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Chromosomes in the chronic phase of chronic granulocytic leukemia
Chromosomes in the Chronic Phase of Chronic Granulocytic Leukemia Naoki Sadamori, Masako Matsunaga, Eiichi Yao, Kenji Nishino, Yu Tomonaga, Masuko Tagawa, Miyuki Kusano, and Michito Ichimaru
ABSTRACT Chromosomal analysis of 86 cases with chronic granulocytic leukemia (CGL) was performed using Q- and G-banding techniques. In 65 of the 66 cases, a Philadelphia [Ph’) chromosome with evidence of translocation was observed. Karyotypic abnormalities in addition to the Ph’ were found in 21 cases. In 6 of these 21 cases, the chronic phase persisted for more than 1 year after the first appearance of these abnormalities; in the remaining 15 cases, the abnormalities appeared shortly before or during the acute phase. Of the 6 cases with karyotypic abnormalities in addition to the Ph’ in the chronic phase, 3 cases had trisomy #8 and the other 3 cases had a double Ph’, a translocation between chromosomes X4 and X9, and loss of the Y chromosome, respectively. In contmst to the 3 cases where trisomy #8 wasfound in the chronic phase and exhibited no other abnormalities besides the extra #8 and a Ph’, a115 cases whose trisomy #8 was found in the acute phase showed one or more additional chromosomal changes besides the extra #8 and the Ph’. In the former 3 cases, the percentage of metaphases with trisomy #8 increased gradually throughout the chronic phase during low dose busulfan therapy. One patient in the chronic phase for more than 14 months had only one extra PI?. In contrast, a11 of the 7 cases with double Ph’ chromosomes shortly before or in the acute phase showed one or more additional chromosomes in addition to double Ph’. The significance of a double double Ph’ in the chronic and acute phases appears to be similar to that of trisomy #8 in the respective phases. The observation of trisomy #8 and double Ph’ might prove to be a useful clinical indicator of the progression of CGL.
INTRODUmON Cytogenetically, chronic granulocytic leukemia (CGL) is an interesting disease because of the presence of Philadelphia (Phi) chromosome and the appearance of new additional chromosomal changes throughout the course of the disease in more than 75% of the CGL cases studied [l]. As the majority of additional changes occur shortly before or during the acute phase, they are used as one of the predictive or diagnostic indicators of the acute crisis. However, some cases remain in the chronic phase in spite of the presence of additional chromosomal changes. If differences in additional chromosomal changes
From the Department of Hematology, Atomic Disease Institute, Nagasaki University School of Medicine, and St. Mary’s Hospital, Fukue, Nagasaki Prefecture 853, Japan.
Address requests for reprints to: Dr. Naoki Sadamori, Department of Hematology, Atomic Diseases Institute, Nagasaki University School of Medicine, 7-l Sakamoto-Machi, Nagasaki 852,Japan. Received
May 15,1979;
@Elsevier North Holland, Inc., 1980 0165-4606/60/02029912$02.25
accepted
July 30,1979.
Cancer Genetics and Cytogenetics 1,299-
310 (1960)
299
N. Sadamori
300
et al.
between the chronic and acute phases can be elucidated, chromosomal examination may prove to be useful for the understanding of the progression of the disease. MATERIALS
AND METHODS
The majority of the 66 CGL cases studied, composed of 39 males and 27 females ranging in age from 6 to 83 years, were inpatients of the Department of Hematology, Nagasaki University. Chromosomal analysis was performed on 41 cases in the chronic phase only, on 14 cases in the acute phase only or shortly before the acute crisis, and on 11 cases both in the chronic and acute phases; 2 cases studied in the acute phase only presented themselves with no prior history of CGL. The acute phase was thought to exist when the percentage of myeloblasts in the bone marrow exceeded 20% and/or when the combined percentage of myeloblasts, promyelocytes, and promonocytes in the bone marrow exceeded 30%. For the determination of neutrophil alkaline phosphatase (NAP), Tomonaga’s method [2] (normal range, rate 84 5 13% activity score 227 2 68) was used. The chromosomes in the cells of bone marrow, blood, and pleural effusion were analyzed by the direct method without phytohemagglutinin (PHA). For chromosome banding studies, Q [3] and G banding [4] were used. In each specimen, lo- 50 metaphases were analyzed. An abnormal clone was defined as two cells with the same extra chromosome or structural rearrangement, or three cells with the same missing chromosome. In order to avoid frequent lengthy description of additional chromosomal changes in the karyotypes, we have used the term “simple type trisomy 8” to indicate the presence of trisomy 8 in addition to a Phr chromosome, “complex type trisomy 8” to indicate the presence of one or more additional changes besides trisomy 8 and a Phr chromosome, “simple type double Ph”’ to represent the presence of only double Phr chromosomes, and “complex type double Ph’” to show the presence of one or more additional changes besides the double Ph’ chromosomes. CASE REPORTS Six cases with additional
chromosomes
in the chronic phase are described below.
Case 1 An II-year-old boy was noticed to have facial pallor in November 1964 and was first seen in October 1965 at the Obama National Hospital, Minami Takaki-gun, Nagasaki Prefecture, where he was found to have anemia and leukocytosis. He was referred and admitted to the Department of Hematology, Nagasaki University. Physical and hematological examinations revealed marked splenomegaly, anemia (Hb 7.2 g/dl), leukocytosis (174,60O/mm”), and a low level of NAP. With the diagnosis of CGL established, low dose busulfan therapy was started. As of December 1978, he remained in the chronic phase. Case 2 A 25-year-old male noticed a tumor on his left hypochondrium. In January 1976 he was admitted to the Nagasaki Citizens Hospital where splenomegaly, leukocytosis (188,200/mm3) and a low level of NAP were found. He responded to low doses of busulfan therapy until January 1977, when progressive anemia and enlargement of the spleen appeared. He was referred and admitted to the Department of Hematology, Nagasaki University. In February 1977, when he was diagnosed to be in an acute cris-
Chromosomes in the Chronic Phase of CGL
301
is of CGL, myelofibrosis was revealed by needle biopsy. He was treated with irradiation (a total of 3100 R) to the spleen and with prednisolone. A bloody pleural effusion was revealed by chest x-ray in June 1977;smears of the cells contained peroxidasepositive premature granulocytes. The patient died in July 1977. Case 3 A 4%year-old male complaining of pain in the left hypochondrium, in May 1971, was first seen at the Sasebo Citizens Hospital, Sasebo, Nagasaki Prefecture and found to have marked splenomegaly, leukocytosis (97,000/mm3), a low level of NAP (rate 42, activity score 105), and a hyperplastic myeloid bone marrow. He remained in the chronic phase with low dose busulfan therapy till December 1974,when he died following the development of an acute crisis and complications with progressive multifocal leukoencephalopathy. Case 4 A X&year-old male was injured in a traffic accident in September 1977 and was admitted to the Sasebo Citizens Hospital. He was found to have splenomegaly, leukocytosis (52,000/mm3) and a low level of NAP (rate 10, score 15).With low dose busulfan therapy, he remains in the chronic phase as of December 1978. Case 5 A 30-year-old female noted pain in the left hypochondrium during her first (1970) and second (1972) pregnancies. In November 1974 she noticed a tumor on her left hypochondrium. She was admitted to the Department of Hematology, Kitazato University, Sagamihara, Kanagawa where she was found to have marked splenomegaly, slight anemia (Hb 11.3 gldl), leukocytosis (404,000/mm3), and a low level of NAP. Following the diagnosis of CGL, she was treated with low dose dibromomanitol therapy. Subsequent to July 1975 she followed up at the Second Department of Internal Medicine, Kumamoto University, Kumamoto. In June 1978, slight fever, progression of the anemia, and increased blast cells in the blood and bone marrow were noted. With the diagnosis of acute crisis she was treated with the DCVP (daunorubicin, cytosine arabinoside, 6 MP, and prednisolone) regimen. However, the disease was resistant to chemotherapy and the patient died in November 1978. Case 6 An 83-year-old male was exposed to the atomic bomb at 2.5 km from the hypocenter in Hiroshima in August 1945. He suffered from severe diarrhea for 3 months after the A-bomb exposure. In November 1976 and June 1977 he was found to have leukocytosis following a health check for atomic bomb survivors. He visited the Atomic Bomb Hospital, Nagasaki in June 1977 and was found to have leukocytosis (76,000/mm3) and a low level of NAP (rate 33, score 76). Under low dose busulfan therapy he still remains in the chronic phase as of December 1978. RESULTS
Sixty-five of the 66 cases studied with banding techniques had a Phl with evidence of translocation. Of these, 64 cases had the usual type of Phl translocation and one an unusual type between chromosomes #17 and #22, reported elsewhere by Matsunaga et al. [5]. One Phi-negative case without chromosome changes was observed in the
N. Sadamori
302 Table 1
The phases studied and chromosomal 65 Phi-positive cases of CGL No. of cases
Phase studied
with Ph’ only
findings
No. of cases with additional changes
et al.
in
Total
Chronic only Shortly before or in acute only Chronic and Acute 65
Total
chronic phase only. The phases and chromosomal findings of the 65 Ph’-positive cases are summarized in Table 1.Three (cases 1,4, and 6) of the 41 cases studied in the chronic phase only, including the Phr-negative case, showed a chromosomal change in addition to the Phi. Eight of the 14 cases studied in the acute phase only, including those shortly before the acute crisis, showed additional changes. Two cases who presented themselves in the acute phase only did not show any additional chromosomes besides the Phi. Three (cases 2,3, and 5) of the 11 cases studied in both the chronic and acute phases showed an additional chromosome in the chronic
Table
z
Chromosomal of CGL
findings
in 6 cases with additional
changes
in the chronic
Case
Age
Sex
Date
Phase
Source
No. of metaphases
1. S.N.
11
M
8-18-76 11-24-78
chronic chronic
marrow marrow
32 50
47, XY, +8, Ph’ 47, XY, +8, Ph’
Predominant
abnormal
karyotype
94
25
M
l-22-76 2-24-76 6-22-76 6-27-77
chronic chronic chronic acute
marrow marrow marrow pleural effusion
14 22 21 18
47,XY, +8, Ph’ 47, XY, +8, Ph’ 47, XY, i-8, Ph’ 47, XY, +8, i(17q), Ph*
3. M.F.
43
M
5-14-71 12-12-72 9-13-74 12- 5-74 12-24-74
chronic chronic chronic acute acute
marrow marrow marrow marrow marrow
50" 34" 25 13 10
46,XY, Ph’
ll- 2-77 chronic 3-29-78 chronic
merrow marrow
28 37
47, XY, +Ph’, Ph’ 47, XY, +Ph’, Ph’
6-21-76 chronic
marrow blood marrow
26 25 16
46,XX, t(4p+;9q-),
marrow marrow
18 20
45, X,-Y, 45, X, -Y,
4.Y.U.
32
M
5.F.O.
30
F
4-27-77 9-26-78
6.T.H.
83
M
chronic acute
6-11-77 chronic 2-23-78
chronic
“All metaphases were studied only by conventional staining method.
XY, XY, XY, XY,
21 59 62 78
100
+C, +8, f8, +8,
46, XX 45, XX, -7,
%
100
2. K.Y.
47, 47, 47, 47,
phase
Ph’ Ph’ Ph’ Ph’
71 100 100 100 18 46 Ph’
t(4pt;9q-), Ph’ Ph’
Ph’
77 88 31 83 95
Chromosomes
in the Chronic Phase of CGL
303
phase. In 2 (cases 2 and 5) of these 3 cases, new additional changes, i.e., i(li’q) and missing 7, respectively, appeared in the acute phase. Seven of the 8 cases who had no additional chromosomes except Phi in the chronic phase showed additional chromosomal changes in the acute phase. Of the 6 cases (cases 1,2, 3,4, 5, and 6) who were in the chronic phase for one or more years after the first appearance of the additional chromosome in the chronic phase, 3 (cases 1, 2 and 3) had trisomy 8 and 3 others had a double Phl (case 4), a translocation between chromosomes #4 and #9 (case 5), and loss of the Y (case 6), respectively. Chromosomal follow-up study of these 6 cases was performed and the results are summarized in Table 2. CYTOGENETIC FINDINGS Case 1 In August 1976 the first chromosomal analysis showed 94% of the metaphases to have an extra #8 in addition to Phi, i.e., a simple type trisomy 8, as shown in Figure 1. In November 1978 100% of the metaphases had simple type trisomy 8. The patient has been in the chronic phase for 158 months after diagnosis of the disease and for 29 months after the finding of trisomy 8. Figure 1 Karyotype of case 1 showing (arrows) trisomy 8 and a PW translocation between chromosomes #9 and #22, i.e., 47,XY,+8,t(9;22) (9;22) (q34;qll).
304
N. Sadamori
et al.
Case 2 At diagnosis in January 1976, chromosomal analysis showed 21% of metaphases to have a simple type trisomy 8. The percentage of metaphases with trisomy 8 rose to 59% in February 1976 and to 62% in June 1976. During the acute phase in June 1977, chromosomal analysis by means of direct method without PHA of the cells in a bloody pleural effusion showed 78% of the metaphases to have trisomy 8 and an i (17q), in addition to Phi, i.e., a complex type trisomy 8, as shown in Figure 2. This case had a simple type trisomy 8 at diagnosis and developed a complex type trisomy 8 in the acute phase. Case 3 In May 1971 the first chromosomal analysis, showed no evidence of additional chromosomes except for the Phr. In December 1972 the second chromosomal analysis, based on the conventional staining method, showed 71% of the metaphases having an extra C in addition to the Phi. In September 1974, in the chronic phase, the third analysis showed 100% of the metaphases to have a simple type trisomy 8, as was the case in the fourth and fifth analyses in December 1974, when the patient developed an acute crisis. There had been no evidence of any additional chromosomes at an early stage of the disease. The metaphases with a simple type trisomy 8 increased gradually and steadily under low dose busulfan therapy throughout the chronic phase. No new additional chromosomes were seen in the acute phase. Case 4 In November 1977 the first chromosomal analysis showed 18% of the metaphases to have only an extra Ph’. In March 1978 analysis showed 46% of the metaphases to have a simple type double Ph’. In this case the double Ph’ appeared at diagnosis of the disease and increased gradually under low dose busulfan therapy. The patient has remained in the chronic phase for more than 14 months.
Case 5 In June 1976 the first chromosomal analysis using banding techniques showed a translocation between chromosomes #4 and #9 in addition to the Phl due to translocation between chromosomes #9 and #22, as shown in Figure 3. In April 1977 chromosomal analysis of blood stimulated with PHA showed a normal female karyotype without any translocations. In September 1978 when she was in the acute phase, chromosomal analysis showed monosomy 7 in addition to translocation between chromosomes #4 and #9 and a Ph’. All the metaphases with a Phl had the translocation between chromosomes #4 and #9. On the other hand, all metaphases of lymphocytes stimulated with PHA had a normal karyotype. Case 6 In June 1977 and February 1978 chromosomal analysis showed loss of the Y and the presence of a Phr. The majority of the metaphases examined at diagnosis had lacked the Y, but did have a Phi. It should be emphasized that he was the oldest case in this series and an atomic bomb survivor of Hiroshima. Karyotypes of 15 cases with additional chromosomal changes which occurred shortly before or during the acute phase are summarized in Table 3. It includes 5 cases with trisomy 8 and 7 cases with double Phi. These data will be discussed in comparison with additional chromosomal changes in the chronic phase.
Chromosomes
in the Chronic Phase of CGL
305
306
N. Sadamori
et al.
Figure 3 Karyotype of case 5 showing (arrows) a translocation between chromosomes #4 and #9 and a Ph’ translocation between chromosomes #9 and #22, i.e., 46,XX,t(4;9) (p16;q32), t(9;22)(q34;qll).
DISCUSSION Sonta and Sandberg [6] analyzed a large number of cells (llO- 500) and demonstrated the presence of a small percentage of aneuploid metaphases in the bone marrow during the chronic phase of the CGL in 5 of the 6 cases studied. However, the number of metaphases analyzed in routine chromosomal examinations is usually from 10 to 50 [6]. In this series, we defined an abnormal clone as one that was verified in routine examination. More than 75% of CGL cases show additional chromosomal changes during the course of the disease [l]. Additional chromosomal changes in the majority of cases appeared shortly before or during the acute phase and could be used as indicators of the acute crisis. On the other hand, the number of cases with additional chromosomal changes in the chronic phase is not small. Lawler [7] reported the incidence of additional chromosomal changes in the chronic phase as follows: trisomy 8 (3%), trisomy 8 accompanied by a double Phl (S%), a double Phl (3%), stahle rearrangements (8%), and loss of Y (8%). Strictly speaking, the definition of the chronic phase of the disease is not universally the same. However, it is certain that some cases exist with additional chromosomal changes present in the chronic phase for several years under low dose maintenance therapy. This fact prompted us to differentiate the additional
Chromosomes in the Chronic Phase of CGL Table 3
307
Chromosomal findings in 15 cases with additional changes shortly before or in the acute phase of CGL
Case’
Age
Sex
Phase acute acute acute acute acute 3.7 months before acute crisis acute acute acute 0.6 months before acute crisis acute acute acute acute acute
7. 8. 9. 10. 11. 12.
U.K. K.K. SM. M.W. U.M. H.K.
8 33 22 46 25 34
M M M M F M
13. 14. 15. 16.
C.H. I.Y. R.E. M.W.
57 49 24 6
F M F F
17. 16. 19. 20. 21.
M.U. M.K. SK. H.K. M.H.
56 35 46 24 19
M F F F F
Predominant abnormal keryotype 52, 52, 50, 49, 49, 48,
XY, +8, +14, +15, +19, +21, +Ph’, Ph’ XY, +l, $8, +lO, +ll, +21, +Ph’, Ph’ XYY, +8, +19, +Ph’, Ph’ XY, +6, +8, +Ph’, lp+, 14q+, Ph’ XX, f17, +19, +Ph’, Ph’ XY, +21, +Ph’, t(3p;4p+), Ph’
47, 47, 47, 46,
XX, XY, XX, XX,
+8, 16q+, i(l7q), Phi +Ph’, 4p+, Ph’ +15, Phi t(5q-;21q+), Ph’
46, XY, r(ll), Phl 46, XX, t(2q+;4q-), Ph’ 46, XX, t(17p+;18q-), Ph’ 44, X, -?X, -3, lq+, Ph’ Multiple changes
4n cases 7,8,10,11,12,13,14,17 end 18, the kayotype of 46,XX or XY, Ph’ is confirmed in the chronic phase.
chromosomal changes in the chronic phase from those in the acute phase. Thus, we analyzed 21 cases with additional chromosomal changes in our series and a3 cases with abnormalities found by banding techniques in the literature [5, a- 141, in an attempt to elucidate this differentiation. Trisomy a It is well-known that the additional chromosomal changes in CGL cases are commonly trisomy a and a double Ph’. Some investigators [a,q are inclined to think that the appearance of trisomy a is a poor prognostic sign. One the other hand, Hayata et al. [lo] suggested that the prognosis is probably more favorable for cases with a simple type trisomy a than for cases with other numerical changes in the karyotype. Some controversy still exists as to whether or not trisomy a is associated with progression of the disease. In this series, we found that the 3 cases (cases 1, 2, and 3) with trisomy a in the chronic phase had a simple type trisomy a, whereas the 5 cases (cases 7, a, 9, 10, and 13) with trisomy a in the acute phase had a complex type of trisomy a.
Table 4
The phases studied and the state of trisomy a in 31 cases of CGL in the literature
Phase studied
No. of cases with simple type trtsomy 8
No. of cases with complex type trisomy 8
Total
Chronic Acute
2 1
3” 25
5 26
Total
3
28
31
Two of 3 casesshow tetmsomy 8 end trisomy 8 with double Ph’, respectively.
N. Sadamori
308 Table 5
The phases studied and the state of the double Ph’ in of CGL in the literature
et al.
24 cases
Phase studied
No. of cases with simple type double Ph’
No. of cases with complex type double Phl
Total
Chronic Acute
3 2
2a 17
5 19
Total
5
19
24
=One of 2 cases shows trisomy 8 with double Phi.
The 31 cases with trisomy 8 in the literature were analyzed (Table 4). The probability of x2= 6.27 with one degree of freedom was 2.5%, which is conclusive evidence that the phase of the disease is not independent of the type of trisomy 8 (simple vs. complex). Double Ph’ Whang-Peng et al. [15] concluded that a double Phl as an initial aneuploidy does not necessarily represent or lead to an acute crisis. However, a double Phl is an additional chromosomal change commonly observed in the acute phase. Nevertheless, there is still some controversy as to whether or not a double Ph’ constitutes an indicator of the progression of the disease. In this series, case 4 had a simple double Phl in the chronic phase, whereas the 7 cases (cases 7,8,9,10,11,12, and 14) with a complex double Phl were shown to have this anomaly shortly before or in the acute phase of CGL. The 24 cases with a double Phi in the literature were analyzed (Table 5). The probability of x2 = 5.87 with one degree of freedom was 2.5%, which is conclusive evidence that the phase of CGL is not independent of the type of double Ph’ (simple vs. complex). Translocations In case 5 all metaphases with the translocation between chromosomes #4 and #9 were accompanied by a Phl. The karyotypes of lymphocytes stimulated with PHA were normal without any translocation or Phi. These findings suggest that the translocation between chromosomes #4 and #9 occurred in a CGL cell line with a Phi. Case 5 showed a new karyotype with monosomy 7 in the acute crisis. This fact may indicate that metaphases with a translocation in addition to that of the Ph’ in the chronic phase might be inclined to develop further karyotypic evolution relative to the acute crisis. However, more cases with translocations in the chronic phase will have to be studied before reaching any final conclusion. Loss of the Y Sakurai and Sandberg [16] advanced the hypothesis that cells which lack a Y are generally resistant to the development of further chromosomal abnormalities. As shown in Table 6, the finding that 7 of 10 cases with a missing Y in the literature [9, 14, IS-Xl] did not show any further chromosomal abnormalities in the cell lines lacking the Y but with a Phl after developing an acute crisis, supports the hypothesis of Sakurai and Sandberg [16]. In general, in the majority of cases which lack a Y, the Y is lost during the chronic phase and, thus, the loss of the Y is not an adequate predictive or diagnostic indicator of the acute crisis.
Chromosomes
in the Chronic
Table 6
Phase
Karyotypes in the chronic and acute phases lackine the Y uublished in the literature
27 24 27 43 59 64 61 43
45, x, -Y, Ph’ 45,X, -Y, Ph’ 45,X,-Y, Ph’ 45,X, -Y, Ph’ 45,X, -Y, Ph’ 45,x, -Y, Ph’ 45,X,-Y, Ph’ 45,x, -Y, Ph’ 146, XY, Ph’ 45, X,-Y, Ph’ 45, X,-Y, Ph’
50 48
in 10 cases with
Karyotype in the acute phase
Karyotype in the chronic phase
Age
309
of CGL
59-64 and 65, XY, Ph’ 45,x,-Y, Ph’ additional abnormalities 45,X,-Y, Ph’ 45,x, -Y, Ph’ 45,x,-Y, Ph’ 45,X, -Y, Ph’ 45,X, -Y, Ph’l46, XY, Ph’ 145-47, XY, *a, Ph’ 45-46, X,-Y, +m, 2lq+, Ph’ 43, X, -Y, -16,-17, Ph’145, X, -Y, Ph’
CGL
References Motomura 8 Ogi [ 171 Furusawa et al. [18] Lawler et al. [19] Lawler et al. [19] Lawler et al. [19] Whittaker et al. [20] Whittaker et al. [20] Sakurai 8 Sandberg [16] Sharp et al. (91 Sonta & Sandberg
[14]
The authors wish to express their thanks to Drs. Shotaro Neriishi and Takeo Honda and Mr. Hitoshi Tokai of the Radiation Effects Research Foundation, Nagasaki, for their help. The skillful technical assistance of Mrs. Sayoko Mori and Mrs. Tamiko Sakaguchi is greatly appreciated.
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M, Sasaki T, Okusaki M (1963): Studies on leukocyte alkaline phosphatase. I. naphthol AS-MX phosphate-fast blue RR staining method. Acta Haematol Jap 26, T, Zech L, Jahansson C (1970): Differential banding of alkylating fluorochmmes chromosomes. Exp Cell Res 60,315 -319.
4. Sumner AT, Evans HJ, Buckland RA (1971): New techniques chromosomes. Nature 232,31-32.
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5. Matsunaga M, Sadamori N, Tomonaga Y, Tagawa M, Ichimaru M (1976): Chronic myelogenous leukemia with an unusual karyotype: 46,XY,t(17q+;22q-). NEngl J Med 295,1537. 6. Sonta S, Sandberg AA (1977): Chromosomes and causation of human cancer and leukemia. XXVIII. Value of detailed chromosome studies on large numbers of cells in CML. Am J Hematol3,121-126. 7. Lawler SD (1977): The cytogenetics 55 - 75.
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8. Gahrton G, Lindsten J, Zech L (1974): Involvement of chromosomes 8,9,19, and 22 in Ph’ positive and Ph’ negative chronic myelocytic leukemia in the chronic or blastic stage. Acta Med Stand 196,355 -360. 9. Sharp JC, Potter AM, Keith Wood J (1976): Non-random and random chromosome abnormalities in transformed chronic granulocytic leukemia. Stand J Haematol 16, 5 - 12. 10. Hayata I, Sakurai M, Kakati S, Sandberg AA (1975): Chromosomes and causation of human cancer and leukemia. XVI. Banding studies of chronic myelocytic leukemia, including five unusual Ph’translocations. Cancer 36,1177-1191. 11. Lawler SD, O’Malley F, Lobb DS (1976): Chromosome banding studies in Philadelphia chromosome positive myeloid leukemia. Stand J Haematoll7,17-28. 12. Prigogina EL, Fleischman EW, Volkova MA, Frenkel MA (1978): Chromosome abnonnalities and clinical and morphologic manifestations of chronic myeloid leukemia. Hum Genet 41,143-156. 13. Hossfeld DK (1974): Identification of chromosome anomalies in the blastic phase of chronic myelocytic leukemia (CML) by Giemsa- and quinacrine-banding techniques. Hum Genet 23,111-118.
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14. Sonta S, Sandberg AA (1978): Chromosomes and causation of human cancer and leukemia. XXIX. Further studies on karyotypic progression in CML. Cancer 41,153 - 163. 15. Whang-Peng J, Canellos GP, Carbone PP, Tjio JH (1988): Clinical implications variants in chronic myelocytic leukemia (CML). Blood 32,755 - 766.
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ABSTRACT Chromosomal analysis of 86 cases with chronic granulocytic leukemia (CGL) was performed using Q- and G-banding techniques. In 65 of the 66 cases, a Philadelphia [Ph’) chromosome with evidence of translocation was observed. Karyotypic abnormalities in addition to the Ph’ were found in 21 cases. In 6 of these 21 cases, the chronic phase persisted for more than 1 year after the first appearance of these abnormalities; in the remaining 15 cases, the abnormalities appeared shortly before or during the acute phase. Of the 6 cases with karyotypic abnormalities in addition to the Ph’ in the chronic phase, 3 cases had trisomy #8 and the other 3 cases had a double Ph’, a translocation between chromosomes X4 and X9, and loss of the Y chromosome, respectively. In contmst to the 3 cases where trisomy #8 wasfound in the chronic phase and exhibited no other abnormalities besides the extra #8 and a Ph’, a115 cases whose trisomy #8 was found in the acute phase showed one or more additional chromosomal changes besides the extra #8 and the Ph’. In the former 3 cases, the percentage of metaphases with trisomy #8 increased gradually throughout the chronic phase during low dose busulfan therapy. One patient in the chronic phase for more than 14 months had only one extra PI?. In contrast, a11 of the 7 cases with double Ph’ chromosomes shortly before or in the acute phase showed one or more additional chromosomes in addition to double Ph’. The significance of a double double Ph’ in the chronic and acute phases appears to be similar to that of trisomy #8 in the respective phases. The observation of trisomy #8 and double Ph’ might prove to be a useful clinical indicator of the progression of CGL.
INTRODUmON Cytogenetically, chronic granulocytic leukemia (CGL) is an interesting disease because of the presence of Philadelphia (Phi) chromosome and the appearance of new additional chromosomal changes throughout the course of the disease in more than 75% of the CGL cases studied [l]. As the majority of additional changes occur shortly before or during the acute phase, they are used as one of the predictive or diagnostic indicators of the acute crisis. However, some cases remain in the chronic phase in spite of the presence of additional chromosomal changes. If differences in additional chromosomal changes
From the Department of Hematology, Atomic Disease Institute, Nagasaki University School of Medicine, and St. Mary’s Hospital, Fukue, Nagasaki Prefecture 853, Japan.
Address requests for reprints to: Dr. Naoki Sadamori, Department of Hematology, Atomic Diseases Institute, Nagasaki University School of Medicine, 7-l Sakamoto-Machi, Nagasaki 852,Japan. Received
May 15,1979;
@Elsevier North Holland, Inc., 1980 0165-4606/60/02029912$02.25
accepted
July 30,1979.
Cancer Genetics and Cytogenetics 1,299-
310 (1960)
299
N. Sadamori
300
et al.
between the chronic and acute phases can be elucidated, chromosomal examination may prove to be useful for the understanding of the progression of the disease. MATERIALS
AND METHODS
The majority of the 66 CGL cases studied, composed of 39 males and 27 females ranging in age from 6 to 83 years, were inpatients of the Department of Hematology, Nagasaki University. Chromosomal analysis was performed on 41 cases in the chronic phase only, on 14 cases in the acute phase only or shortly before the acute crisis, and on 11 cases both in the chronic and acute phases; 2 cases studied in the acute phase only presented themselves with no prior history of CGL. The acute phase was thought to exist when the percentage of myeloblasts in the bone marrow exceeded 20% and/or when the combined percentage of myeloblasts, promyelocytes, and promonocytes in the bone marrow exceeded 30%. For the determination of neutrophil alkaline phosphatase (NAP), Tomonaga’s method [2] (normal range, rate 84 5 13% activity score 227 2 68) was used. The chromosomes in the cells of bone marrow, blood, and pleural effusion were analyzed by the direct method without phytohemagglutinin (PHA). For chromosome banding studies, Q [3] and G banding [4] were used. In each specimen, lo- 50 metaphases were analyzed. An abnormal clone was defined as two cells with the same extra chromosome or structural rearrangement, or three cells with the same missing chromosome. In order to avoid frequent lengthy description of additional chromosomal changes in the karyotypes, we have used the term “simple type trisomy 8” to indicate the presence of trisomy 8 in addition to a Phr chromosome, “complex type trisomy 8” to indicate the presence of one or more additional changes besides trisomy 8 and a Phr chromosome, “simple type double Ph”’ to represent the presence of only double Phr chromosomes, and “complex type double Ph’” to show the presence of one or more additional changes besides the double Ph’ chromosomes. CASE REPORTS Six cases with additional
chromosomes
in the chronic phase are described below.
Case 1 An II-year-old boy was noticed to have facial pallor in November 1964 and was first seen in October 1965 at the Obama National Hospital, Minami Takaki-gun, Nagasaki Prefecture, where he was found to have anemia and leukocytosis. He was referred and admitted to the Department of Hematology, Nagasaki University. Physical and hematological examinations revealed marked splenomegaly, anemia (Hb 7.2 g/dl), leukocytosis (174,60O/mm”), and a low level of NAP. With the diagnosis of CGL established, low dose busulfan therapy was started. As of December 1978, he remained in the chronic phase. Case 2 A 25-year-old male noticed a tumor on his left hypochondrium. In January 1976 he was admitted to the Nagasaki Citizens Hospital where splenomegaly, leukocytosis (188,200/mm3) and a low level of NAP were found. He responded to low doses of busulfan therapy until January 1977, when progressive anemia and enlargement of the spleen appeared. He was referred and admitted to the Department of Hematology, Nagasaki University. In February 1977, when he was diagnosed to be in an acute cris-
Chromosomes in the Chronic Phase of CGL
301
is of CGL, myelofibrosis was revealed by needle biopsy. He was treated with irradiation (a total of 3100 R) to the spleen and with prednisolone. A bloody pleural effusion was revealed by chest x-ray in June 1977;smears of the cells contained peroxidasepositive premature granulocytes. The patient died in July 1977. Case 3 A 4%year-old male complaining of pain in the left hypochondrium, in May 1971, was first seen at the Sasebo Citizens Hospital, Sasebo, Nagasaki Prefecture and found to have marked splenomegaly, leukocytosis (97,000/mm3), a low level of NAP (rate 42, activity score 105), and a hyperplastic myeloid bone marrow. He remained in the chronic phase with low dose busulfan therapy till December 1974,when he died following the development of an acute crisis and complications with progressive multifocal leukoencephalopathy. Case 4 A X&year-old male was injured in a traffic accident in September 1977 and was admitted to the Sasebo Citizens Hospital. He was found to have splenomegaly, leukocytosis (52,000/mm3) and a low level of NAP (rate 10, score 15).With low dose busulfan therapy, he remains in the chronic phase as of December 1978. Case 5 A 30-year-old female noted pain in the left hypochondrium during her first (1970) and second (1972) pregnancies. In November 1974 she noticed a tumor on her left hypochondrium. She was admitted to the Department of Hematology, Kitazato University, Sagamihara, Kanagawa where she was found to have marked splenomegaly, slight anemia (Hb 11.3 gldl), leukocytosis (404,000/mm3), and a low level of NAP. Following the diagnosis of CGL, she was treated with low dose dibromomanitol therapy. Subsequent to July 1975 she followed up at the Second Department of Internal Medicine, Kumamoto University, Kumamoto. In June 1978, slight fever, progression of the anemia, and increased blast cells in the blood and bone marrow were noted. With the diagnosis of acute crisis she was treated with the DCVP (daunorubicin, cytosine arabinoside, 6 MP, and prednisolone) regimen. However, the disease was resistant to chemotherapy and the patient died in November 1978. Case 6 An 83-year-old male was exposed to the atomic bomb at 2.5 km from the hypocenter in Hiroshima in August 1945. He suffered from severe diarrhea for 3 months after the A-bomb exposure. In November 1976 and June 1977 he was found to have leukocytosis following a health check for atomic bomb survivors. He visited the Atomic Bomb Hospital, Nagasaki in June 1977 and was found to have leukocytosis (76,000/mm3) and a low level of NAP (rate 33, score 76). Under low dose busulfan therapy he still remains in the chronic phase as of December 1978. RESULTS
Sixty-five of the 66 cases studied with banding techniques had a Phl with evidence of translocation. Of these, 64 cases had the usual type of Phl translocation and one an unusual type between chromosomes #17 and #22, reported elsewhere by Matsunaga et al. [5]. One Phi-negative case without chromosome changes was observed in the
N. Sadamori
302 Table 1
The phases studied and chromosomal 65 Phi-positive cases of CGL No. of cases
Phase studied
with Ph’ only
findings
No. of cases with additional changes
et al.
in
Total
Chronic only Shortly before or in acute only Chronic and Acute 65
Total
chronic phase only. The phases and chromosomal findings of the 65 Ph’-positive cases are summarized in Table 1.Three (cases 1,4, and 6) of the 41 cases studied in the chronic phase only, including the Phr-negative case, showed a chromosomal change in addition to the Phi. Eight of the 14 cases studied in the acute phase only, including those shortly before the acute crisis, showed additional changes. Two cases who presented themselves in the acute phase only did not show any additional chromosomes besides the Phi. Three (cases 2,3, and 5) of the 11 cases studied in both the chronic and acute phases showed an additional chromosome in the chronic
Table
z
Chromosomal of CGL
findings
in 6 cases with additional
changes
in the chronic
Case
Age
Sex
Date
Phase
Source
No. of metaphases
1. S.N.
11
M
8-18-76 11-24-78
chronic chronic
marrow marrow
32 50
47, XY, +8, Ph’ 47, XY, +8, Ph’
Predominant
abnormal
karyotype
94
25
M
l-22-76 2-24-76 6-22-76 6-27-77
chronic chronic chronic acute
marrow marrow marrow pleural effusion
14 22 21 18
47,XY, +8, Ph’ 47, XY, +8, Ph’ 47, XY, i-8, Ph’ 47, XY, +8, i(17q), Ph*
3. M.F.
43
M
5-14-71 12-12-72 9-13-74 12- 5-74 12-24-74
chronic chronic chronic acute acute
marrow marrow marrow marrow marrow
50" 34" 25 13 10
46,XY, Ph’
ll- 2-77 chronic 3-29-78 chronic
merrow marrow
28 37
47, XY, +Ph’, Ph’ 47, XY, +Ph’, Ph’
6-21-76 chronic
marrow blood marrow
26 25 16
46,XX, t(4p+;9q-),
marrow marrow
18 20
45, X,-Y, 45, X, -Y,
4.Y.U.
32
M
5.F.O.
30
F
4-27-77 9-26-78
6.T.H.
83
M
chronic acute
6-11-77 chronic 2-23-78
chronic
“All metaphases were studied only by conventional staining method.
XY, XY, XY, XY,
21 59 62 78
100
+C, +8, f8, +8,
46, XX 45, XX, -7,
%
100
2. K.Y.
47, 47, 47, 47,
phase
Ph’ Ph’ Ph’ Ph’
71 100 100 100 18 46 Ph’
t(4pt;9q-), Ph’ Ph’
Ph’
77 88 31 83 95
Chromosomes
in the Chronic Phase of CGL
303
phase. In 2 (cases 2 and 5) of these 3 cases, new additional changes, i.e., i(li’q) and missing 7, respectively, appeared in the acute phase. Seven of the 8 cases who had no additional chromosomes except Phi in the chronic phase showed additional chromosomal changes in the acute phase. Of the 6 cases (cases 1,2, 3,4, 5, and 6) who were in the chronic phase for one or more years after the first appearance of the additional chromosome in the chronic phase, 3 (cases 1, 2 and 3) had trisomy 8 and 3 others had a double Phl (case 4), a translocation between chromosomes #4 and #9 (case 5), and loss of the Y (case 6), respectively. Chromosomal follow-up study of these 6 cases was performed and the results are summarized in Table 2. CYTOGENETIC FINDINGS Case 1 In August 1976 the first chromosomal analysis showed 94% of the metaphases to have an extra #8 in addition to Phi, i.e., a simple type trisomy 8, as shown in Figure 1. In November 1978 100% of the metaphases had simple type trisomy 8. The patient has been in the chronic phase for 158 months after diagnosis of the disease and for 29 months after the finding of trisomy 8. Figure 1 Karyotype of case 1 showing (arrows) trisomy 8 and a PW translocation between chromosomes #9 and #22, i.e., 47,XY,+8,t(9;22) (9;22) (q34;qll).
304
N. Sadamori
et al.
Case 2 At diagnosis in January 1976, chromosomal analysis showed 21% of metaphases to have a simple type trisomy 8. The percentage of metaphases with trisomy 8 rose to 59% in February 1976 and to 62% in June 1976. During the acute phase in June 1977, chromosomal analysis by means of direct method without PHA of the cells in a bloody pleural effusion showed 78% of the metaphases to have trisomy 8 and an i (17q), in addition to Phi, i.e., a complex type trisomy 8, as shown in Figure 2. This case had a simple type trisomy 8 at diagnosis and developed a complex type trisomy 8 in the acute phase. Case 3 In May 1971 the first chromosomal analysis, showed no evidence of additional chromosomes except for the Phr. In December 1972 the second chromosomal analysis, based on the conventional staining method, showed 71% of the metaphases having an extra C in addition to the Phi. In September 1974, in the chronic phase, the third analysis showed 100% of the metaphases to have a simple type trisomy 8, as was the case in the fourth and fifth analyses in December 1974, when the patient developed an acute crisis. There had been no evidence of any additional chromosomes at an early stage of the disease. The metaphases with a simple type trisomy 8 increased gradually and steadily under low dose busulfan therapy throughout the chronic phase. No new additional chromosomes were seen in the acute phase. Case 4 In November 1977 the first chromosomal analysis showed 18% of the metaphases to have only an extra Ph’. In March 1978 analysis showed 46% of the metaphases to have a simple type double Ph’. In this case the double Ph’ appeared at diagnosis of the disease and increased gradually under low dose busulfan therapy. The patient has remained in the chronic phase for more than 14 months.
Case 5 In June 1976 the first chromosomal analysis using banding techniques showed a translocation between chromosomes #4 and #9 in addition to the Phl due to translocation between chromosomes #9 and #22, as shown in Figure 3. In April 1977 chromosomal analysis of blood stimulated with PHA showed a normal female karyotype without any translocations. In September 1978 when she was in the acute phase, chromosomal analysis showed monosomy 7 in addition to translocation between chromosomes #4 and #9 and a Ph’. All the metaphases with a Phl had the translocation between chromosomes #4 and #9. On the other hand, all metaphases of lymphocytes stimulated with PHA had a normal karyotype. Case 6 In June 1977 and February 1978 chromosomal analysis showed loss of the Y and the presence of a Phr. The majority of the metaphases examined at diagnosis had lacked the Y, but did have a Phi. It should be emphasized that he was the oldest case in this series and an atomic bomb survivor of Hiroshima. Karyotypes of 15 cases with additional chromosomal changes which occurred shortly before or during the acute phase are summarized in Table 3. It includes 5 cases with trisomy 8 and 7 cases with double Phi. These data will be discussed in comparison with additional chromosomal changes in the chronic phase.
Chromosomes
in the Chronic Phase of CGL
305
306
N. Sadamori
et al.
Figure 3 Karyotype of case 5 showing (arrows) a translocation between chromosomes #4 and #9 and a Ph’ translocation between chromosomes #9 and #22, i.e., 46,XX,t(4;9) (p16;q32), t(9;22)(q34;qll).
DISCUSSION Sonta and Sandberg [6] analyzed a large number of cells (llO- 500) and demonstrated the presence of a small percentage of aneuploid metaphases in the bone marrow during the chronic phase of the CGL in 5 of the 6 cases studied. However, the number of metaphases analyzed in routine chromosomal examinations is usually from 10 to 50 [6]. In this series, we defined an abnormal clone as one that was verified in routine examination. More than 75% of CGL cases show additional chromosomal changes during the course of the disease [l]. Additional chromosomal changes in the majority of cases appeared shortly before or during the acute phase and could be used as indicators of the acute crisis. On the other hand, the number of cases with additional chromosomal changes in the chronic phase is not small. Lawler [7] reported the incidence of additional chromosomal changes in the chronic phase as follows: trisomy 8 (3%), trisomy 8 accompanied by a double Phl (S%), a double Phl (3%), stahle rearrangements (8%), and loss of Y (8%). Strictly speaking, the definition of the chronic phase of the disease is not universally the same. However, it is certain that some cases exist with additional chromosomal changes present in the chronic phase for several years under low dose maintenance therapy. This fact prompted us to differentiate the additional
Chromosomes in the Chronic Phase of CGL Table 3
307
Chromosomal findings in 15 cases with additional changes shortly before or in the acute phase of CGL
Case’
Age
Sex
Phase acute acute acute acute acute 3.7 months before acute crisis acute acute acute 0.6 months before acute crisis acute acute acute acute acute
7. 8. 9. 10. 11. 12.
U.K. K.K. SM. M.W. U.M. H.K.
8 33 22 46 25 34
M M M M F M
13. 14. 15. 16.
C.H. I.Y. R.E. M.W.
57 49 24 6
F M F F
17. 16. 19. 20. 21.
M.U. M.K. SK. H.K. M.H.
56 35 46 24 19
M F F F F
Predominant abnormal keryotype 52, 52, 50, 49, 49, 48,
XY, +8, +14, +15, +19, +21, +Ph’, Ph’ XY, +l, $8, +lO, +ll, +21, +Ph’, Ph’ XYY, +8, +19, +Ph’, Ph’ XY, +6, +8, +Ph’, lp+, 14q+, Ph’ XX, f17, +19, +Ph’, Ph’ XY, +21, +Ph’, t(3p;4p+), Ph’
47, 47, 47, 46,
XX, XY, XX, XX,
+8, 16q+, i(l7q), Phi +Ph’, 4p+, Ph’ +15, Phi t(5q-;21q+), Ph’
46, XY, r(ll), Phl 46, XX, t(2q+;4q-), Ph’ 46, XX, t(17p+;18q-), Ph’ 44, X, -?X, -3, lq+, Ph’ Multiple changes
4n cases 7,8,10,11,12,13,14,17 end 18, the kayotype of 46,XX or XY, Ph’ is confirmed in the chronic phase.
chromosomal changes in the chronic phase from those in the acute phase. Thus, we analyzed 21 cases with additional chromosomal changes in our series and a3 cases with abnormalities found by banding techniques in the literature [5, a- 141, in an attempt to elucidate this differentiation. Trisomy a It is well-known that the additional chromosomal changes in CGL cases are commonly trisomy a and a double Ph’. Some investigators [a,q are inclined to think that the appearance of trisomy a is a poor prognostic sign. One the other hand, Hayata et al. [lo] suggested that the prognosis is probably more favorable for cases with a simple type trisomy a than for cases with other numerical changes in the karyotype. Some controversy still exists as to whether or not trisomy a is associated with progression of the disease. In this series, we found that the 3 cases (cases 1, 2, and 3) with trisomy a in the chronic phase had a simple type trisomy a, whereas the 5 cases (cases 7, a, 9, 10, and 13) with trisomy a in the acute phase had a complex type of trisomy a.
Table 4
The phases studied and the state of trisomy a in 31 cases of CGL in the literature
Phase studied
No. of cases with simple type trtsomy 8
No. of cases with complex type trisomy 8
Total
Chronic Acute
2 1
3” 25
5 26
Total
3
28
31
Two of 3 casesshow tetmsomy 8 end trisomy 8 with double Ph’, respectively.
N. Sadamori
308 Table 5
The phases studied and the state of the double Ph’ in of CGL in the literature
et al.
24 cases
Phase studied
No. of cases with simple type double Ph’
No. of cases with complex type double Phl
Total
Chronic Acute
3 2
2a 17
5 19
Total
5
19
24
=One of 2 cases shows trisomy 8 with double Phi.
The 31 cases with trisomy 8 in the literature were analyzed (Table 4). The probability of x2= 6.27 with one degree of freedom was 2.5%, which is conclusive evidence that the phase of the disease is not independent of the type of trisomy 8 (simple vs. complex). Double Ph’ Whang-Peng et al. [15] concluded that a double Phl as an initial aneuploidy does not necessarily represent or lead to an acute crisis. However, a double Phl is an additional chromosomal change commonly observed in the acute phase. Nevertheless, there is still some controversy as to whether or not a double Ph’ constitutes an indicator of the progression of the disease. In this series, case 4 had a simple double Phl in the chronic phase, whereas the 7 cases (cases 7,8,9,10,11,12, and 14) with a complex double Phl were shown to have this anomaly shortly before or in the acute phase of CGL. The 24 cases with a double Phi in the literature were analyzed (Table 5). The probability of x2 = 5.87 with one degree of freedom was 2.5%, which is conclusive evidence that the phase of CGL is not independent of the type of double Ph’ (simple vs. complex). Translocations In case 5 all metaphases with the translocation between chromosomes #4 and #9 were accompanied by a Phl. The karyotypes of lymphocytes stimulated with PHA were normal without any translocation or Phi. These findings suggest that the translocation between chromosomes #4 and #9 occurred in a CGL cell line with a Phi. Case 5 showed a new karyotype with monosomy 7 in the acute crisis. This fact may indicate that metaphases with a translocation in addition to that of the Ph’ in the chronic phase might be inclined to develop further karyotypic evolution relative to the acute crisis. However, more cases with translocations in the chronic phase will have to be studied before reaching any final conclusion. Loss of the Y Sakurai and Sandberg [16] advanced the hypothesis that cells which lack a Y are generally resistant to the development of further chromosomal abnormalities. As shown in Table 6, the finding that 7 of 10 cases with a missing Y in the literature [9, 14, IS-Xl] did not show any further chromosomal abnormalities in the cell lines lacking the Y but with a Phl after developing an acute crisis, supports the hypothesis of Sakurai and Sandberg [16]. In general, in the majority of cases which lack a Y, the Y is lost during the chronic phase and, thus, the loss of the Y is not an adequate predictive or diagnostic indicator of the acute crisis.
Chromosomes
in the Chronic
Table 6
Phase
Karyotypes in the chronic and acute phases lackine the Y uublished in the literature
27 24 27 43 59 64 61 43
45, x, -Y, Ph’ 45,X, -Y, Ph’ 45,X,-Y, Ph’ 45,X, -Y, Ph’ 45,X, -Y, Ph’ 45,x, -Y, Ph’ 45,X,-Y, Ph’ 45,x, -Y, Ph’ 146, XY, Ph’ 45, X,-Y, Ph’ 45, X,-Y, Ph’
50 48
in 10 cases with
Karyotype in the acute phase
Karyotype in the chronic phase
Age
309
of CGL
59-64 and 65, XY, Ph’ 45,x,-Y, Ph’ additional abnormalities 45,X,-Y, Ph’ 45,x, -Y, Ph’ 45,x,-Y, Ph’ 45,X, -Y, Ph’ 45,X, -Y, Ph’l46, XY, Ph’ 145-47, XY, *a, Ph’ 45-46, X,-Y, +m, 2lq+, Ph’ 43, X, -Y, -16,-17, Ph’145, X, -Y, Ph’
CGL
References Motomura 8 Ogi [ 171 Furusawa et al. [18] Lawler et al. [19] Lawler et al. [19] Lawler et al. [19] Whittaker et al. [20] Whittaker et al. [20] Sakurai 8 Sandberg [16] Sharp et al. (91 Sonta & Sandberg
[14]
The authors wish to express their thanks to Drs. Shotaro Neriishi and Takeo Honda and Mr. Hitoshi Tokai of the Radiation Effects Research Foundation, Nagasaki, for their help. The skillful technical assistance of Mrs. Sayoko Mori and Mrs. Tamiko Sakaguchi is greatly appreciated.
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