Hum. Genet. 37, 141--148 (1977) © by Springer-Verlag 1977
Variation in the Philadelphia Chromosome Jessie L. Watt 1, Peter J. Hamilton 2, and Brenda M. Page 1 Departments of Genetics ~and Haematology2, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, United Kingdom
Summary. Cytogenetic study of 17 cases of chronic myeloid leukaemia has shown that the Philadelphia chromosome is a variable entity, differing in size and banding pattern between individuals.
Introduction The Philadelphia chromosome (Ph'), originally described by Nowell and Hungerford (1960), is recognised as a consistent but by no means universal marker present in the great majority of patients with chronic myeloid leukaemia (Lawler et al., 1976). Rowley (1973) demonstrated translocation of the deleted material from chromosome 22 to chromosome 9; other authors have subsequently shown alternative, or even no, recipient chromosomes (Mitelman and Levan, 1976). The genesis of the Philadelphia chromosome itself has been considered recently and the breakpoint pinpointed to the 22ql 1/q12 band interphase (Whang-Peng et al., 1974; Pravtcheya and Manolov, 1975). In investigating 17 patients with chronic myeloid leukaemia we have found four patients with unusual variants of the Philadelphia chromosome, and on G-banding the breakpoint has proved to be within band 22q12 or at the 22q12/13 band interphase.
Material and Methods Patients. Bone marrow and blood were obtained from 17 patients with chronic myeloid
leukaemia whose clinical and haematological data are summarised in Table 1. Cell Culture and Chromosome Preparation. RPMI 1640 (Gibco-Biocult Labs. Ltd.) was used for all cultures, supplemented by 20% calf serum or human plasma. Cells were arrested at metaphase by addition of colcemid (4 ~tg/ml). Cell harvesting was standard with hypotonic pretreatment in 0.075 M KC1 followed by several fixes in 3 parts methanol : 1 part acetic acid. The final cell suspension was dropped onto cold wet slides and air dried. Aceto-orcein staining was used, with additional C-banding (Chandley and Fletcher, 1973) and G-banding (Stephen, 1977) in most cases.
Table 1. Hb (g/dl)
White cells × 109/1
Platelets 1012/1
LAP a
total
eosinophils
F
10.4
212
2.1
266
Low
78
M
10.8
343
850
Increased
3
51
F
11.8
141
3.0
15.0
480
Low
4
55
M
10.0
325
33.0
12.0
1209
Low
5
31
M
8.7
448
5.3
172
Low
6
63
F
14.4
43
0.4
4.3
636
Normal
7
61
F
12.0
105
3.2
3.2
96
Low
8
47
M
8.1
210
8.4
16.8
344
Low
9
52
M
7.2
450
4.5
45.0
1800
Low
10
33
F
9.8
107
8.5
3.2
204
Low
11
70
F
12.3
97
--
4.8
450
Low
12
66
F
5.7
67
1.3
4.5
55
Low
13
78
F
7.8
250
10.0
25.0
272
Low
14
22
M
11.2
274
2.7
21.9
655
Low
15
40
F
13.7
225
2.5
4.5
366
Low
16
52
F
10.2
337
3.3
19.8
216
Low
17
75
M
14.6
16
--
0.8
288
Low
Case No.
Age (years)
Sex
1
40
2
a LAP = Leucocyte Alkaline Phosphatase
basophils 4.2
At presentation Splenic enlargementb
Treatment
Survival years
Clinical comment
Marked
Busulphan
7
Alive
Well: No clinical signs but persistent basophilia
Nil
Busulphan
2
Dead
Sudden cardiac death. Previously well controlled but X-ray myelosclerosis
Mild
Busulphan Splenectomy Busulphan Dibromannitol
3
Alive
Well: Persistent eosinophilia and basophilia--occasional bone pain
Marked
Busulphan
292
Alive
Complete clinical and haematological remission
Marked
Busulphan Splenectomy Busulphan Dibromannitol
2i/2
Alive
Subjectively well but some liver and lymph node enlargement and occasional myeloblast seen recently
Nil
Busulphan Vincristine Prednisolone
11/2
Dead
No clinical or haematological remission; terminal lymphoblastic crisis
Mild
Busulphan
2
Alive
Well: Recent increasing resistance to therapy with haematological relapse
Moderate
Busulphan Splenectomy
1
Dead
Initial excellent response; acute myeloblastic transformation
Marked
Busulphan 2 Splenic irradiation, 32p Busulphan Dibromannitol
Alive
Recurrent vascular problems with poor disease control and persistent hepato-splenomegaly
Moderate
Busulphan Splenic irradiation
3
Dead
Well for 1 year but prominent basophilia, myelofibrosis and terminal marrow aplasia
Nil
Busulphan
1
Alive
Well: Persistent thrombocytosis but normal white count
Marked
Busulphan Splenic irradiation
11/2
Alive
Well: Haematological remission but persistent mild splenomegaly
Moderate
Busulphan
19z
Alive
Well: Complete haematological remission, X-ray myelosclerosis
Marked
Busulphan Splenectomy Busulphan
11/2
Alive
Clinically well but recent haematological relapse
Nil
Busulphan Dibromannitol
6
Alive
Clinically well with good haematological control
Moderate
Busulphan
492
Alive
Some intermittent sepsis with recent haematological relapse
Mild
Nil
0
Alive
Recently presented with prostatism
b Splenic enlargement - - Mild = easily palpable; Moderate = to the umbilicus; Marked = into the iliac fossa
144
J.L. Watt et al.
Table 2 Case number
Number of samples
Total number of cells analysed (bone marrow + blood - - PHA)
Average % of cells with Philadelphia chromosome
Comments on karyotype
1
3
100
50%
."Typical Ph' chromosome present in all 3 samples. The 3rd sample revealed an isochromosome long arm of 17 in addition to the Ph' chromosome
2
1
60
60%
Typical Ph' chromosome present. Three cells had 2 Ph' chromosomes that were identical in size
3
2
70
55%
Atypical form of Ph' chromosome present in both samples
4
2
70
75%
a Typical Ph' chromosome present in both samples
5
2
50
50%
Typical Ph' chromosome present in both samples
6
3
100
63%
Atypical form of Ph' chromosome present in all 3 samples
7
2
70
48%
a Typical Ph' chromosome present in both samples
8
3
155
82%
Typical Ph' chromosome present in the 1st 2 samples. The 3rd sample revealed an additional Ph' chromosome, of the atypical variety and 2 extra C-group chromosomes
9
2
75
89%
Atypical form of Ph' chromosome present in both samples
10
2
100
91%
Typical Ph' chromosome present in both samples
11
1
50
0%
Ph'-negative
12
1
60
0%
Ph'-negative
13
1
50
18%
Typical Ph' chromosome present
14
2
120
60%
Typical Ph' chromosome present in both samples
15
1
50
52%
Typical Ph' chromosome present
16
3
150
34%
Typical Ph' chromosome present in all 3 samples
17
1
150
100%
"Typical Ph' chromosome present. Also Y chromosome absent from all bone marrow metaphases
a Chromosome 9 was unambiguously identified as the recipient chromosome involved in the translocation (9q+22q-)
Variation in the Philadelphia Chromosome
145
Results
The cytogenetic results are summarised in Table 2. Only two of the 17 patients in this study are Ph'-negative. In the other patients the most common form of the Ph' chromosome had the typical appearance of a tiny metacentric element. This is shown most effectively with the conventional orcein staining (see Fig. 1). Cbanding depicts the chromosome as being monocentric, while G-banding locates the breakpoint to the 22ql 1/q12 band interphase as reported. Chromosome 9 was identified as the recipient chromosome in five cases where a clear decision could be made (see Fig. 2). However, four other patients had atypical forms of the Ph' chromosome, where the breakpoint proved to be within band 22q12 or at the 22q12/13 band interphase. No recipient chromosome was identified in these cases. A fragile region was observed in one chromosome 22 in a single cell from Case 3 at a position corresponding to the breakpoint leading to atypical Philadelphia chromosome formation (see Fig. 3).
Fig. 1. Partial karyotypes of G-group chromosomes showing typical Ph' chromosomes by Orcein staining (a), C-banding (b) and G-banding (c and d), and typical Ph' chromosomes by G-banding (e, f, and g)
146
J.L. Watt et al.
Fig. 2. Karyotype from Case 1 showing the most usual form of translocation in Ph' chromosome formation: 9q+22qFig. 3. Partial metaphase from Case 3 showing fragile region on chromosome 22 at the 22q12/13 band interphase
Variation in the Philadelphia Chromosome
147
Discussion It is not difficult to explain why variation in the Ph' chromosome has previously escaped detailed banding analysis: A very small part of chromosome 22 is involved and one requires long thin chromosomes that are well banded to distinguish differences in the amount of deleted material. Such preparations are notoriously difficult to obtain from h u m a n bone marrow, and this difficulty is often compounded by patients having been treated with anti-mitotic d r u g s prior to bone marrow aspiration. This study provides convincing evidence that variants of the Philadelphia chromosome exist. Previous reports of differences in size have been ascribed to technical handling procedures and to the stage in mitosis (Makino, 1975). Makino also suggested that the Philadelphia chromosome was larger in males than in females. In this study, at least two samples were studied from each of the patients with the atypical Ph' chromosome, with consistent findings, and two of these patients were female. Case 8 is of particular interest since he presented in 1974 with a typical Ph' chromosome, and in 1975 at blast transformation, he acquired a second Ph' chromosome and two additional C-group chromosomes (49,XY). The second Ph' chromosome was of the longer type and distinctly dissimilar to the first. He died soon afterwards. In a recent review by Mitelman and Levan (1976) 16 publications are discussed, detailing 33 cases of chronic myeloid leukaemia where a second Ph' chromosome has been identified. In all 33 cases the two Ph' chromosomes seem to be identical in size and banding pattern. However, a report by Rowley (1973) mentions a case where the second Ph' chromosome is different from the first. Such cases where the two Ph' chromosomes are not identical would shed some doubt on the assumption that the second Ph' chromosome arises through nondisjunction of the first (Sharp et al., 1975). Of the other three patients with the atypical chromosome 22, one has died in an unusual blast cell transformation, and the remaining two are difficult to control with adequate drug therapy. It must be noted that these three patients were shown to have atypical Ph' chromosomes at the time of initial presentation so that it is not a result of treatment. Fitzgerald (1976) recently reported a family with a balanced translocation resulting in a Ph'-like chromosome element with the breakpoint at the 22q12/13 band interphase and one chromosome 11 as a recipient. This family showed no evidence of leukaemia or any myeloproliferative disorder. Comparison between this familial marker and the Ph' chromosome suggested to the authors that band 22q12 is concerned with the uncontrolled cell proliferation seen in chronic myeloid leukaemia. The present findings must cast some doubt on this inference. Neither Fitzgerald's familial marker nor the typical Philadelphia chromosome have been clearly shown to be reciprocal translocations although it is a general belief that most translocations are reciprocal according to Muller's (1940) telomere hypothesis. It is probable that the transfer of an undetectable amount of material from the recipient chromosome to chromosome 22 occurs. The possibility cannot therefore be excluded that the presence of part or all of band 22q12 in our patients with atypical Ph' chromosomes might represent a 'mimic' band
148
J.L. Watt et al.
from a n o t h e r c h r o m o s o m e p a r t i c i p a t i n g in reciprocal translocation. This reasoning can be further applied to the situation in Ph'-negative chronic myeloid leukaemia. H o w e v e r the fragile region depicted in Figure 3 does n o t f a v o u r this possibility.
Acknowledgements. We thank Dr. A. Dawson for her interest and for permission to report clinical details of the patients in this study. This work was supported by SRC Research Studentship Reference Number B/74/208.
References
Chandley, A. C., Fletcher, J. M.: Centromeric staining and meiosis in man. Humangenetik 18, 247--252 (1973) Fitzgerald, P. H.: Evidence that chromosome band 22q12 is concerned with cell proliferation in CML. Hum. Genet. 33, 269--274 (1976) Lawler, S. D., O'Malley, F., Lobb, D. S.: Chromosome banding studies in Philadelphia chromosome positive myeloid leukaemia. Scand. J. Haematol. 17, 17--28 (1976) Makmo, S.: Human chromosomes. Tokyo: Igaku shoin 1975 Muller, H. J.: An analysis of the process of structural change in the chromosomes of Drosophila. J. Genet. 40, 1--66 (1940) Mitelman, F., Levan, G.: Clustering of aberrations to specific chromosomes in human neoplasms. II. A survey of 287 neoplasms. Hereditas 82, 167--174 (1976) Nowell, P. C., Hungerford, D. A.: A minute chromosomes in human chronic granulocytic leukaemia. Science 132, 1497 (1960) Pravtcheva, D., Manolov, G.: Genesis of the Philadelphia chromosome. Possible points of breakage in chromosome number 22. Hereditas 79, 301--303 (1975) Rowley, J. D.: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and giemsa staining. Nature 243, 290--293 (1973) Sharp, J. C., Potter, A. M., Guyer, R. J.: Karyotypic abnormalities in transformed chronic granulocytic leukaemia. Br. J. Haemat. 29, 587--591 (1975) Stephen, G. S.: Mammalian chromosomes G-banded in four minutes. Genetica (1977, in press) Whang-Peng, J., Lee, E. C., Knutsen, T. A.: Genesis of the Ph' chromosome. J. Natl. Cancer Inst. 52, 1035--1036 (1974)
Received January 3, 1977
Note Added in ProoL Further analysis on a sample from Case 1 prior to her recent death revealed
that 100% of the bone marrow metaphases had in addition to the described t(9;22), an additional 8, a missing 16 and 17 and a t dic (16;17)(101lpl 1). On reflection it would seem probable that Fig. 2 could be interpreted as an early indication of this clone.
Variation in the Philadelphia Chromosome Jessie L. Watt 1, Peter J. Hamilton 2, and Brenda M. Page 1 Departments of Genetics ~and Haematology2, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, United Kingdom
Summary. Cytogenetic study of 17 cases of chronic myeloid leukaemia has shown that the Philadelphia chromosome is a variable entity, differing in size and banding pattern between individuals.
Introduction The Philadelphia chromosome (Ph'), originally described by Nowell and Hungerford (1960), is recognised as a consistent but by no means universal marker present in the great majority of patients with chronic myeloid leukaemia (Lawler et al., 1976). Rowley (1973) demonstrated translocation of the deleted material from chromosome 22 to chromosome 9; other authors have subsequently shown alternative, or even no, recipient chromosomes (Mitelman and Levan, 1976). The genesis of the Philadelphia chromosome itself has been considered recently and the breakpoint pinpointed to the 22ql 1/q12 band interphase (Whang-Peng et al., 1974; Pravtcheya and Manolov, 1975). In investigating 17 patients with chronic myeloid leukaemia we have found four patients with unusual variants of the Philadelphia chromosome, and on G-banding the breakpoint has proved to be within band 22q12 or at the 22q12/13 band interphase.
Material and Methods Patients. Bone marrow and blood were obtained from 17 patients with chronic myeloid
leukaemia whose clinical and haematological data are summarised in Table 1. Cell Culture and Chromosome Preparation. RPMI 1640 (Gibco-Biocult Labs. Ltd.) was used for all cultures, supplemented by 20% calf serum or human plasma. Cells were arrested at metaphase by addition of colcemid (4 ~tg/ml). Cell harvesting was standard with hypotonic pretreatment in 0.075 M KC1 followed by several fixes in 3 parts methanol : 1 part acetic acid. The final cell suspension was dropped onto cold wet slides and air dried. Aceto-orcein staining was used, with additional C-banding (Chandley and Fletcher, 1973) and G-banding (Stephen, 1977) in most cases.
Table 1. Hb (g/dl)
White cells × 109/1
Platelets 1012/1
LAP a
total
eosinophils
F
10.4
212
2.1
266
Low
78
M
10.8
343
850
Increased
3
51
F
11.8
141
3.0
15.0
480
Low
4
55
M
10.0
325
33.0
12.0
1209
Low
5
31
M
8.7
448
5.3
172
Low
6
63
F
14.4
43
0.4
4.3
636
Normal
7
61
F
12.0
105
3.2
3.2
96
Low
8
47
M
8.1
210
8.4
16.8
344
Low
9
52
M
7.2
450
4.5
45.0
1800
Low
10
33
F
9.8
107
8.5
3.2
204
Low
11
70
F
12.3
97
--
4.8
450
Low
12
66
F
5.7
67
1.3
4.5
55
Low
13
78
F
7.8
250
10.0
25.0
272
Low
14
22
M
11.2
274
2.7
21.9
655
Low
15
40
F
13.7
225
2.5
4.5
366
Low
16
52
F
10.2
337
3.3
19.8
216
Low
17
75
M
14.6
16
--
0.8
288
Low
Case No.
Age (years)
Sex
1
40
2
a LAP = Leucocyte Alkaline Phosphatase
basophils 4.2
At presentation Splenic enlargementb
Treatment
Survival years
Clinical comment
Marked
Busulphan
7
Alive
Well: No clinical signs but persistent basophilia
Nil
Busulphan
2
Dead
Sudden cardiac death. Previously well controlled but X-ray myelosclerosis
Mild
Busulphan Splenectomy Busulphan Dibromannitol
3
Alive
Well: Persistent eosinophilia and basophilia--occasional bone pain
Marked
Busulphan
292
Alive
Complete clinical and haematological remission
Marked
Busulphan Splenectomy Busulphan Dibromannitol
2i/2
Alive
Subjectively well but some liver and lymph node enlargement and occasional myeloblast seen recently
Nil
Busulphan Vincristine Prednisolone
11/2
Dead
No clinical or haematological remission; terminal lymphoblastic crisis
Mild
Busulphan
2
Alive
Well: Recent increasing resistance to therapy with haematological relapse
Moderate
Busulphan Splenectomy
1
Dead
Initial excellent response; acute myeloblastic transformation
Marked
Busulphan 2 Splenic irradiation, 32p Busulphan Dibromannitol
Alive
Recurrent vascular problems with poor disease control and persistent hepato-splenomegaly
Moderate
Busulphan Splenic irradiation
3
Dead
Well for 1 year but prominent basophilia, myelofibrosis and terminal marrow aplasia
Nil
Busulphan
1
Alive
Well: Persistent thrombocytosis but normal white count
Marked
Busulphan Splenic irradiation
11/2
Alive
Well: Haematological remission but persistent mild splenomegaly
Moderate
Busulphan
19z
Alive
Well: Complete haematological remission, X-ray myelosclerosis
Marked
Busulphan Splenectomy Busulphan
11/2
Alive
Clinically well but recent haematological relapse
Nil
Busulphan Dibromannitol
6
Alive
Clinically well with good haematological control
Moderate
Busulphan
492
Alive
Some intermittent sepsis with recent haematological relapse
Mild
Nil
0
Alive
Recently presented with prostatism
b Splenic enlargement - - Mild = easily palpable; Moderate = to the umbilicus; Marked = into the iliac fossa
144
J.L. Watt et al.
Table 2 Case number
Number of samples
Total number of cells analysed (bone marrow + blood - - PHA)
Average % of cells with Philadelphia chromosome
Comments on karyotype
1
3
100
50%
."Typical Ph' chromosome present in all 3 samples. The 3rd sample revealed an isochromosome long arm of 17 in addition to the Ph' chromosome
2
1
60
60%
Typical Ph' chromosome present. Three cells had 2 Ph' chromosomes that were identical in size
3
2
70
55%
Atypical form of Ph' chromosome present in both samples
4
2
70
75%
a Typical Ph' chromosome present in both samples
5
2
50
50%
Typical Ph' chromosome present in both samples
6
3
100
63%
Atypical form of Ph' chromosome present in all 3 samples
7
2
70
48%
a Typical Ph' chromosome present in both samples
8
3
155
82%
Typical Ph' chromosome present in the 1st 2 samples. The 3rd sample revealed an additional Ph' chromosome, of the atypical variety and 2 extra C-group chromosomes
9
2
75
89%
Atypical form of Ph' chromosome present in both samples
10
2
100
91%
Typical Ph' chromosome present in both samples
11
1
50
0%
Ph'-negative
12
1
60
0%
Ph'-negative
13
1
50
18%
Typical Ph' chromosome present
14
2
120
60%
Typical Ph' chromosome present in both samples
15
1
50
52%
Typical Ph' chromosome present
16
3
150
34%
Typical Ph' chromosome present in all 3 samples
17
1
150
100%
"Typical Ph' chromosome present. Also Y chromosome absent from all bone marrow metaphases
a Chromosome 9 was unambiguously identified as the recipient chromosome involved in the translocation (9q+22q-)
Variation in the Philadelphia Chromosome
145
Results
The cytogenetic results are summarised in Table 2. Only two of the 17 patients in this study are Ph'-negative. In the other patients the most common form of the Ph' chromosome had the typical appearance of a tiny metacentric element. This is shown most effectively with the conventional orcein staining (see Fig. 1). Cbanding depicts the chromosome as being monocentric, while G-banding locates the breakpoint to the 22ql 1/q12 band interphase as reported. Chromosome 9 was identified as the recipient chromosome in five cases where a clear decision could be made (see Fig. 2). However, four other patients had atypical forms of the Ph' chromosome, where the breakpoint proved to be within band 22q12 or at the 22q12/13 band interphase. No recipient chromosome was identified in these cases. A fragile region was observed in one chromosome 22 in a single cell from Case 3 at a position corresponding to the breakpoint leading to atypical Philadelphia chromosome formation (see Fig. 3).
Fig. 1. Partial karyotypes of G-group chromosomes showing typical Ph' chromosomes by Orcein staining (a), C-banding (b) and G-banding (c and d), and typical Ph' chromosomes by G-banding (e, f, and g)
146
J.L. Watt et al.
Fig. 2. Karyotype from Case 1 showing the most usual form of translocation in Ph' chromosome formation: 9q+22qFig. 3. Partial metaphase from Case 3 showing fragile region on chromosome 22 at the 22q12/13 band interphase
Variation in the Philadelphia Chromosome
147
Discussion It is not difficult to explain why variation in the Ph' chromosome has previously escaped detailed banding analysis: A very small part of chromosome 22 is involved and one requires long thin chromosomes that are well banded to distinguish differences in the amount of deleted material. Such preparations are notoriously difficult to obtain from h u m a n bone marrow, and this difficulty is often compounded by patients having been treated with anti-mitotic d r u g s prior to bone marrow aspiration. This study provides convincing evidence that variants of the Philadelphia chromosome exist. Previous reports of differences in size have been ascribed to technical handling procedures and to the stage in mitosis (Makino, 1975). Makino also suggested that the Philadelphia chromosome was larger in males than in females. In this study, at least two samples were studied from each of the patients with the atypical Ph' chromosome, with consistent findings, and two of these patients were female. Case 8 is of particular interest since he presented in 1974 with a typical Ph' chromosome, and in 1975 at blast transformation, he acquired a second Ph' chromosome and two additional C-group chromosomes (49,XY). The second Ph' chromosome was of the longer type and distinctly dissimilar to the first. He died soon afterwards. In a recent review by Mitelman and Levan (1976) 16 publications are discussed, detailing 33 cases of chronic myeloid leukaemia where a second Ph' chromosome has been identified. In all 33 cases the two Ph' chromosomes seem to be identical in size and banding pattern. However, a report by Rowley (1973) mentions a case where the second Ph' chromosome is different from the first. Such cases where the two Ph' chromosomes are not identical would shed some doubt on the assumption that the second Ph' chromosome arises through nondisjunction of the first (Sharp et al., 1975). Of the other three patients with the atypical chromosome 22, one has died in an unusual blast cell transformation, and the remaining two are difficult to control with adequate drug therapy. It must be noted that these three patients were shown to have atypical Ph' chromosomes at the time of initial presentation so that it is not a result of treatment. Fitzgerald (1976) recently reported a family with a balanced translocation resulting in a Ph'-like chromosome element with the breakpoint at the 22q12/13 band interphase and one chromosome 11 as a recipient. This family showed no evidence of leukaemia or any myeloproliferative disorder. Comparison between this familial marker and the Ph' chromosome suggested to the authors that band 22q12 is concerned with the uncontrolled cell proliferation seen in chronic myeloid leukaemia. The present findings must cast some doubt on this inference. Neither Fitzgerald's familial marker nor the typical Philadelphia chromosome have been clearly shown to be reciprocal translocations although it is a general belief that most translocations are reciprocal according to Muller's (1940) telomere hypothesis. It is probable that the transfer of an undetectable amount of material from the recipient chromosome to chromosome 22 occurs. The possibility cannot therefore be excluded that the presence of part or all of band 22q12 in our patients with atypical Ph' chromosomes might represent a 'mimic' band
148
J.L. Watt et al.
from a n o t h e r c h r o m o s o m e p a r t i c i p a t i n g in reciprocal translocation. This reasoning can be further applied to the situation in Ph'-negative chronic myeloid leukaemia. H o w e v e r the fragile region depicted in Figure 3 does n o t f a v o u r this possibility.
Acknowledgements. We thank Dr. A. Dawson for her interest and for permission to report clinical details of the patients in this study. This work was supported by SRC Research Studentship Reference Number B/74/208.
References
Chandley, A. C., Fletcher, J. M.: Centromeric staining and meiosis in man. Humangenetik 18, 247--252 (1973) Fitzgerald, P. H.: Evidence that chromosome band 22q12 is concerned with cell proliferation in CML. Hum. Genet. 33, 269--274 (1976) Lawler, S. D., O'Malley, F., Lobb, D. S.: Chromosome banding studies in Philadelphia chromosome positive myeloid leukaemia. Scand. J. Haematol. 17, 17--28 (1976) Makmo, S.: Human chromosomes. Tokyo: Igaku shoin 1975 Muller, H. J.: An analysis of the process of structural change in the chromosomes of Drosophila. J. Genet. 40, 1--66 (1940) Mitelman, F., Levan, G.: Clustering of aberrations to specific chromosomes in human neoplasms. II. A survey of 287 neoplasms. Hereditas 82, 167--174 (1976) Nowell, P. C., Hungerford, D. A.: A minute chromosomes in human chronic granulocytic leukaemia. Science 132, 1497 (1960) Pravtcheva, D., Manolov, G.: Genesis of the Philadelphia chromosome. Possible points of breakage in chromosome number 22. Hereditas 79, 301--303 (1975) Rowley, J. D.: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and giemsa staining. Nature 243, 290--293 (1973) Sharp, J. C., Potter, A. M., Guyer, R. J.: Karyotypic abnormalities in transformed chronic granulocytic leukaemia. Br. J. Haemat. 29, 587--591 (1975) Stephen, G. S.: Mammalian chromosomes G-banded in four minutes. Genetica (1977, in press) Whang-Peng, J., Lee, E. C., Knutsen, T. A.: Genesis of the Ph' chromosome. J. Natl. Cancer Inst. 52, 1035--1036 (1974)
Received January 3, 1977
Note Added in ProoL Further analysis on a sample from Case 1 prior to her recent death revealed
that 100% of the bone marrow metaphases had in addition to the described t(9;22), an additional 8, a missing 16 and 17 and a t dic (16;17)(101lpl 1). On reflection it would seem probable that Fig. 2 could be interpreted as an early indication of this clone.
