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Near-triploid Ph-positive leukemia
Near-Triploid Ph-Positive Leukemia Kazuo Ohtaki, Rokuo Abe, Cameron K. Tebbi, Rafael de los Santos, Tin Han, and Avery A. Sandberg
ABSTRACT: Tetraploid populations have been observed in various types of leukemia, but relatively few reports exist of triploid cell populations in acute or chronic leukemia. We report two cases of Ph-positive leukemia with a modal triploid cell population. Examination of peripheral blood from a 3-year-old boy with Ph-positive acute lymphoblastic leukemia (ALL) and a 68-year-old male with Ph-positive chronic myelocytic leukemia (CIVIL) in blastic crisis revealed modal populations of 72 and 63 chromosomes, respectively. G-banding analysis of both cases revealed the following: karyotypic instability (no clonality), dominant trisomy, and the random association of the Ph chromosome with gains and losses of chromosomes involved in this translocation. The cytogenetic evidence obtained suggests that the triploid cell populations were not derived from a duplication of a hypodiploid cell population, but resuited from random loss of chromosomes from tetraplaid cell populations derived from duplication of pseudodiploid cells. INTRODUCTION Cases of l e u k e m i a w i t h h a p l o i d or near-haploid and tetraploid or near-tetraploid c h r o m o s o m e numbers, though relatively rare, have been reported [1]. Near-haploid ceils have been observed only in acute l y m p h o b l a s t i c l e u k e m i a {ALL} or in the blastic phase of chronic myelocytic leukemia (CML). Tetraploid cell p o p u l a t i o n s have been reported in various leukemias, but few reports exist of triploid cell populations in such diseases. We report two cases of Ph-positive leukemia, one of CML and another of ALL, with triploid cell populations. CASE REPORT Patient P. A.
This 31/2-year-old black boy was first seen in January 1982 because of easy bruising, petechiae, purpura, and vomiting. The patient's h e m o g l o b i n at diagnosis was 5.3 g/dl, WBC 164,000/mm 3, and platelets 27,O00/mm 3. More than 90% of the leukocytes in the blood smear were lymphoblasts. A bone marrow aspiration p r o v e d the patient to have ALL w i t h L1 morphology, according to the FAB classification [2]. The
From Roswell Park Memorial Institute, Buffalo,NY. Address requests for reprints to Dr. Avery A. Sandberg, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263. Received May 2, 1984; accepted November 28, 1984.
113 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderhilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 18, 113-121 (1985) 0165-4608/85/$03.30
114
K. Ohtaki et al. surface marker determination disclosed the blasts to be of T cell origin. Cytogenetic analysis was performed on peripheral blood cells on January 26, 1982. He was treated with prednisone, L-asparaginase, and vincristine (VCR), followed by methotrexate (MTX) and high doses of cyclophosphamide (CTX). He also received prophylactic central nervous system (CNS) therapy with injections of MTX, cytosine arabinoside (Ara-C), and hydrocortisone. After 6 weeks of the above therapy, the patient had failed to achieve a remission and was then placed on a regimen consisting of high doses of CTX and MTX along with doses of VCR and prednisone. After a brief remission, the patient relapsed in April 1982. He was then treated with various regimens, i.e., Ara-C and VM-26, POMP (prednisone, VCR, MTX, and 6mercaptopurine), and adriamycin and methylglyoxal Bis (guanylhydrazone) (MGBG), but remission could not be induced. He eventually had a short-lived remission in December 1982 with Mitoxantrone; however, he died in April 1983.
Patient C. J. This 68-year-old white male was admitted to the hospital on April 19, 1977 and was diagnosed as having CML. At that time, he presented with a hemoglobin of 10.7 g/dl, WBC 125,000/mm 3, with 4% myeloblasts, 7% promyelocytes, 24% myelocytes, and 62% grannlocytes, and a platelet count of 515,000/mm 3. He had no lymphadenopathy; his spleen was 10 cm below the lower costal margin. He was treated with Ara-C and CCNU, with a good initial response. In January 1978, he was placed on oral myleran. In January 1979, he was switched to 6-thioguanine and myleran until September 1982, when he was given hydroxyurea due to an increasing WBC and splenomegaly. Serum alkaline phosphatase and lactate dehydrogenase (LDH) levels increased from 1981. In December 1982, he was admitted to the hospital with back pain, constipation, and gait disturbance. A myelogram showed complete block at T9-T10, and he was treated with radiation therapy (3000 R) to the involved area. His WBC was 50,900/ mm 3, with 6% myeloblasts, 1% promyelocytes, and 3% myelocytes; his hemoglobin was 9.5 g/dl and platelets 210,000/mm 3. Spinal tap revealed pleocytosis with increased blasts, which is consistent with CML involvement. For this he received weekly intrathecal Ara-C with reduction of the WBC count in the spinal fluid from 358 to 8, with no blasts seen. In spite of this treatment, his condition continued to deteriorate, his peripheral blood blast count increased to 20%, and he became progressively thrombocytopenic. He was treated with VCR and prednisone without response, then with higher doses of hydroxyurea, but his WBC continued to rise. He was treated empirically with brain radiation, without improvement. Cytogenetic analysis was performed on peripheral blood cells on January 20, 1983. The patient died on January 26, 1983. No autopsy was performed. MATERIALS AND METHODS Cytogenetic studies were performed on peripheral blood cells in both cases. Peripheral blood specimens were cultured in RPMI 1640 medium with 16.7% fetal bovine serum and penicillin-streptomycin for 24 hr without PHA and for 72 hr with PHA. Specimens were treated with 0.01 ~g/ml of colcemid for 2 hr and then placed in 0.075 M KC1 for 15 min. After several changes of methanol-acetic acid fixative, slides were prepared by flame-drying and trypsin G-banded. Karyotypic analyses were performed, and the chromosome identified according to ISCN (1978) [3].
Near-Triploid Ph-Positive Leukemia
115
RESULTS The peripheral blood cells cultured for 24 and 72 hr contained similar cell populations in both cases; hence, the karyotypic results of the 24-hr and 72-hr cultures were combined. The cells of patient P. A. contained 63-75 chromosomes [modal chromosome number, 71); 62 cells were analyzed. In patient C. J., the cells contained 50-123 chromosomes (modal chromosome number, 63); 76 cells were also analyzed with conventional staining (Table 1). Only 9 cells with 69-71 chromosomes of patient P. A. were karyotyped by G-banding because of the relatively poor spreading of the chromosomes. In patient C. J., 50 of the 78 cells that contained 57-66 chromosomes were analyzed by G-banding. Representative chromosome complements with neartriploid chromosome numbers in both cases are shown in Table 2. Figures 1 and 2 show representative karyotypes. In both cases, karyotype analysis revealed instability, i.e., absence of a clone with the same chromosome complement and aberrations. However, both cases had a Ph-chromosome in the cells due to t(9;22), and in patient C. J., other aberrations, such as 3 q - and 11p + were observed in the cells. Sixteen chromosomes were trisomic in the nine analyzed cells of patient P. A., with the remaining chromosomes being disomic or tetrasomic. In the 50 analyzed cells of patient C. J., each chromosome showed a wide variation from monosomy to hexasomy [Table 3). Both cases had Ph-positive leukemia with t(9;22). Table 4 shows the various combinations of chromosomes #9 and #22 in 50 cells of patient C. J. One or two Ph chromosomes were detected in 16 cells. Thirty-one cells had three Ph chromosomes. As shown in Table 4, the combination of Ph chromosomes and chromosomes #9 and #22 showed a random association with gains and losses of chromosomes involved in this translocation. Nine cells analyzed in patient P. A. revealed results similar to those of patient C. J. DISCUSSION Few reports exist of triploid cell populations in acute or chronic leukemia [4-6]. Several mechanisms for developing triploid cell populations have been hypothesized: (a) duplication of near-haploid cells [4], (b) loss of some chromosomes from tetraploid cells [5], and (c) triploid cells developing as a counterpart of haploid cells after the tetraploid ceils had undergone multipolar mitoses [7]. According to the first possibility, disomy or tetrasomy of the chromosomes should be seen, with trisomy being very rare unless due to loss of chromosomes after duplication. Triploid cells may have two copies of translocations, e.g., between a chromosome #9 and a chromosome #22, when the hypodiploid ceils had one translocation. Pederson et al. [4] reported two cytogenetically distinct populations of marrow ceils in CML: one population with 35-36 chromosomes, the other with 66-72 chromosomes. In all pairs, except for chromosomes #10, #12, #14, and #20, the triploid population contained approximately twice the number of homologs present in the hypodiploid population. The cells with triploid chromosome numbers appeared to develop from an extremely hypodiploid cell population through duplication of the chromosome complement. In cases with haploid cell populations, a cell population with twice the chromosome number due to duplication of the haploid set was also simultaneously observed [8-10]. According to the second possibility, tetraploid cells first develop from duplica-
2
2
57
2
58
4
59
2
60
in two
5
61
cases
12
62
with
1 23
63 3 10
64
9
65 3 1
66
1
67 6
68 10
69
numbers
of chromosomes
chromosome
Number
near-triploid
13
70 22
71 2
72
1 2 3 4 5
°M, marker chromosome.
c.l.
P.A.
Patient
Metaphase examined
3 3 3 3 3 3
3 3 3 3 3
1
3 3 3 3 3 3
3 3 3 3 3
2
3 3 3 4 2 2
2 3 3 4 3
3
2 3 3 3 3 3
3 3 3 3 3
4
3 1 1 2 2 2
4 4 3 4 3
5
4 3 3 4 4 3
3 3 4 3 3
6
2 2 2 2 3 2
3 3 3 2 3
7
3 3 3 3 2 4
3 3 2 3 3
8
2 3 3 2 3 3
3 3 4 4 3
9
3 3 3 3 3 4
4 3 3 3 4
10
4 4 4 3 6 4
2 3 2 4 3
11
2 2 2 3 1 2
3 3 3 3 3
12
3 4 4 3 4 3
3 3 3 3 3
13
2 2 1 2 2 2
3 3 3 3 3
14
Chromosome
3 3 3 3 2 2
3 3 3 3 3
15
number
2 1 1 3 2 3
3 2 2 3 2
16
2 2 2 1 1 3
3 2 3 2 3
17
2 1 2 2 2 3
3 3 3 3 3
18
3 4 4 3 3 2
2 3 2 2 3
19
2 2 2 2 3 2
4 3 3 2 3
20
2 4 4 4 3 2
3 3 6 3 4
21
Table 2 Chromosome complements representative of metaphases with near-triploid chromosome number
1
54
of cells observed
°Number of counted cells.
1
P.A. C.J.
53
Number
50
1
Patient
Table
1
73
5 5 5 4 4 4
3 5 4 4 3
22
1
1 1 1 1 1 0
2 2 2 1 2
X
75
1 1 1 0 1 2
0 1 0 2 2
Y
1
89
1 0 0 0 0 0
1 0 0 0 0
Ma
1
122
62 a 78 a
63 63 63 63 63 63
69 70 70 70 71
Total chromosome number
1
123
117
Near-Triploid Ph-Positive Leukemia
I1¢t111
2
3
4
///
5
-|tl 1 | f - I i t - t ¢ ¢ I i if-11 l - t ll6
7
8
9
tl-l
-ni--jli-l-ll 13
14
10
15
16
//
11
12
l-il--tt-m17
18
-1t1.-19
20
21
22
XY
Figure 1 Representative karyotype in patient P. A.: 71,XXYY,+1,+2,+3,+4,+5,+6,+7, + 8,+inv(9)(pllq13),+ 10,+ 10,+ 11,+ 12,+ 13,+ 14,+ 15,+ 17,+ 18,+ 19,+ 20,+ 21,+ 21,+ 22, + X,+Y,t(9;22}(q34;q11},t(9;22}(q34;q11), showing a mixture of di-, tri-, tetrasomies and two copies of Ph. An inverted #9 was observed in every cell. Arrows indicate structural abnormalities.
tion of d i p l o i d cells, and triploid cells occur after loss of some chromosomes from tetraploid cells. In a case of erythroleukemia, Douglass et al. [5] found a m o d a l c h r o m o s o m e number of 72 (68-92 chromosomes}, w i t h tetraploidy of some c h r o m o s o m e s and d u p l i c a t i o n of markers. Weh et al. [6] reported one case of AML to be h y p o t e t r a p l o i d (70-80 chromosomes} by a direct preparation method. In the third possibility, t r i p l o i d cells occur as the counterpart to h a p l o i d cells w h e n the tetraploid cells undergo m u l t i p o l a r cell division. Oksala et al. [7] compared 10 h u m a n fetal fallopian tubes w i t h 23 cases of cancer of the same organ. In the n o n m a l i g n a n t tissues, the metaphase/prophase ratio varied b e t w e e n 0.57 a n d 1.07, and in the malignant neoplasias, between 1.7 and 36.6. Multipolar divisions occurred in more than half of cancer cases, whereas none was found in the fetal tissues. Three cell lines were identified in a bone marrow from a patient w i t h chronic granulocytic l e u k e m i a [11]. The major cell line {60%) was h y p e r d i p l o i d , w h i c h was shown to be 5 0 , X X , + 8 , + 1 1 , + 1 3 , + 2 2 , t(9;22},t(9;22}. The other line {20%} was tetraploid [92,XXXX,t(9;22),t(9;22)]. The r e m a i n i n g 20% cells were d i p l o i d w i t h a Ph [46,XX,t(9;22}]. The authors suggested that the a d d i t i o n a l c h r o m o s o m e s m a y have been p r o d u c e d either b y selective c h r o m o s o m e loss from a tetraploid cell line or through m u l t i p o l a r mitoses.
0 1 2 3 4 5 6
"M, marker chromosome.
c.j.
P.A.
Patient
N u m b e r of co pie s
8 86 6
89 11
1
4 22 72 2
22 67 11
2
6 34 54 6
44 44 11
3
2 38 56 4
22 56 22
4
14 60 26
22 78
5
6 44 42 8
67 33
6
2 74 20 4
11 56 33
7
14 34 36 16
22 56 22
8
4 44 48 4
22 44 33
9
4 36 54 6
56 44
10
2
4 10 84
44 33 22
11
4 86 8 2
22 78
12
2 32 64 2
89 11
13
14 72 14
11 78 11
14
4 20 66 10
11 78 11
15
Chromosome number
8 82 10
67 33
16
2 14 76 8
33 67
17
10 68 20 2
11 78 11
18
26 28 46
11 56 33
19
2 2 68 26 2
11 56 33
20
22 34 44
11
11 44 33
21
4 42 44 10
22 22 56
22
12 82 6
33 67
X
14 78 8
33 33 33
Y
72 24 4
89 11
M°
Table 3 Percentage of metaphases with multiple copies of certain chromosomes in patient P.A. (9 cells examined) and patient C.J. (50 cells examined)
~0
°N, normal chromosome.
q+ N, N N, q + q+, q+ N,N,q+ N,q+ ,q+ N,N,q + ,q + Total
1
1
P h + 3N a
2
2
2Ph + N
Association of the Ph chromosome
Chromosome #9
Table 4
2 1 1 10
1 2 3
2Ph + 2N
1 3
1
1
2Ph + 3N
and #9 chromosome
10
2 4 2
2
3Ph + N
Chromosome #22
17
1 5 1 6 4
3 P h + 2N
i n 5 0 c e l l s i n p a t i e n t C.J.
4
1 1
2
3 P h + 3N
2
1 1
4Ph + N
1
1
4 P h + 2N
2 8 11 3 15 9 2 50
Total
c.O
12 0
K. Ohtaki et al.
Figure 2 Representative karyotype in patient C.J.: 6 3 , X Y , + 2 , + 3 q - , + 4 , + 6 , + 8 , + 1 0 , + 11p +, + 11p +, + 13, + 13, + 15, + 19, + 19, + 21, + 22q - , + 22q - , + 22q - ,t (9;22)(q34;q11),del (3)(qllq26), two copies of 11p + (unknown origin), and showing a mixture of di-, tri-, tetra-, and pentasomies. This case had one or more Ph chromosomes and other structural changes; 3 q - in 23 cells and two copies of 11p+ were detected in 49 of 50 cells. Arrows indicate structural abnormalities.
G-banding analysis of the two cases reported here revealed the following: (a) karyotypic instability (no clonality) with modal n u m b e r s of 71 (63-75) and 63 (50123); (b) high frequency of trisomy in autosomes (16/22 or 9/22), with each cell having a wide variation from m o n o s o m y to tetrasomy in each chromosome set; and (c) r a n d o m association of the Ph chromosomes with # 9 chromosomes, with gains and losses of chromosome involved in this translocation. The near-triploid populations seen by us were not produced through duplication of a haploid cell population, but seem to be due to r a n d o m loss of chromosomes from tetraploid cell populations derived from duplication of p s e u d o d i p l o i d cells.
REFERENCES 1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier NorthHolland, New York. 2. Bennet JM, Catovsky D, Daniel M-T, Flandrin G, Galton DAG, Gralnick HR, Sultan C (FAB Co-operative Group) (1976): Proposals for the classification of the acute leukaemias. Br J Haematol 33:451-458.
Near-Triploid Ph-Positive Leukemia
121
3. ISCN (1978): An International System for Human Cytogenetic Nomenclature (1978). Cytogenet Cell Genet 21:309-404. 4. Pederson B, Boesen AM (1983): Extreme hypodiploidy in a case of myelomonocytic crisis of chronic myelogenous leukemia. Cancer Genet Cytogenet 9:101-112. 5. Douglass EC, Freeman DL (1983): Hypotetraploidy in erythroleukemia. Cancer Ganet Cytogenet 8:231-234. 6. Weh HJ, von Paleske A, Hossfeld DK (1983): Disappearance of hypotetraploid clones after short-term culture of leukemic cells. A case report. Cancer Genet Cytogenet 10:237-240. 7. Oksala T, Therman E (1974): Mitotic abnormalities and cancer. In: Chromosomes and Cancer, J German, ed. John Wiley, New York, pp 239-263. 8. Oshimura M, Freeman AI, Sandberg AA (1977): Chromosomes and causation of human cancer and leukemia. XXIII. Near-haploidy in acute leukemia. Cancer 40:1143-1148. 9. Shabtai F, Lewinski UH, Har-Zahav L, Gaffer U, Halbrecht I, Djaldetti M (1979): A hypo~ diploid clone and its duplicate in acute lymphoblastic leukemia. Am J Clin Pathol 72:1018-1024. 10. Kaneko Y, Sakurai M (1980): Acute lymphocytic leukemia (ALL) with near-haploidy--A unique subgroup of ALL? Cancer Genet Cytogenet 2:13-18. 11. Rajasekariah P, Illes I, Garson OM (1982): Double 9;22 translocation with hyperdiploidy appearing in blastic transformation of chronic granulocytic leukemia. Cancer Genet Cytogenet 7:85-88.
ABSTRACT: Tetraploid populations have been observed in various types of leukemia, but relatively few reports exist of triploid cell populations in acute or chronic leukemia. We report two cases of Ph-positive leukemia with a modal triploid cell population. Examination of peripheral blood from a 3-year-old boy with Ph-positive acute lymphoblastic leukemia (ALL) and a 68-year-old male with Ph-positive chronic myelocytic leukemia (CIVIL) in blastic crisis revealed modal populations of 72 and 63 chromosomes, respectively. G-banding analysis of both cases revealed the following: karyotypic instability (no clonality), dominant trisomy, and the random association of the Ph chromosome with gains and losses of chromosomes involved in this translocation. The cytogenetic evidence obtained suggests that the triploid cell populations were not derived from a duplication of a hypodiploid cell population, but resuited from random loss of chromosomes from tetraplaid cell populations derived from duplication of pseudodiploid cells. INTRODUCTION Cases of l e u k e m i a w i t h h a p l o i d or near-haploid and tetraploid or near-tetraploid c h r o m o s o m e numbers, though relatively rare, have been reported [1]. Near-haploid ceils have been observed only in acute l y m p h o b l a s t i c l e u k e m i a {ALL} or in the blastic phase of chronic myelocytic leukemia (CML). Tetraploid cell p o p u l a t i o n s have been reported in various leukemias, but few reports exist of triploid cell populations in such diseases. We report two cases of Ph-positive leukemia, one of CML and another of ALL, with triploid cell populations. CASE REPORT Patient P. A.
This 31/2-year-old black boy was first seen in January 1982 because of easy bruising, petechiae, purpura, and vomiting. The patient's h e m o g l o b i n at diagnosis was 5.3 g/dl, WBC 164,000/mm 3, and platelets 27,O00/mm 3. More than 90% of the leukocytes in the blood smear were lymphoblasts. A bone marrow aspiration p r o v e d the patient to have ALL w i t h L1 morphology, according to the FAB classification [2]. The
From Roswell Park Memorial Institute, Buffalo,NY. Address requests for reprints to Dr. Avery A. Sandberg, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263. Received May 2, 1984; accepted November 28, 1984.
113 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderhilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 18, 113-121 (1985) 0165-4608/85/$03.30
114
K. Ohtaki et al. surface marker determination disclosed the blasts to be of T cell origin. Cytogenetic analysis was performed on peripheral blood cells on January 26, 1982. He was treated with prednisone, L-asparaginase, and vincristine (VCR), followed by methotrexate (MTX) and high doses of cyclophosphamide (CTX). He also received prophylactic central nervous system (CNS) therapy with injections of MTX, cytosine arabinoside (Ara-C), and hydrocortisone. After 6 weeks of the above therapy, the patient had failed to achieve a remission and was then placed on a regimen consisting of high doses of CTX and MTX along with doses of VCR and prednisone. After a brief remission, the patient relapsed in April 1982. He was then treated with various regimens, i.e., Ara-C and VM-26, POMP (prednisone, VCR, MTX, and 6mercaptopurine), and adriamycin and methylglyoxal Bis (guanylhydrazone) (MGBG), but remission could not be induced. He eventually had a short-lived remission in December 1982 with Mitoxantrone; however, he died in April 1983.
Patient C. J. This 68-year-old white male was admitted to the hospital on April 19, 1977 and was diagnosed as having CML. At that time, he presented with a hemoglobin of 10.7 g/dl, WBC 125,000/mm 3, with 4% myeloblasts, 7% promyelocytes, 24% myelocytes, and 62% grannlocytes, and a platelet count of 515,000/mm 3. He had no lymphadenopathy; his spleen was 10 cm below the lower costal margin. He was treated with Ara-C and CCNU, with a good initial response. In January 1978, he was placed on oral myleran. In January 1979, he was switched to 6-thioguanine and myleran until September 1982, when he was given hydroxyurea due to an increasing WBC and splenomegaly. Serum alkaline phosphatase and lactate dehydrogenase (LDH) levels increased from 1981. In December 1982, he was admitted to the hospital with back pain, constipation, and gait disturbance. A myelogram showed complete block at T9-T10, and he was treated with radiation therapy (3000 R) to the involved area. His WBC was 50,900/ mm 3, with 6% myeloblasts, 1% promyelocytes, and 3% myelocytes; his hemoglobin was 9.5 g/dl and platelets 210,000/mm 3. Spinal tap revealed pleocytosis with increased blasts, which is consistent with CML involvement. For this he received weekly intrathecal Ara-C with reduction of the WBC count in the spinal fluid from 358 to 8, with no blasts seen. In spite of this treatment, his condition continued to deteriorate, his peripheral blood blast count increased to 20%, and he became progressively thrombocytopenic. He was treated with VCR and prednisone without response, then with higher doses of hydroxyurea, but his WBC continued to rise. He was treated empirically with brain radiation, without improvement. Cytogenetic analysis was performed on peripheral blood cells on January 20, 1983. The patient died on January 26, 1983. No autopsy was performed. MATERIALS AND METHODS Cytogenetic studies were performed on peripheral blood cells in both cases. Peripheral blood specimens were cultured in RPMI 1640 medium with 16.7% fetal bovine serum and penicillin-streptomycin for 24 hr without PHA and for 72 hr with PHA. Specimens were treated with 0.01 ~g/ml of colcemid for 2 hr and then placed in 0.075 M KC1 for 15 min. After several changes of methanol-acetic acid fixative, slides were prepared by flame-drying and trypsin G-banded. Karyotypic analyses were performed, and the chromosome identified according to ISCN (1978) [3].
Near-Triploid Ph-Positive Leukemia
115
RESULTS The peripheral blood cells cultured for 24 and 72 hr contained similar cell populations in both cases; hence, the karyotypic results of the 24-hr and 72-hr cultures were combined. The cells of patient P. A. contained 63-75 chromosomes [modal chromosome number, 71); 62 cells were analyzed. In patient C. J., the cells contained 50-123 chromosomes (modal chromosome number, 63); 76 cells were also analyzed with conventional staining (Table 1). Only 9 cells with 69-71 chromosomes of patient P. A. were karyotyped by G-banding because of the relatively poor spreading of the chromosomes. In patient C. J., 50 of the 78 cells that contained 57-66 chromosomes were analyzed by G-banding. Representative chromosome complements with neartriploid chromosome numbers in both cases are shown in Table 2. Figures 1 and 2 show representative karyotypes. In both cases, karyotype analysis revealed instability, i.e., absence of a clone with the same chromosome complement and aberrations. However, both cases had a Ph-chromosome in the cells due to t(9;22), and in patient C. J., other aberrations, such as 3 q - and 11p + were observed in the cells. Sixteen chromosomes were trisomic in the nine analyzed cells of patient P. A., with the remaining chromosomes being disomic or tetrasomic. In the 50 analyzed cells of patient C. J., each chromosome showed a wide variation from monosomy to hexasomy [Table 3). Both cases had Ph-positive leukemia with t(9;22). Table 4 shows the various combinations of chromosomes #9 and #22 in 50 cells of patient C. J. One or two Ph chromosomes were detected in 16 cells. Thirty-one cells had three Ph chromosomes. As shown in Table 4, the combination of Ph chromosomes and chromosomes #9 and #22 showed a random association with gains and losses of chromosomes involved in this translocation. Nine cells analyzed in patient P. A. revealed results similar to those of patient C. J. DISCUSSION Few reports exist of triploid cell populations in acute or chronic leukemia [4-6]. Several mechanisms for developing triploid cell populations have been hypothesized: (a) duplication of near-haploid cells [4], (b) loss of some chromosomes from tetraploid cells [5], and (c) triploid cells developing as a counterpart of haploid cells after the tetraploid ceils had undergone multipolar mitoses [7]. According to the first possibility, disomy or tetrasomy of the chromosomes should be seen, with trisomy being very rare unless due to loss of chromosomes after duplication. Triploid cells may have two copies of translocations, e.g., between a chromosome #9 and a chromosome #22, when the hypodiploid ceils had one translocation. Pederson et al. [4] reported two cytogenetically distinct populations of marrow ceils in CML: one population with 35-36 chromosomes, the other with 66-72 chromosomes. In all pairs, except for chromosomes #10, #12, #14, and #20, the triploid population contained approximately twice the number of homologs present in the hypodiploid population. The cells with triploid chromosome numbers appeared to develop from an extremely hypodiploid cell population through duplication of the chromosome complement. In cases with haploid cell populations, a cell population with twice the chromosome number due to duplication of the haploid set was also simultaneously observed [8-10]. According to the second possibility, tetraploid cells first develop from duplica-
2
2
57
2
58
4
59
2
60
in two
5
61
cases
12
62
with
1 23
63 3 10
64
9
65 3 1
66
1
67 6
68 10
69
numbers
of chromosomes
chromosome
Number
near-triploid
13
70 22
71 2
72
1 2 3 4 5
°M, marker chromosome.
c.l.
P.A.
Patient
Metaphase examined
3 3 3 3 3 3
3 3 3 3 3
1
3 3 3 3 3 3
3 3 3 3 3
2
3 3 3 4 2 2
2 3 3 4 3
3
2 3 3 3 3 3
3 3 3 3 3
4
3 1 1 2 2 2
4 4 3 4 3
5
4 3 3 4 4 3
3 3 4 3 3
6
2 2 2 2 3 2
3 3 3 2 3
7
3 3 3 3 2 4
3 3 2 3 3
8
2 3 3 2 3 3
3 3 4 4 3
9
3 3 3 3 3 4
4 3 3 3 4
10
4 4 4 3 6 4
2 3 2 4 3
11
2 2 2 3 1 2
3 3 3 3 3
12
3 4 4 3 4 3
3 3 3 3 3
13
2 2 1 2 2 2
3 3 3 3 3
14
Chromosome
3 3 3 3 2 2
3 3 3 3 3
15
number
2 1 1 3 2 3
3 2 2 3 2
16
2 2 2 1 1 3
3 2 3 2 3
17
2 1 2 2 2 3
3 3 3 3 3
18
3 4 4 3 3 2
2 3 2 2 3
19
2 2 2 2 3 2
4 3 3 2 3
20
2 4 4 4 3 2
3 3 6 3 4
21
Table 2 Chromosome complements representative of metaphases with near-triploid chromosome number
1
54
of cells observed
°Number of counted cells.
1
P.A. C.J.
53
Number
50
1
Patient
Table
1
73
5 5 5 4 4 4
3 5 4 4 3
22
1
1 1 1 1 1 0
2 2 2 1 2
X
75
1 1 1 0 1 2
0 1 0 2 2
Y
1
89
1 0 0 0 0 0
1 0 0 0 0
Ma
1
122
62 a 78 a
63 63 63 63 63 63
69 70 70 70 71
Total chromosome number
1
123
117
Near-Triploid Ph-Positive Leukemia
I1¢t111
2
3
4
///
5
-|tl 1 | f - I i t - t ¢ ¢ I i if-11 l - t ll6
7
8
9
tl-l
-ni--jli-l-ll 13
14
10
15
16
//
11
12
l-il--tt-m17
18
-1t1.-19
20
21
22
XY
Figure 1 Representative karyotype in patient P. A.: 71,XXYY,+1,+2,+3,+4,+5,+6,+7, + 8,+inv(9)(pllq13),+ 10,+ 10,+ 11,+ 12,+ 13,+ 14,+ 15,+ 17,+ 18,+ 19,+ 20,+ 21,+ 21,+ 22, + X,+Y,t(9;22}(q34;q11},t(9;22}(q34;q11), showing a mixture of di-, tri-, tetrasomies and two copies of Ph. An inverted #9 was observed in every cell. Arrows indicate structural abnormalities.
tion of d i p l o i d cells, and triploid cells occur after loss of some chromosomes from tetraploid cells. In a case of erythroleukemia, Douglass et al. [5] found a m o d a l c h r o m o s o m e number of 72 (68-92 chromosomes}, w i t h tetraploidy of some c h r o m o s o m e s and d u p l i c a t i o n of markers. Weh et al. [6] reported one case of AML to be h y p o t e t r a p l o i d (70-80 chromosomes} by a direct preparation method. In the third possibility, t r i p l o i d cells occur as the counterpart to h a p l o i d cells w h e n the tetraploid cells undergo m u l t i p o l a r cell division. Oksala et al. [7] compared 10 h u m a n fetal fallopian tubes w i t h 23 cases of cancer of the same organ. In the n o n m a l i g n a n t tissues, the metaphase/prophase ratio varied b e t w e e n 0.57 a n d 1.07, and in the malignant neoplasias, between 1.7 and 36.6. Multipolar divisions occurred in more than half of cancer cases, whereas none was found in the fetal tissues. Three cell lines were identified in a bone marrow from a patient w i t h chronic granulocytic l e u k e m i a [11]. The major cell line {60%) was h y p e r d i p l o i d , w h i c h was shown to be 5 0 , X X , + 8 , + 1 1 , + 1 3 , + 2 2 , t(9;22},t(9;22}. The other line {20%} was tetraploid [92,XXXX,t(9;22),t(9;22)]. The r e m a i n i n g 20% cells were d i p l o i d w i t h a Ph [46,XX,t(9;22}]. The authors suggested that the a d d i t i o n a l c h r o m o s o m e s m a y have been p r o d u c e d either b y selective c h r o m o s o m e loss from a tetraploid cell line or through m u l t i p o l a r mitoses.
0 1 2 3 4 5 6
"M, marker chromosome.
c.j.
P.A.
Patient
N u m b e r of co pie s
8 86 6
89 11
1
4 22 72 2
22 67 11
2
6 34 54 6
44 44 11
3
2 38 56 4
22 56 22
4
14 60 26
22 78
5
6 44 42 8
67 33
6
2 74 20 4
11 56 33
7
14 34 36 16
22 56 22
8
4 44 48 4
22 44 33
9
4 36 54 6
56 44
10
2
4 10 84
44 33 22
11
4 86 8 2
22 78
12
2 32 64 2
89 11
13
14 72 14
11 78 11
14
4 20 66 10
11 78 11
15
Chromosome number
8 82 10
67 33
16
2 14 76 8
33 67
17
10 68 20 2
11 78 11
18
26 28 46
11 56 33
19
2 2 68 26 2
11 56 33
20
22 34 44
11
11 44 33
21
4 42 44 10
22 22 56
22
12 82 6
33 67
X
14 78 8
33 33 33
Y
72 24 4
89 11
M°
Table 3 Percentage of metaphases with multiple copies of certain chromosomes in patient P.A. (9 cells examined) and patient C.J. (50 cells examined)
~0
°N, normal chromosome.
q+ N, N N, q + q+, q+ N,N,q+ N,q+ ,q+ N,N,q + ,q + Total
1
1
P h + 3N a
2
2
2Ph + N
Association of the Ph chromosome
Chromosome #9
Table 4
2 1 1 10
1 2 3
2Ph + 2N
1 3
1
1
2Ph + 3N
and #9 chromosome
10
2 4 2
2
3Ph + N
Chromosome #22
17
1 5 1 6 4
3 P h + 2N
i n 5 0 c e l l s i n p a t i e n t C.J.
4
1 1
2
3 P h + 3N
2
1 1
4Ph + N
1
1
4 P h + 2N
2 8 11 3 15 9 2 50
Total
c.O
12 0
K. Ohtaki et al.
Figure 2 Representative karyotype in patient C.J.: 6 3 , X Y , + 2 , + 3 q - , + 4 , + 6 , + 8 , + 1 0 , + 11p +, + 11p +, + 13, + 13, + 15, + 19, + 19, + 21, + 22q - , + 22q - , + 22q - ,t (9;22)(q34;q11),del (3)(qllq26), two copies of 11p + (unknown origin), and showing a mixture of di-, tri-, tetra-, and pentasomies. This case had one or more Ph chromosomes and other structural changes; 3 q - in 23 cells and two copies of 11p+ were detected in 49 of 50 cells. Arrows indicate structural abnormalities.
G-banding analysis of the two cases reported here revealed the following: (a) karyotypic instability (no clonality) with modal n u m b e r s of 71 (63-75) and 63 (50123); (b) high frequency of trisomy in autosomes (16/22 or 9/22), with each cell having a wide variation from m o n o s o m y to tetrasomy in each chromosome set; and (c) r a n d o m association of the Ph chromosomes with # 9 chromosomes, with gains and losses of chromosome involved in this translocation. The near-triploid populations seen by us were not produced through duplication of a haploid cell population, but seem to be due to r a n d o m loss of chromosomes from tetraploid cell populations derived from duplication of p s e u d o d i p l o i d cells.
REFERENCES 1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier NorthHolland, New York. 2. Bennet JM, Catovsky D, Daniel M-T, Flandrin G, Galton DAG, Gralnick HR, Sultan C (FAB Co-operative Group) (1976): Proposals for the classification of the acute leukaemias. Br J Haematol 33:451-458.
Near-Triploid Ph-Positive Leukemia
121
3. ISCN (1978): An International System for Human Cytogenetic Nomenclature (1978). Cytogenet Cell Genet 21:309-404. 4. Pederson B, Boesen AM (1983): Extreme hypodiploidy in a case of myelomonocytic crisis of chronic myelogenous leukemia. Cancer Genet Cytogenet 9:101-112. 5. Douglass EC, Freeman DL (1983): Hypotetraploidy in erythroleukemia. Cancer Ganet Cytogenet 8:231-234. 6. Weh HJ, von Paleske A, Hossfeld DK (1983): Disappearance of hypotetraploid clones after short-term culture of leukemic cells. A case report. Cancer Genet Cytogenet 10:237-240. 7. Oksala T, Therman E (1974): Mitotic abnormalities and cancer. In: Chromosomes and Cancer, J German, ed. John Wiley, New York, pp 239-263. 8. Oshimura M, Freeman AI, Sandberg AA (1977): Chromosomes and causation of human cancer and leukemia. XXIII. Near-haploidy in acute leukemia. Cancer 40:1143-1148. 9. Shabtai F, Lewinski UH, Har-Zahav L, Gaffer U, Halbrecht I, Djaldetti M (1979): A hypo~ diploid clone and its duplicate in acute lymphoblastic leukemia. Am J Clin Pathol 72:1018-1024. 10. Kaneko Y, Sakurai M (1980): Acute lymphocytic leukemia (ALL) with near-haploidy--A unique subgroup of ALL? Cancer Genet Cytogenet 2:13-18. 11. Rajasekariah P, Illes I, Garson OM (1982): Double 9;22 translocation with hyperdiploidy appearing in blastic transformation of chronic granulocytic leukemia. Cancer Genet Cytogenet 7:85-88.