity due to increased granulopoiesis and megakaryopoiesis. There was a moderate diffused increase in marrow fibrosis. The hematologic and trephine findings corresponded with a diagnosis of CML. Myleran therapy was initiated, and when the peripheral blood count returned to normal, maintenance chemotherapy was resumed, using intermittent courses of Myleran. The repeat marrow trephine biopsy findings in May 1980 corresponded to those observed in 1978. In August 1983, the patient was readmitted to the hospital because of bronchopneumonia with a greatly elevated white cell count of 311 x 109/L. Differential count showed 66% myeloblasts, 1% promyelocyte, 2% myelocytes, 8% metamyelocytes, 9% bands, 13% segmented neutrophils, and 1% eosinophils. Marrow trephine biopsy showed a hypercellular marrow with moderate diffuse fibrosis, confirming the acute blastic phase of CML. Small, discrete cervical, axillary, and inguinal lymph nodes were palpable, and both liver and spleen were enlarged 4 cm below the costal margin. A diagnosis was made of CML in blast crisis. Treatment with vincristine and prednisone was without effect, and the patient died 5 days after admission. Postmortem findings revealed changes in the sternum, femur, and vertebra that corresponded to an acute transformation of CML with complete overgrowth of the bone marrow by myeloblasts. Enlarged lymph nodes in axillary, cervical, and inguinal regions, splenomegaly (1055 g), hepatomegaly (3120 g), and enlarged kidneys (each 200 g) were found with leukemic infiltration. Both lungs showed confluent bronchopneumonia, with leukemic infiltration in the parenchyma and on the visceral pleura.
Cytogenetic Studies Chromosome analyses were carried out on bone marrow aspirates using the direct method and on peripheral blood cultures after 24 hr of incubation. Heparinized blood and bone marrow samples were centrifuged, and white blood cells were washed twice in a medium without fetal calf serum. The cells then were processed for direct preparation or cultured in RPMI 1640 medium supplemented with 15% fetal calf serum for 24 hr. Colchicine was added only during the hypotonic treatment at a final concentration of 0.1 p.g/ml. After fixing, air-dried preparations were made, and GTG- and GBG-bandings were performed. RESULTS AND DISCUSSION Eighty-one metaphases from unstimulated blood cultures were analyzed using GTGbanding; 38 metaphases from bone marrow were also analyzed using this method. The karyotyping of bone marrow, as well as of peripheral blood cells, revealed a simple clonal evolution following a linear pathway (Table 1). The first step was a classical translocation between chromosomes #9 and #22. In the second step, subsequent Ph chromosomes underwent abnormal replication, resulting in the first isodicentric Ph chromosome, which further replicated abnormally, resulting in the second and third isochromosomes 2 2 q - . The banding pattern of isodicentric Ph chromosomes was identical in all cells analyzed (Fig. 1A). When the metaphases were stained with CBG-technique, it was obvious that these isochromosomes were dicentric (Fig. 1B). The translocated segment on chromosome #9 was not further
31
Isodicentric Ph Chromosomes in CML
Table 1
Chromosome aberrations in bone marrow and peripheral blood cells analyzed with G-banding No. of cells
Stemline Sideline 1 Sideline 2 Sideline 3
Karyotypes
Bone marrow
Blood
47,XY,9q +, - 22, + 2idicPh 46,XY,t(9;22) 46,XY,9q+,-22,+idicPh 48,XY,9q+,-22,+3idicPh
22 3 5 8
43 9 11 18
increased, and no additional chromosome material was detected on any other chromosomes. Two isodicentric Ph chromosomes were found in 55% of metaphases; thus, the stemline karyotype was 47,XY,9q + , - 2 2 , + 2idicPh. Isodicentric Ph chromosomes present in this case are apparently identical to those found by Whang-Peng et al. [1]. These authors postulated that the isodicentric Ph chromosome represents two Ph chromosomes "joined at the satellite region of the short arms by either fusion or translocation." According to this hypothesis, the Ph c h r o m o s o m e must be first duplicated, triplicated, or futher replicated, and must then undergo centric fusion or translocation, resulting in iso- or isodicentric Ph chromosomes. A Ph c h r o m o s o m e with isodicentric Ph chromosome(s) appearing in the same cell must also occur. This p a t h w a y of the genesis of isochromosomes does not seem to be possible w h e n v i e w e d against the clonal evolution in our case: there was no evidence for the appearance of both a Ph c h r o m o s o m e and an isodicentric Ph chromosome in the same cells. Instead, it is possible that an isoPh chromosome arises through " m i s d i v i s i o n " of the centromere (the centromere divides crosswise) or through homologous adjacent symmetrical exchange at the centromeric region of a Ph c h r o m o s o m e [5, 6]. The second and third isochromosomes could be p r o d u c e d through n o n d i s j u n c t i o n or selective endoreduplication. In the stemline karyotype of the case presented here, there were four copies of the rearranged region of chromosome #22. However, in 22% of the cells analyzed, the karyotype revealed six copies of this region. This extreme increase in the amount of the Ph chromosome occurs very rarely in CML; only a few cases have been reported with more than two Ph chromosomes. In one of five cases described by Whang-Peng et al., two dicentric Ph chromosomes occurred in 12% of the cells [1]. Furthermore, Stoll and Oberling [7] reported a case with three Ph chromosomes, and a h y p o t r i p l o i d stemline with four Ph chromosomes was described in an erythroleukemic acute phase of CML [8]. To our knowledge, the present case of CML is the first to reveal three isodicentric Ph chromosomes, and in this way, six copies of the rearranged sequence of chromosome #22. Recently, it has been demonstrated by recombinant DNA technology that the Ph translocation is reciprocal [9]. The h u m a n c-abl oncogene has been found consistently on chromosome #22, even in c o m p l e x Ph translocations [10]. Furthermore, the presence of a specific chromosomal breakpoint on chromosome #22 w i t h i n a limited region has been described [11]. It appears that the translocation of the c-abl oncogene in its transcriptionally active site on c h r o m o s o m e #22 is more important in the promotion, as well as in the progression, of the leukemic process than the rearrangement on chromosome #9. Two or more copies of the abnormal chromosome # 9 have been very rarely reported; however, the d u p l i c a t i o n of the Ph chromosome is the more c o m m o n additional chromosome aberration in the acute phase of CML.
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Isodicentric Ph Chromosomes in CML
33
This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ge 121/21-1). We would like to thank Miss Anja Stiirmer for excellent technical assistance.
REFERENCES 1. Whang-Peng J, Knutsen TA, Lee EC (1973): Brief communication: Dicentric Ph I chromosome. J Natl Cancer Inst 51:2009-2012. 2. Prigogina EL, Fleischman EW, Volkova MA, Frenkel MA (1978): Chromosome abnormalities and clinical and morphologic manifestations of chronic myeloid leukemia. Hum Genet 41:143-156. 3. Mitelman F, Levan G, Nilsson PG, Brandt L (1976): Non-random karyotypic evolution in chronic myeloid leukemia. Int J Cancer 18:24-30. 4. Hagemeijer A, Stenfert Kroeze WF, Abels J (1980): Cytogenetic follow-up of patients with nonlymphocytic leukemia. I. Philadelphia chromosome positive chronic myeloid leukemia. Cancer Genet Cytogenet 2:317-326. 5. Darlington CD (1939): Misdivision and the genetics of the centromere. J Genet 37:341364. 6. Oshimura M, Kakati S, Sandberg AA (1977): Possible mechanisms for the genesis of common chromosome abnormalities, including isochromosomes and the Philadelphia chromosome. Cancer Res 37:3501-3507. 7. Stoll C, Oberling F (1979): Non-random clonal evolution in 45 cases of chronic myeloid leukemia. Leuk Res 3:61-66. 8. Hjorth M, Mark J, Tibblin E (1980): A hypotriploid stemline with 4 Ph 1 chromosomes in erythroleukemic blast crisis of a CML-patient with a long survival time. Hereditas 93:333336. 9. Groffen J, Heisterkamp N, Stephenson JR, Geurts van Kessel A, de Klein A, Grosveld G, Bootsma D (1983): c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia. J Exp Med 158:9-15. 10. de Klein A, Geurts van Kessel A, Grosveld G, Bartram CR, Hagemeijer A, Bootsma D, Spurr NK, Heisterkamp N, Groffen J, Stephenson JR (1982): A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300:765767. 11. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93-99.