Hum. Genet. 32, 229~232 (1976) © by Springer-Verlag 1976
A New Translocation in Chronic Myelogenous Leukemia D. P r a v t e h e v a 1, P. A n d r e e v a 2, a n d R. T s a n e v a ~ 1 Centre of Oncology, Sofia, ~ Institute of Hematology and Blood Transfusion, Sofia Received January 7, 1976
Summary. A reciprocal transloeation involving chromosomes Nos. 3 and 22 has been found in a patient with seemingly Ph'-negative chronic myelogenous leukemia (CML). G-band analysis revealed, that deletion in No. 22 occurred at the same point, as in the typical eases of the disease. I t was concluded, that breakage in No. 22 at a specific site with spatial disjunction of the resulting segments might be the crucial eytogenetic event in the genesis of CML, the Philadelphia chromosome not being an obligatory result of the rearrangement. I n 1973 R o w l e y described an a d d i t i o n a l s e g m e n t on t h e long a r m of chromosome No. 9 in p a t i e n t s w i t h P h ' - p o s i t i v e CML a n d suggested t h a t i t was identical to t h e missing p o r t i o n o f t h e d e l e t e d No. 22 (Ph') chromosome. I t t u r n e d o u t later, t h a t No. 9 is t h e m o s t f r e q u e n t l y f o u n d b u t n o t u n i q u e r e c i p i e n t of t h e m a t e r i a l , d e t a c h e d from No. 22. Several eases h a v e been r e p o r t e d in which t h e t r a n s l o e a t i o n t o o k place onto o t h e r c h r o m o s o m e s : No. 2 ( H a y a t a et al., 1973), No. 11 (Mnldal et al., 1975), No. 13 ( H a y a t a et al., 1975), No. 19 ( G a h r t o n et al., 1974). Of special i n t e r e s t is an a p p a r e n t l y P h ' - n e g a t i v e , m i n u s 22 ease of chronic m y e l o i d leukemia, r e p o r t e d b y E n g e l et al. (1974) in which t h e b a n d i n g analysis r e v e a l e d a No. 17 c a r r y i n g t h e two segments of t h e missing No. 22 on b o t h s h o r t a n d long arms. One of t h e segments was t h e e q u i v a l e n t of t h e P h ' chromosome. H e r e we r e p o r t a case of u n u s u a l t r a n s l o e a t i o n in CML b e t w e e n chromosomes No. 3 a n d 22. I n t h e p r e s e n t w o r k Dr. D. P r a v t e h e v a is responsible for t h e chromosome s t u d y a n d Dr. P. A n d r e e v a a n d Dr. R. T s a n e v a for t h e clinical p a r t of t h e work.
Case History The patient, a 55-year-old female, was first diagnosed as having CML 2 years ago on the basis of leukoeytosis, myeloid immaturity, enlarged spleen and liver. Myelosan therapy was started and she remained in good general condition until April, 1975 when her condition rapidly deteriorated. On admission, prominent hepatosplenomegaly was found: the spleen extended to the symphysis and the liver was palpable 10 cm below the right costal margin. The Hb value was 5.9 g/100 ml, Er 2150000, Thr 680000. The white blood cell count ranged from 96,000 to 24,000 during therapy with 60--80~o of the cells being abnormal myeloblasts. About one third of the nucleated cells in the peripheral blood were atypical mononuclears, resembling both morphologically and eytoehemieally mieromegakaryoblasts and mieromegakaryocytes (Huhn et al., 1975). The bone marrow was hypocellular, with erythroblastie elements reduced in number. All naturation stages of the granulocytie system were present, with marked preponderance of myeloblasts and myelocytes. Of the bone marrow cells 20--30~o were atypical mononuelears, identical to those in the peripheral blood.
230
D. Pravtcheva et al.
It was concluded, that the patient developed an unusual variant of blastic phase in CML---a micromegakaryoblastic-micromegakaryoeytic crisis. No significant changes in her condition nor in the blood picture could be obtained despite intensive therapy. She was discharged in August, 1975. Material and Methods The cytogenetic investigation was carried out on direct bone marrow preparations, as well as on peripheral blood leukocyte cultuers with and without PHA. Colcemid in a final concentration of 0.6 mkg/ml was added for 2 h to the bone marrow sample and to the culture medium. Hypotonization in 0.075 M KC1 was followed by triple fixation in Carnoy's mixture (3:1 methanol acetic acid). The trypsin technic of Wang and Fedoroff (1972) was used to achieve G-banding. The chromosomes are described according to the criteria of the Paris Conference (1971). Results and Discussion I n the initial survey of c o n v e n t i o n a l l y stained bone marrow preparations, o n l y 3 n o r m a l G-group chromosomes a n d no P h ' chromosome could be f o u n d i n all metaphases examined. The t r y p s i n G - b a n d i n g revealed (Fig. 1), t h a t the missing chromosome was a No. 22. I n s t e a d , a new aerocentrie chromosome was present, a b o u t the size of D-group members. I t s short arm, centromeric region, a n d proximal p a r t of the long a r m closely resembled the corresponding parts on the n o r m a l 22, b u t a n a d d i t i o n a l segment, including a n intensive proximal dark b a n d , a n
Fig. 1. Karyotype of patient, showing the rearranged No. 3 and 22 chromosomes (arrows)
Transloeation in Chronic Myelogenous Leukemia
231
Fig. 2. Normal and rearranged No. 3 and normal and rearranged No. 22 chromosomes of 3 different metaphases
intermediate light band, and a smaller and less colored terminal dark band, was connected to the distal part of the long arm. I n all metaphases bearing this chromosome an altered No. 3 was also present with short arm lacking the bands p22--pter. There is good reason to believe, t h a t the segment attached on No. 22 is identical to the deleted part of No. 3. Several assumptions could be made concerning the mechanism of transloeation. The most simple variant would be realized by the breaking of No. 3 near band 3p22 and the attachment of the deleted material on the terminal p a r t of the long arm of No. 22. I-Iowever, the terminal negative band on the short arm of the deleted No. 3 is longer t h a n band p21 on the normal No. 3 and the distance from the eentromere to the proximal dark band of the additional segment on the rearranged No. 22 is shorter than the long arm of the normal 22 (Fig. 2). I t m a y be concluded therefore, t h a t both No. 3 and 22 have lost and gained material i.e., the transloeation is reeiproeal. I n such a ease, the breaking point in No. 22 is of special interest. I n the commonly found 9/22 translocation the deleted and translocated No. 22 portion includes the dark band 22q12 (it can be seen on chromosome 9 q + ) , hence the breakage occurs above this band. In our case band 22q12 is detected on the short arm of No. 3 at some distance from tile dark band 3p14. I t is better observed in suboptimally trypsinized chromosomes. A longer exposure to enzymatic action reduces its stainability and this, along with the adjacent position of the dark band 3p14 makes its discovery difficult. This finding discards the possibility of breakage in No. 22 below 22q12 (in which case this band would be expected to be seen on the rearranged No. 22) and points to band 2 2 q l l as the most probable site of breakage in No. 22. The break point in No. 3 might be located in 3p21 near 3p14. In a small percentage of the rearranged No. 22 chromosomes, a weak dark band near the eentromere can be seen. Because of its inconstant expression and more proximal position we consider it as being the morphologie manifestation of the point of junction (Manolov et al., 1971) of No. 22 and No. 3 parts, rather than being identical to 22q12. The above chromosome changes have been observed in 100% of the metaphases in bone marrow (28 cells) as well as in peripheral blood leukocyte cultures without PI-IA (20 cells). I n the PHA-stimulated cultures, 41~o (33 cells) shouted normal female karyotype, while the remaining 59~o (48 cells) had the abnormal k a r y o t y p e described a b o v e - - 4 6 , X X , t (3 ; 22) (p21 ; ql 1). 10 °/o of the metaphases bearing the rearranged No. 3 and 22 chromosomes formed a 48 chromosome clone, accounted for by trisomy of both No. 19 and 21. 1 Yo of 500 bone marrow metaphases examined at low power were tetraploid. The same percentage was found in the peripheral blood cultures. This finding,
232
D. Pravtcheva et al.
c o m p a r e d t o t h e high n u m b e r of m e g a k a r y o b l a s t s in t h e bone m a r r o w a n d perip h e r a l blood suggests t h a t these cells are diploid, which is consistent w i t h t h e i r m o r p h o l o g y - - m i e r o m e g a k a r y o b l a s t s . The G - b a n d analysis of 3 t e t r a p l o i d rectaphases r e v e a l e d in all of t h e m t h e a l t e r e d No. 3 a n d 22 chromosomes in duplicate. Our case of u n u s u a l t r a n s l o c a t i o n i n CML as well as those r e p o r t e d before, gives f u r t h e r s u p p o r t to t h e concept t h a t t h e a l t e r a t i o n of No. 22 p l a y s a m a j o r role in t h e genesis of CML. A c o m m o n f e a t u r e to all cases is b r e a k a g e in No. 22 a t a specific p o i n t a n d s p a t i a l d i s j u n c t i o n of t h e resulting s e g m e n t s - - a t y p i c a l P h ' chromosome n o t being a n o b l i g a t o r y resu]t of t h e r e a r r a n g e m e n t . I t is impossible to decide w h e t h e r t h e disease is a consequence of such a s p a t i a l d i s j u n c t i o n of chromosome parts, d i s t u r b i n g t h e i r c o o r d i n a t e d function, w h e t h e r i t is due to changes in t h e genetic a c t i v i t y , a t t r i b u t a b l e to t h e p o s i t i o n effect, or to a m o r p h o ]ogically u n d e t e c t a b l e loss of genetic m a t e r i a l .
Acknowledgements. We are greatful to Dr. G. Manolov for helping our work in every way possible.
References Engel, E., McGee, B. J., Flexner, J. M., Russell, M. T., Myers, B. J. : Philadelphia chromosome (Ph') translocation in an apparently Ph' negative, minus G22 case of chronic myeloid leukemia. New Engl. J. Med. 291, 154 (1974) Gahrton, G., Zech, L., Lindsten, J.: A new variant translocation (19q+, 22q--) in chronic myelocytic leukemia. Exp. Cell Res. 86, 214--2t6 (1974) Hayata, I., Kakati, S., Sandberg, A.A.: A new translocation related to the Philadelphia chromosome. Lancet 1973 II, 1385 Hayata, I., Kakati, S., Sandberg, A. A. : Another translocation related to the Philadelphia chromosome. Lancet 1975 I, 1300 Huhn, D., Ascher, S.: Mikrokaryoblastenschub bei chroniseher Myelose. Acta haemat. (Basel) 58, 183--190 (1975) Manolov, G., Manolova, Y., Levan, A. : Experiments with fluorescent chromosome staining in Burkitt tumors. I-Iereditas (Lund) 68, 235--244 (1971) Muldal, S., Mir, M.A., Freeman, C. B., Geary, C. G. : A new translocation associated with the Ph' chromosome and an acute course of chronic granulocytic leukemia. Brit. J. Cancer 31, 364--368 (1975) Nowell, P. C., Jensen, g , Gardner, F.: Two complex translocations in chronic granulocytic leukemia involving chromosomes 22, 9 and a third chromosome, ttumangenetik 80, 13--21 (1975) Paris Conference (1971): Standardization in human cytogenetics. Birth Defects: Original Article Series VIII, 7 (1972). The National Foundation, New York Rowley, J. D. : A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature 43, 290--293 (1973) Wang, H. C., Fedoroff, S. : Banding in human chromosomes treated with trypsin. Nature (Loud.) New Biol. 285, 52--54 (1972) Dr. D. Pravteheva Centre of Oneology Sofia 56, Bulgaria
Addendum. After completion of this manuscript we read the paper of Nowell et al. (1975), dealing with 2 cases of complex trans!ocations in CML between No. 22, 9, and a third chromosome. In one of the cases, the third chromosome involved was a No. 3.
A New Translocation in Chronic Myelogenous Leukemia D. P r a v t e h e v a 1, P. A n d r e e v a 2, a n d R. T s a n e v a ~ 1 Centre of Oncology, Sofia, ~ Institute of Hematology and Blood Transfusion, Sofia Received January 7, 1976
Summary. A reciprocal transloeation involving chromosomes Nos. 3 and 22 has been found in a patient with seemingly Ph'-negative chronic myelogenous leukemia (CML). G-band analysis revealed, that deletion in No. 22 occurred at the same point, as in the typical eases of the disease. I t was concluded, that breakage in No. 22 at a specific site with spatial disjunction of the resulting segments might be the crucial eytogenetic event in the genesis of CML, the Philadelphia chromosome not being an obligatory result of the rearrangement. I n 1973 R o w l e y described an a d d i t i o n a l s e g m e n t on t h e long a r m of chromosome No. 9 in p a t i e n t s w i t h P h ' - p o s i t i v e CML a n d suggested t h a t i t was identical to t h e missing p o r t i o n o f t h e d e l e t e d No. 22 (Ph') chromosome. I t t u r n e d o u t later, t h a t No. 9 is t h e m o s t f r e q u e n t l y f o u n d b u t n o t u n i q u e r e c i p i e n t of t h e m a t e r i a l , d e t a c h e d from No. 22. Several eases h a v e been r e p o r t e d in which t h e t r a n s l o e a t i o n t o o k place onto o t h e r c h r o m o s o m e s : No. 2 ( H a y a t a et al., 1973), No. 11 (Mnldal et al., 1975), No. 13 ( H a y a t a et al., 1975), No. 19 ( G a h r t o n et al., 1974). Of special i n t e r e s t is an a p p a r e n t l y P h ' - n e g a t i v e , m i n u s 22 ease of chronic m y e l o i d leukemia, r e p o r t e d b y E n g e l et al. (1974) in which t h e b a n d i n g analysis r e v e a l e d a No. 17 c a r r y i n g t h e two segments of t h e missing No. 22 on b o t h s h o r t a n d long arms. One of t h e segments was t h e e q u i v a l e n t of t h e P h ' chromosome. H e r e we r e p o r t a case of u n u s u a l t r a n s l o e a t i o n in CML b e t w e e n chromosomes No. 3 a n d 22. I n t h e p r e s e n t w o r k Dr. D. P r a v t e h e v a is responsible for t h e chromosome s t u d y a n d Dr. P. A n d r e e v a a n d Dr. R. T s a n e v a for t h e clinical p a r t of t h e work.
Case History The patient, a 55-year-old female, was first diagnosed as having CML 2 years ago on the basis of leukoeytosis, myeloid immaturity, enlarged spleen and liver. Myelosan therapy was started and she remained in good general condition until April, 1975 when her condition rapidly deteriorated. On admission, prominent hepatosplenomegaly was found: the spleen extended to the symphysis and the liver was palpable 10 cm below the right costal margin. The Hb value was 5.9 g/100 ml, Er 2150000, Thr 680000. The white blood cell count ranged from 96,000 to 24,000 during therapy with 60--80~o of the cells being abnormal myeloblasts. About one third of the nucleated cells in the peripheral blood were atypical mononuclears, resembling both morphologically and eytoehemieally mieromegakaryoblasts and mieromegakaryocytes (Huhn et al., 1975). The bone marrow was hypocellular, with erythroblastie elements reduced in number. All naturation stages of the granulocytie system were present, with marked preponderance of myeloblasts and myelocytes. Of the bone marrow cells 20--30~o were atypical mononuelears, identical to those in the peripheral blood.
230
D. Pravtcheva et al.
It was concluded, that the patient developed an unusual variant of blastic phase in CML---a micromegakaryoblastic-micromegakaryoeytic crisis. No significant changes in her condition nor in the blood picture could be obtained despite intensive therapy. She was discharged in August, 1975. Material and Methods The cytogenetic investigation was carried out on direct bone marrow preparations, as well as on peripheral blood leukocyte cultuers with and without PHA. Colcemid in a final concentration of 0.6 mkg/ml was added for 2 h to the bone marrow sample and to the culture medium. Hypotonization in 0.075 M KC1 was followed by triple fixation in Carnoy's mixture (3:1 methanol acetic acid). The trypsin technic of Wang and Fedoroff (1972) was used to achieve G-banding. The chromosomes are described according to the criteria of the Paris Conference (1971). Results and Discussion I n the initial survey of c o n v e n t i o n a l l y stained bone marrow preparations, o n l y 3 n o r m a l G-group chromosomes a n d no P h ' chromosome could be f o u n d i n all metaphases examined. The t r y p s i n G - b a n d i n g revealed (Fig. 1), t h a t the missing chromosome was a No. 22. I n s t e a d , a new aerocentrie chromosome was present, a b o u t the size of D-group members. I t s short arm, centromeric region, a n d proximal p a r t of the long a r m closely resembled the corresponding parts on the n o r m a l 22, b u t a n a d d i t i o n a l segment, including a n intensive proximal dark b a n d , a n
Fig. 1. Karyotype of patient, showing the rearranged No. 3 and 22 chromosomes (arrows)
Transloeation in Chronic Myelogenous Leukemia
231
Fig. 2. Normal and rearranged No. 3 and normal and rearranged No. 22 chromosomes of 3 different metaphases
intermediate light band, and a smaller and less colored terminal dark band, was connected to the distal part of the long arm. I n all metaphases bearing this chromosome an altered No. 3 was also present with short arm lacking the bands p22--pter. There is good reason to believe, t h a t the segment attached on No. 22 is identical to the deleted part of No. 3. Several assumptions could be made concerning the mechanism of transloeation. The most simple variant would be realized by the breaking of No. 3 near band 3p22 and the attachment of the deleted material on the terminal p a r t of the long arm of No. 22. I-Iowever, the terminal negative band on the short arm of the deleted No. 3 is longer t h a n band p21 on the normal No. 3 and the distance from the eentromere to the proximal dark band of the additional segment on the rearranged No. 22 is shorter than the long arm of the normal 22 (Fig. 2). I t m a y be concluded therefore, t h a t both No. 3 and 22 have lost and gained material i.e., the transloeation is reeiproeal. I n such a ease, the breaking point in No. 22 is of special interest. I n the commonly found 9/22 translocation the deleted and translocated No. 22 portion includes the dark band 22q12 (it can be seen on chromosome 9 q + ) , hence the breakage occurs above this band. In our case band 22q12 is detected on the short arm of No. 3 at some distance from tile dark band 3p14. I t is better observed in suboptimally trypsinized chromosomes. A longer exposure to enzymatic action reduces its stainability and this, along with the adjacent position of the dark band 3p14 makes its discovery difficult. This finding discards the possibility of breakage in No. 22 below 22q12 (in which case this band would be expected to be seen on the rearranged No. 22) and points to band 2 2 q l l as the most probable site of breakage in No. 22. The break point in No. 3 might be located in 3p21 near 3p14. In a small percentage of the rearranged No. 22 chromosomes, a weak dark band near the eentromere can be seen. Because of its inconstant expression and more proximal position we consider it as being the morphologie manifestation of the point of junction (Manolov et al., 1971) of No. 22 and No. 3 parts, rather than being identical to 22q12. The above chromosome changes have been observed in 100% of the metaphases in bone marrow (28 cells) as well as in peripheral blood leukocyte cultures without PI-IA (20 cells). I n the PHA-stimulated cultures, 41~o (33 cells) shouted normal female karyotype, while the remaining 59~o (48 cells) had the abnormal k a r y o t y p e described a b o v e - - 4 6 , X X , t (3 ; 22) (p21 ; ql 1). 10 °/o of the metaphases bearing the rearranged No. 3 and 22 chromosomes formed a 48 chromosome clone, accounted for by trisomy of both No. 19 and 21. 1 Yo of 500 bone marrow metaphases examined at low power were tetraploid. The same percentage was found in the peripheral blood cultures. This finding,
232
D. Pravtcheva et al.
c o m p a r e d t o t h e high n u m b e r of m e g a k a r y o b l a s t s in t h e bone m a r r o w a n d perip h e r a l blood suggests t h a t these cells are diploid, which is consistent w i t h t h e i r m o r p h o l o g y - - m i e r o m e g a k a r y o b l a s t s . The G - b a n d analysis of 3 t e t r a p l o i d rectaphases r e v e a l e d in all of t h e m t h e a l t e r e d No. 3 a n d 22 chromosomes in duplicate. Our case of u n u s u a l t r a n s l o c a t i o n i n CML as well as those r e p o r t e d before, gives f u r t h e r s u p p o r t to t h e concept t h a t t h e a l t e r a t i o n of No. 22 p l a y s a m a j o r role in t h e genesis of CML. A c o m m o n f e a t u r e to all cases is b r e a k a g e in No. 22 a t a specific p o i n t a n d s p a t i a l d i s j u n c t i o n of t h e resulting s e g m e n t s - - a t y p i c a l P h ' chromosome n o t being a n o b l i g a t o r y resu]t of t h e r e a r r a n g e m e n t . I t is impossible to decide w h e t h e r t h e disease is a consequence of such a s p a t i a l d i s j u n c t i o n of chromosome parts, d i s t u r b i n g t h e i r c o o r d i n a t e d function, w h e t h e r i t is due to changes in t h e genetic a c t i v i t y , a t t r i b u t a b l e to t h e p o s i t i o n effect, or to a m o r p h o ]ogically u n d e t e c t a b l e loss of genetic m a t e r i a l .
Acknowledgements. We are greatful to Dr. G. Manolov for helping our work in every way possible.
References Engel, E., McGee, B. J., Flexner, J. M., Russell, M. T., Myers, B. J. : Philadelphia chromosome (Ph') translocation in an apparently Ph' negative, minus G22 case of chronic myeloid leukemia. New Engl. J. Med. 291, 154 (1974) Gahrton, G., Zech, L., Lindsten, J.: A new variant translocation (19q+, 22q--) in chronic myelocytic leukemia. Exp. Cell Res. 86, 214--2t6 (1974) Hayata, I., Kakati, S., Sandberg, A.A.: A new translocation related to the Philadelphia chromosome. Lancet 1973 II, 1385 Hayata, I., Kakati, S., Sandberg, A. A. : Another translocation related to the Philadelphia chromosome. Lancet 1975 I, 1300 Huhn, D., Ascher, S.: Mikrokaryoblastenschub bei chroniseher Myelose. Acta haemat. (Basel) 58, 183--190 (1975) Manolov, G., Manolova, Y., Levan, A. : Experiments with fluorescent chromosome staining in Burkitt tumors. I-Iereditas (Lund) 68, 235--244 (1971) Muldal, S., Mir, M.A., Freeman, C. B., Geary, C. G. : A new translocation associated with the Ph' chromosome and an acute course of chronic granulocytic leukemia. Brit. J. Cancer 31, 364--368 (1975) Nowell, P. C., Jensen, g , Gardner, F.: Two complex translocations in chronic granulocytic leukemia involving chromosomes 22, 9 and a third chromosome, ttumangenetik 80, 13--21 (1975) Paris Conference (1971): Standardization in human cytogenetics. Birth Defects: Original Article Series VIII, 7 (1972). The National Foundation, New York Rowley, J. D. : A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature 43, 290--293 (1973) Wang, H. C., Fedoroff, S. : Banding in human chromosomes treated with trypsin. Nature (Loud.) New Biol. 285, 52--54 (1972) Dr. D. Pravteheva Centre of Oneology Sofia 56, Bulgaria
Addendum. After completion of this manuscript we read the paper of Nowell et al. (1975), dealing with 2 cases of complex trans!ocations in CML between No. 22, 9, and a third chromosome. In one of the cases, the third chromosome involved was a No. 3.
