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Chronic granulocytic leukemia of childhood
7 1 0 T h e Journal of P E D I A T R I C S
Chronic granulocytic leukemia of chiMbood C l i n i c a l a n d cytogenetic studies
Chronic granulocytie leukemia in iufancy differs in some clinical and hematologic aspects from the disease seen in older children and adults. Cytogenetic studies were performed on cells from peripheral blood culture and direct marrow preparations in 9 children with chronic granulocytic leukemia. The 5 children with the adult type of disease demonstrated the Phi-chromosome, but none of the 4 infants studied had this abnormal chromosome. However, 2 infants, as well as one child in the older group, had aneuploid stem-lines with a modal number of 47, the extra chromosomes being " m i n u t e " elements.
Leonard E. Reisman, M.D., r and Joss M. Trujillo, M.D. D UARTE~ CALIF.
T H ~ infrequency of chronic granulocytic leukemia in childhood and its rarity in infancy are reflected by a rather sparse literature. Eisenberg and Wallerstein 1 found only 30 cases of childhood chronic leukemia in the world literature when they reported a case in 1934, and the incidence of the disease, as reported from some large centers ranges from 1 to 5 per cent of all cases of leukemia in children. 2-~
From the Departments of Pediatrics and Experimental Pathology, City of Hope Medical Center, Duarte, Calif. Presented in part at the Fifth Annual Meeting of the American Society of Hematology, Columbus, Ohio, Nov. 25 to 27, 1962. This study was supported in part by Grant C-5138 from the National Cancer Institute, United States Pu.blic Health Service. ":~Present address, The Chitd Research Center of Mi~'M~,an, 660 Frederick Street, Detroit 2, Mich.
Cook& in 1953 managed to gather 15 cases from over 20 years of experience. The infants and younger children in his series had a relatively acute course and manifested hemorrhagic manifestations associated with marked thrombocytopenia at early stages of the disease. T h e disease in older children resembled chronic granulocytic leukemia in adults, clinically and hematologically, having normal or increased numbers of platelets at the onset of the illness and a generally more benign course. Several other authors have documented also variations from the typical features of chronic granulocytic leukemia seen in the infantile form of the disease. 7-9 The recent demonstration of a chromosomal anomaly associated with chronic granulocytic leukemia 1~ has added new impetus to the study of this disorder. However, chronic granulocytic leukemia in childhood
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has attracted scant attention. There have been no specific cytogenetic studies in children although this form of leukemia is the only acquired disease in which a chromosomal abnormality has been described with any consistency. The studies of Nowell and Hungerford 1~ included 2 children, aged 9 and 10, in both of whom a minute aeroeentric chromosome (Phi-chromosome) was found in peripheral blood and marrow preparations. No other karyotypic abnormalities were found. The 2 groups of patients studied by Tough and his co_authors,12, 18 consisted of adults, and the other studies reported thus far have not included children. 1~-1~ The purpose of this paper is to outline 9 cases of chronic granulocytic leukemia in children, together with the chromosome findings in peripheral blood cultures and direct bone marrow preparations. Six of the cases were studied at this institution and represent 3.75 per cent of the total number of 160 patients with childhood leukemia seen since 1956. Three children were patients in the Department of Pediatrics of Los Angeles County General Hospital. METHODS
Chromosome counts and analyses were made from peripheral blood cultures with only slight modification of the method of Moorhead and his associates 1~ and from direct bone marrow preparations. Briefly, the technique used for marrow preparations is as follows. 15 milliliters Hank's solution was prewarmed to room temperature and 0.05 ml. of an 0.04 per cent colchicine solution and 0.1 ml. of heparin were added. Then 0.5 to 1.0 ml. of marrow aspirate, usually obtained from the posterior iliac spine, was placed immediately into the solution. After an incubation period of 1 hour at 37 ~ C., the specimen was collected and prepared. The hypotonic treatment, fixation in Carnoy's, and final slide preparation followed the air dry procedure as described by Moorhead 17 and others. The slides were stained with Giemsa stain. Although most of the peripheral blood cul-
Chronic granulocytic leukemia
711
tures were harvested at the end of a period which ranged from 68 to 72 hours, they were watched closely and were harvested before the third day if there was evidence of rapid change of the pH. Photographs were taken of, and analyses performed on at least 5 diploid and 5 aneuploid metaphases of each individual subject who had significant aneuploidy in the peripheral blood or marrow preparations. CASE HISTORIES
Chromosome studies were performed on 9 children. The first group, Cases 1 to 5, were considered to have the "classical" form of chronic granulocytic leukemia, as observed in adults. The second group, Cases 6 to 9, are examples of the infantile form of the disease. Summaries of the relevant clinical and hematologic data in these cases appear in Tables I and II. Case 1. This 8-year-old female of Indonesian descent was first seen in February, 1960, with increasing fatigue and abdominal distention of 6 months' duration. Physical examination revealed a liver 4 cm. below the costal arch and a spleen descending into the pelvis. There was no lymphadenopathy. She has received intermittent busulfan therapy up to the present, generally maintaining normal hematocrit values and a white blood count under 20,000. Case 2. This 10-year-old white male was diagnosed in July, 1958, when he was found to have splenomegaly and refractory anemia. Although his spleen extended to the iliac crest, there was no other visceromegaly, adenopathy, or hemorrhagic manifestations. He was treated with busulfan for 2 years with almost complete remission of the disease. In October, 1960, he developed a hypoplastic marrow with leukopenia and thrombocytopenia. He was maintained by frequent transfusions and with the use of corticosteroids, but the condition progressively deteriorated. He died following generalized hemorrhages and a fulminating staphylococcal pneumonia. Necropsy revealed a markedly enlarged spleen with leukemic infiltration compatible with chronic granulocytic leukemia and a purulent and hemorrhagic pneumonia. Case 3. This 9-year-old white female was seen in May, 1957, with complaints of pains in the lower extremities. Past history was significant in
7 12
Reisman and TrujitIo
May 1963
T a b l e I. S u m m a r y of d a t a o n 9 c h i l d r e n w i t h c h r o n i c g r a n u l o c y t i c l e u k e m i a
Age Case
Sex
onset at
7 ~ years
Whenmade diagnosis Age t Year
Thera#y
Duration o[I illness alter diagnosis Terminal event
1
F
8 years
t960
Busulfan
Still alive
2
M
10 years
10 years
1958
Busulfan X-ray to spieen Corticosteroids Transfusions
35 months
3
F
9 years
9 years
1957
Busulfan
Still alive
4
M
9I/2 years
10 years
1960
Busulfan
Still alive
....
5
M
. 10 months
1 year
1958
Cyclophosphamlde Busulfan
Still aIive
__ _
6
M
7 months
14 months
1961
Busulfan 6-Mercaptopurine X-ray to spleen
Still alive
....
7
F
1 year
15 months
1958
Busulfan 6-Mercaptopurine X-ray to spleen Corticosteroids Transfusions
47 months
Hemorrhage, varicella
8
M
18 months
1962
6-Mercaptopurine X-ray to spleen
Still alive
....
9
M
3 years
1962
6-Mercaptopurine
Still alive
....
15 months 3 years
that she had received radiation therapy to an enlarged thymus in infancy. Physical examination revealed splenomegaly ("at the pelvic brim"). There was no hepatomegaly, adenopathy, or bleeding manifestations. She has been treated intermittently with bulsulfan up to the present and has been generally asymptomatic. Case 4. A 9-year-old white male was seen in January, 1960, with a history of progressive pallor, weight loss, and lethargy. The only abnormal physical finding was a spleen extending to the inguinal region. With the diagnosis of chronic granulocytic leukemia, he was placed on busulfan therapy and has continued in almost complete remission of the disease with intermittent use of busulfan. Case 5. This white male child was seen in September, 1958, at 10 months of age with intermittent vomiting, poor weight g a i ~ h e was generally "not thriving." T h e spleen was palpable 5 cm. below the left costal margin and the liver was barely palpable. No adenopathy or bruising were noted. Intramuscular cyclophosphamide was begun 18 months later, following increase in the size of the spleen. In 1961 a splenectomy was performed and the pathologist reported "extramedullary hematopoiesis." Cyclophosphamide was continued until January, 1962, when the
Staphylococcus pneumonia, hemorrhage
child became thrombopenic and busulfan therapy was begun a month later. Case 6. This 14-month-old white male began bruising at 7 months of age. When seen in November, 1961, he had scattered ecchymoses, moderately enlarged cervical and inguinal nodes, a liver palpable 10 cm. down, and a spleen extending into the pelvis. A course of bulsulfan was ineffective and he received cobalt teletherapy to the spleen (total of 200 rads) with minimal improvement. H e had a good response to 6-mercaptopurine and has been maintained on this therapy. The spleen remains large but platelets and total white blood cells have returned to norreal levels. Case 7. This 15-month-old white female was seen in May, 1958, with a history of a "swollen abdomen" and repeated respiratory infections. Although she had one sibling with Down's syndrome, the patient herself had no ctinicaI signs of Mongolism. She had enlarged cervical nodes, a spleen extending into the pelvis, and a liver palpable 4 cm. below the costal margin. Widespread hemorrhages, including epistaxis, occurred early in the course of the disease. There was no response to a long course of busulfan therapy, but she did have a good clini~;al response to 6mercaptopurine and was maintained on this drug
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intermittently for 4 years. In February, 1962, the bone marrow and peripheral blood became myeloblastic. She contracted varicella during this time and died following widespread bleeding. Autopsy revealed spleen and lymph nodes compatible with chronic granulocytic leukemia and generalized hemorrhages.
Case 8. An 18-month-old Mexican male was seen in March, 1962, because of anorexia, irritability, and abdominal distention of several months. The findings on physical examination included large cervical and inguinal nodes, a liver down 6 cm. at the right costal margin, and a spleen clown 8 era. No bruising was noted. A
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Fig. 2. Karyotype of Case 5 from marrow cell, has 47 chromosomes. Arrows indicate 2 minute chromosomes.
Volume 62 Number 5
Chronic granulocytie leukemia
715
Fig. 3. Metaphase plate (left) and karyotype (right) derived from the same metaphase of marrow cell from Case 7. Cell has 47 chromosomes, including one minute element (arrows). Note the poor quality of aneuploid chromosomes.
Fig. 4. Metaphase plate (left) and karyotype (right) from the same metaphase from peripheral blood of Case 9. Arrows indicate extra minute chromosome. Celt has complement of 47 ehronlosomes.
course of busulfan was initiated and radiation (20 rads) directed to the splenic area was given without clinical or hematologic response. Two weeks following beginning of 6-mercaptopurine therapy, the white blood cells fell to levels of around 10,000, the spleen decreased in size, but the thrombopenia persisted. He has been maintained on 6-mercaptopurine. Case 9. This 3-year-old Negro male was first seen in March, 1962, because of "swelling of the abdomen" and bruising. Physical examination revealed enlarged cervical nodes, liver palpable 6 cm. below the right costal margin arch, and a
spleen down 8 cm. below the left costal margin. Widespread petechiae and ecchymoses were noted. Following the institution of 6-mercaptopurine therapy there was a marked decrease in size of the spleen and return of the white blood cells to normal levels. The thrombopenia has persisted. FINDINGS
T h e chromosome findings are summarized in T a b l e I I I . T h e Phi-chromosome was found in 25 to 90 per cent of the countable
7 16
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Fig. 5. Metaphase plate (left) and karyotype (right) from the same metaphase from peripheral blood culture of Case 6, following splenic irradiation. Arrows indicate 2 chromosome fragments. Other evidence of radiation damage are the many chromatid breaks. metaphases of Cases 1 to 4 (children with the "adult" form of chronic granulocytic leukemia) in both peripheral blood cultures and direct marrow preparations. A diploid chromosome modal n u m b e r was present in all four. An idiogram of Case 2, outlining the Phi-chromosome is shown in Fig. 1. Case 5 presented interesting findings. In addition to cells from blood culture and marrow with diploid chromosome complements (with and without the Ph 1 marker), an aneuploid Chromosomal cell line with a modal number of 47 was identified. This cell type (Fig. 2) constituted about 60 per cent of the cells cultured from peripheral blood and made up one third of the marrow metaphases. The additional chromosome was always minute, probably derived by partial deletion from the 21-22 group. In addition, one of the 21-22 group was often smaller than the others. Thus, cells with 47 chromosomes had either 1 or 2 minute elements which resembled the Phi-chromosome. However, since there is no morphologic criterion to distinguish the Phi-chromosome from other minute elements, it was considered more appropriate for the present to indicate all the abnormally small chromosomes in the aneuploid metaph.ases as "minute" chromosomes. Other aneuploid cells with modal chromosome numbers greater than 49 were
suspected, but were unable to be analyzed because of the poor quality of these aneuploid metaphases. In Cases 6 to 9, the group with the "infantile" form of chronic~ granulocytic leukemia, the Phi-chromosome was not recognized in the metaphases of repeated blood cultures and marrows. However, an aneuploid strain with a modal number of 47 chromosomes, again with the extra chromosome being a minute one, was found in 2 cases. In Case 7, although peripheral blood cultures had only diploid cells with a normal karyotype, close to 38 per cent of the marrow metaphases contained an extra minute element (Fig. 3). Case 9 had a similar aneuploid cell with 47 chromosomes making up 40 per cent of the countable peripheral blood metaphases and about 25 per cent of the total cells counted in the marrow preparations (Fig. 4). Case 6 had an abnormal chromosome complement when studied within 4 weeks following splenic irradiationl A stem-line with 2 minute markers, probably representing fragments of chromosome material made up close to 20 per cent of the countable metaphases (Fig. 5). In this case subsequent studies have revealed a normal chromosome complement. Case 8, also in the infantile group, thus
Volume 62 Number 5
far has demonstrated an apparently normal karyotype and modal number in marrow as well as blood. DISCUSSION
A great majority of the cases of chronic granulocytic leukemia studied cytogenetically have been associated with the finding of an abnormally small chromosome, probably chromosome 21 which has lost about half of its long arm, x2 now commonly referred to as the Pha-chromosome. (Fifteen adults with chronic granulocytic leukemia studied at this institution in all stages of their disease, including the terminal "blastic crisis," showed the Phi-chromosome in metaphase figures of either peripheral blood or marrow.) Five children, Cases 1 to 5, all with typical features of the disease as seen in adults, have indeed demonstrated this "marker" chromosome. In addition, one of these children, Case 5, also showed a high fraction of aneuploid cells, with a modal chromosome number of 47. Although the symptomatology of Case 5 began in infancy (10 months of age), the course and hematologic findings up to the time of our cytogenetic studies were similar to those of the older children. Because of the gradual slope in age distribution of chronic granulocytic leukemia, one woul,d expect to find occasional cases of the adult type o{ disease in infants. Among the four cases of leukemia diagnosed and studied within the first 3 years of life, Cases 6 to 9, all of whom resemble the "infantile" or subacute form of the disease, the Phi-chromosome was not found; and of these four children, 2 revealed an aneuploid stem-line with a modal number of 47, the additional chromosome being a minute one. Thus, the infantile form of chronic granulocytic leukemia was characterized by the absence of the Phi-chromosomal anomaly among the cells with chromosome number of 46. These findings, in relation to the previously mentioned reports (and our own clinical findings) of variances from the adult form of the disease in infants, suggest that the chronic granulocytic leukemia in adults is indeed different from that in infancy.
Chronic granulocytic leukemia
721
The clinical histories and hematologic studies of all the cases were considered to be typical of chronic granutocytic leukemia of either the adult or infant form. Hyperleukocytosis, with a predominance of granulocytes and an increased number of myelocytes and metamyelocytes in both peripheral blood and bone marrow; a relatively small number of blasts and immature cells; splenomegaly, generally of massive proportions; and the absence of an acute toxic onset of the disease were present in both age groups. A decreased leukocyte alkaline phosphatase, as well as an eosinophilia or basophilia, were further diagnostic evidence and were also usually demonstrable in both groups. However, in contrast to the normal or increased platelet counts in the early stages of the disease which are generally observable in the adult disease (and in the children in the older age group), the infantile group presented early with hemorrhagic manifestations, thrombopenia, and decreased or absent megakaryopoiesis. Response to chemotherapy also differed in the two group.s, tha infants responding poorly to busulfan and impressively to 6-mercaptopurine; the older children consistently had prolonged "remissions" with busulfan. While the Phi-chromosome has been present in a great majority of the cases of chronic granulocytic leukemia reported, it must be admitted that a few well studied cases 11, 12 have failed to show this feature. Therefore, fm-ther studies in this field should contribute more evidence to the actual role of this marker in the leukemic cell. The excellent correlation between the finding of the Ph 1 chromosome and chronic granulocytic leukemia in the older children and all adults studied in our laboratories leads us to assume that the Phi-chromosome does play an etiologic role in this disorder. However, since a common finding in both groups of children was the presence of similar aneuploid stemlines, it is a possibility that too much emphasis has been placed on the finding of the Phi-chromosome as the only abnormality in chronic granulocytic leukemia.
722
Reisman and TrufilIo
If one assumes t h a t the hogt conditions in vivo of patients w i t h the a d u l t form of chronic granulocytic l e u k e m i a are f a v o r a b l e for the m a i n t e n a n c e of a continuous "stemline" w i t h the P h i - c h r o m o s o m e as a m a r k e r , it m a y be t h a t in infants the situation is s o m e w h a t different. O n e of the cells which a p p e a r s to have a selective a d v a n t a g e a n d is able to p r o p a g a t e is an a n e u p l o i d stemline. One, again, can only speculate on the relationship of the genetic stability of the c h r o m o s o m e c o m p l e m e n t of a h e m a t o p o i e t i c cell to the type of leukemic process. Evidence of the relationship of a n e u p l o i d y to l e u k e m i a is the finding of a g r e a t l y increased incidence over the e x p e c t e d of acute l e u k e m i a in D o w n ' s s y n d r o m e ? s' 19 A n increase of this disorder in persons with M o n g o l i s m w h o have trisomy of c h r o m o some 21 would certainly be expected if a n e u p l o i d y caused genetic instability and, at the least, an increased susceptibility to leukemia. I n r e g a r d to the a b n o r m a l chromosome constitution f o u n d in 20 per cent of the cells in Case 6 following r a d i a t i o n t h e r a p y , the p r o b a b i l i t y of breaks o c c u r r i n g at the same points in so m a n y cells is h i g h l y remote. I t is t h o u g h t therefore t h a t this was a stemline w h i c h was able to p r o p a g a t e itself for a brief time. It has been previously d e m o n strated by Tough, et a l ? 2 t h a t changes in chromosome m o r p h o l o g y can be i n d u c e d by t h e r a p e u t i c radiation. SUMMARY
N i n e cases of chronic g r a n u l o c y t i c leuk e m i a in childhood are r e p o r t e d , a n d cytogenetic studies on p e r i p h e r a l blood cultures have been performed. I n 5 c h i l d r e n with the a d u l t f o r m of the disease the findings of the Ph z chromosome in diploid cells a r e confirmed. H o w e v e r , in the four children with the infantile form of chronic leukemia, the P h i - c h r o m o s o m e was not present. I n 2 of these children, as well as in one of the first g r o u p of children, a high degree of aneuploidy, p r i m a r i l y of 47 m o d a l n u m b e r a n d w i t h a simitar configuration, was found. T h e significance of these findings is discussed.
May 1963
We are indebted to the Department of Pediatrics of Los Angeles County General Hospital and to Drs. Jacques Nowack and Gerritt d'Ablaing for cooperation in studying their patients; to Drs. Alfred G. Knudson, Jr., Ernest Beutler, and Wolf W. Zuelzer, for their helpful criticism of this manuscript; and to Dr. Susumu Ohno, without whose guidance and assistance this study could not have been accomplished.
REFERENCES
1. Eisenberg, A. A., and Wallerstein, H." Chronic myelosis in children, J. Lab. & Clin. Med. 19: 731, 1934. 2. Cooke, J. V.: The occurrence of leukemia, Blood 9: 340, 1954. 3. Dameshek, W., and Gunz, F.: Leukemia, New York, 1958, Grune & Stratton, Inc., pp. 3637. 4. Rodgers, C. L., Donohue, W. L., and Snelling, C. E.: Leukemia in children, Canad. M. A. J. 65: 548, 1951. 5. Dale, J. H., Jr.: Leukemia in childhood, J. PEDIAT. 34: 421, 1949. 6. Cooke, J. V.: Chronic myelogenous leukemia in children, J. PEDIAT. 42: 537, 1953. 7. Barrett, O., Conrad, M., and Crosby, W. H.: Chronic granulocytic leukemia ir~ childhood, Am. J. Med. Sc. 240: 587, 1960. 8. Keith, tI. M.: Chronic myelogenous leukemia in infancy, Am. J. Dis. Child. 69: 366, 1945. 9. Poncher, H. G., Weir, H. F., and Limarzi, L. R.: Chronic myelogenous leukemia in early infancy, J. PEDIAT. 21: 73, 1942. 10. Nowell, P. C., and Hungerford, D. A.: Chromosome studies in normal and leukemic human leukocytes, J. Nat. Cancer Inst. 25: 85, 1960. 11. Nowell, P. C., and Hungerford, D. A.: Chromosome studies in human leukemia. II. Chronic granulocytic leukemia, J. Nat. Cancer Inst. 27: 1013, 1961. 12. Tough, I. M., Court Brown, W. M., Baikie, A. G., Buckton, K. E., Harnden, D. G., Jacobs, P. A., King, M. H., and McBride, J. A.: Cytogenetic studies in chronic myeloid leukemia and acute leukemia associated with Mongolism, Lancet 1: 4.11, 1961. 13. Tough, I. M., Baikie, A. G., Harnden, D. G., Court Brown, W. M., Buckton, K. E., Jacobs, P. A., and William, J. A.: Chronic myeloid leukemia: cytogenetic studies before and after splenic irradiation, Lancet 2: I15, 1962. 14. Hauschka, T. S.: The chromosomes in ontogeny and oncogeny, Cancer Reg. 21: 957, 1961. 15. Kinlough, M. A., and Robson, H. N.: Study of chromosomes in human leukemia by a direct method, Brit. M. J. 2: 1052, 1961. 16. Adams, A., Fitzgerald, P. H., and Gunz, F. W.: A new chromosome abnormality in
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chronic granulocytic leukemia, Brit. M. J. 2: 1474, 1961. 17. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood, Exper. Cell Res. 20" 613, 1960.
Chronic granulocylic leukemia
723
18. Krivit, W., and Good, R. A.: Simultaneous occurrence of Mongolism and leukemia: Report of a nationwide survey, A. M'. A. J. Dis. Child. 94: 289, 1957. 19. Stewart, A., Webb, J., and Hewitt, D.: A survey of childhood malignancies, Brit. M. J. 1: 1495, 1958.
Chronic granulocytic leukemia of chiMbood C l i n i c a l a n d cytogenetic studies
Chronic granulocytie leukemia in iufancy differs in some clinical and hematologic aspects from the disease seen in older children and adults. Cytogenetic studies were performed on cells from peripheral blood culture and direct marrow preparations in 9 children with chronic granulocytic leukemia. The 5 children with the adult type of disease demonstrated the Phi-chromosome, but none of the 4 infants studied had this abnormal chromosome. However, 2 infants, as well as one child in the older group, had aneuploid stem-lines with a modal number of 47, the extra chromosomes being " m i n u t e " elements.
Leonard E. Reisman, M.D., r and Joss M. Trujillo, M.D. D UARTE~ CALIF.
T H ~ infrequency of chronic granulocytic leukemia in childhood and its rarity in infancy are reflected by a rather sparse literature. Eisenberg and Wallerstein 1 found only 30 cases of childhood chronic leukemia in the world literature when they reported a case in 1934, and the incidence of the disease, as reported from some large centers ranges from 1 to 5 per cent of all cases of leukemia in children. 2-~
From the Departments of Pediatrics and Experimental Pathology, City of Hope Medical Center, Duarte, Calif. Presented in part at the Fifth Annual Meeting of the American Society of Hematology, Columbus, Ohio, Nov. 25 to 27, 1962. This study was supported in part by Grant C-5138 from the National Cancer Institute, United States Pu.blic Health Service. ":~Present address, The Chitd Research Center of Mi~'M~,an, 660 Frederick Street, Detroit 2, Mich.
Cook& in 1953 managed to gather 15 cases from over 20 years of experience. The infants and younger children in his series had a relatively acute course and manifested hemorrhagic manifestations associated with marked thrombocytopenia at early stages of the disease. T h e disease in older children resembled chronic granulocytic leukemia in adults, clinically and hematologically, having normal or increased numbers of platelets at the onset of the illness and a generally more benign course. Several other authors have documented also variations from the typical features of chronic granulocytic leukemia seen in the infantile form of the disease. 7-9 The recent demonstration of a chromosomal anomaly associated with chronic granulocytic leukemia 1~ has added new impetus to the study of this disorder. However, chronic granulocytic leukemia in childhood
Volume 62 Number 5
has attracted scant attention. There have been no specific cytogenetic studies in children although this form of leukemia is the only acquired disease in which a chromosomal abnormality has been described with any consistency. The studies of Nowell and Hungerford 1~ included 2 children, aged 9 and 10, in both of whom a minute aeroeentric chromosome (Phi-chromosome) was found in peripheral blood and marrow preparations. No other karyotypic abnormalities were found. The 2 groups of patients studied by Tough and his co_authors,12, 18 consisted of adults, and the other studies reported thus far have not included children. 1~-1~ The purpose of this paper is to outline 9 cases of chronic granulocytic leukemia in children, together with the chromosome findings in peripheral blood cultures and direct bone marrow preparations. Six of the cases were studied at this institution and represent 3.75 per cent of the total number of 160 patients with childhood leukemia seen since 1956. Three children were patients in the Department of Pediatrics of Los Angeles County General Hospital. METHODS
Chromosome counts and analyses were made from peripheral blood cultures with only slight modification of the method of Moorhead and his associates 1~ and from direct bone marrow preparations. Briefly, the technique used for marrow preparations is as follows. 15 milliliters Hank's solution was prewarmed to room temperature and 0.05 ml. of an 0.04 per cent colchicine solution and 0.1 ml. of heparin were added. Then 0.5 to 1.0 ml. of marrow aspirate, usually obtained from the posterior iliac spine, was placed immediately into the solution. After an incubation period of 1 hour at 37 ~ C., the specimen was collected and prepared. The hypotonic treatment, fixation in Carnoy's, and final slide preparation followed the air dry procedure as described by Moorhead 17 and others. The slides were stained with Giemsa stain. Although most of the peripheral blood cul-
Chronic granulocytic leukemia
711
tures were harvested at the end of a period which ranged from 68 to 72 hours, they were watched closely and were harvested before the third day if there was evidence of rapid change of the pH. Photographs were taken of, and analyses performed on at least 5 diploid and 5 aneuploid metaphases of each individual subject who had significant aneuploidy in the peripheral blood or marrow preparations. CASE HISTORIES
Chromosome studies were performed on 9 children. The first group, Cases 1 to 5, were considered to have the "classical" form of chronic granulocytic leukemia, as observed in adults. The second group, Cases 6 to 9, are examples of the infantile form of the disease. Summaries of the relevant clinical and hematologic data in these cases appear in Tables I and II. Case 1. This 8-year-old female of Indonesian descent was first seen in February, 1960, with increasing fatigue and abdominal distention of 6 months' duration. Physical examination revealed a liver 4 cm. below the costal arch and a spleen descending into the pelvis. There was no lymphadenopathy. She has received intermittent busulfan therapy up to the present, generally maintaining normal hematocrit values and a white blood count under 20,000. Case 2. This 10-year-old white male was diagnosed in July, 1958, when he was found to have splenomegaly and refractory anemia. Although his spleen extended to the iliac crest, there was no other visceromegaly, adenopathy, or hemorrhagic manifestations. He was treated with busulfan for 2 years with almost complete remission of the disease. In October, 1960, he developed a hypoplastic marrow with leukopenia and thrombocytopenia. He was maintained by frequent transfusions and with the use of corticosteroids, but the condition progressively deteriorated. He died following generalized hemorrhages and a fulminating staphylococcal pneumonia. Necropsy revealed a markedly enlarged spleen with leukemic infiltration compatible with chronic granulocytic leukemia and a purulent and hemorrhagic pneumonia. Case 3. This 9-year-old white female was seen in May, 1957, with complaints of pains in the lower extremities. Past history was significant in
7 12
Reisman and TrujitIo
May 1963
T a b l e I. S u m m a r y of d a t a o n 9 c h i l d r e n w i t h c h r o n i c g r a n u l o c y t i c l e u k e m i a
Age Case
Sex
onset at
7 ~ years
Whenmade diagnosis Age t Year
Thera#y
Duration o[I illness alter diagnosis Terminal event
1
F
8 years
t960
Busulfan
Still alive
2
M
10 years
10 years
1958
Busulfan X-ray to spieen Corticosteroids Transfusions
35 months
3
F
9 years
9 years
1957
Busulfan
Still alive
4
M
9I/2 years
10 years
1960
Busulfan
Still alive
....
5
M
. 10 months
1 year
1958
Cyclophosphamlde Busulfan
Still aIive
__ _
6
M
7 months
14 months
1961
Busulfan 6-Mercaptopurine X-ray to spleen
Still alive
....
7
F
1 year
15 months
1958
Busulfan 6-Mercaptopurine X-ray to spleen Corticosteroids Transfusions
47 months
Hemorrhage, varicella
8
M
18 months
1962
6-Mercaptopurine X-ray to spleen
Still alive
....
9
M
3 years
1962
6-Mercaptopurine
Still alive
....
15 months 3 years
that she had received radiation therapy to an enlarged thymus in infancy. Physical examination revealed splenomegaly ("at the pelvic brim"). There was no hepatomegaly, adenopathy, or bleeding manifestations. She has been treated intermittently with bulsulfan up to the present and has been generally asymptomatic. Case 4. A 9-year-old white male was seen in January, 1960, with a history of progressive pallor, weight loss, and lethargy. The only abnormal physical finding was a spleen extending to the inguinal region. With the diagnosis of chronic granulocytic leukemia, he was placed on busulfan therapy and has continued in almost complete remission of the disease with intermittent use of busulfan. Case 5. This white male child was seen in September, 1958, at 10 months of age with intermittent vomiting, poor weight g a i ~ h e was generally "not thriving." T h e spleen was palpable 5 cm. below the left costal margin and the liver was barely palpable. No adenopathy or bruising were noted. Intramuscular cyclophosphamide was begun 18 months later, following increase in the size of the spleen. In 1961 a splenectomy was performed and the pathologist reported "extramedullary hematopoiesis." Cyclophosphamide was continued until January, 1962, when the
Staphylococcus pneumonia, hemorrhage
child became thrombopenic and busulfan therapy was begun a month later. Case 6. This 14-month-old white male began bruising at 7 months of age. When seen in November, 1961, he had scattered ecchymoses, moderately enlarged cervical and inguinal nodes, a liver palpable 10 cm. down, and a spleen extending into the pelvis. A course of bulsulfan was ineffective and he received cobalt teletherapy to the spleen (total of 200 rads) with minimal improvement. H e had a good response to 6-mercaptopurine and has been maintained on this therapy. The spleen remains large but platelets and total white blood cells have returned to norreal levels. Case 7. This 15-month-old white female was seen in May, 1958, with a history of a "swollen abdomen" and repeated respiratory infections. Although she had one sibling with Down's syndrome, the patient herself had no ctinicaI signs of Mongolism. She had enlarged cervical nodes, a spleen extending into the pelvis, and a liver palpable 4 cm. below the costal margin. Widespread hemorrhages, including epistaxis, occurred early in the course of the disease. There was no response to a long course of busulfan therapy, but she did have a good clini~;al response to 6mercaptopurine and was maintained on this drug
Volume 62
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intermittently for 4 years. In February, 1962, the bone marrow and peripheral blood became myeloblastic. She contracted varicella during this time and died following widespread bleeding. Autopsy revealed spleen and lymph nodes compatible with chronic granulocytic leukemia and generalized hemorrhages.
Case 8. An 18-month-old Mexican male was seen in March, 1962, because of anorexia, irritability, and abdominal distention of several months. The findings on physical examination included large cervical and inguinal nodes, a liver down 6 cm. at the right costal margin, and a spleen clown 8 era. No bruising was noted. A
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Volume 62 Number 5
Chronic granulocytie leukemia
715
Fig. 3. Metaphase plate (left) and karyotype (right) derived from the same metaphase of marrow cell from Case 7. Cell has 47 chromosomes, including one minute element (arrows). Note the poor quality of aneuploid chromosomes.
Fig. 4. Metaphase plate (left) and karyotype (right) from the same metaphase from peripheral blood of Case 9. Arrows indicate extra minute chromosome. Celt has complement of 47 ehronlosomes.
course of busulfan was initiated and radiation (20 rads) directed to the splenic area was given without clinical or hematologic response. Two weeks following beginning of 6-mercaptopurine therapy, the white blood cells fell to levels of around 10,000, the spleen decreased in size, but the thrombopenia persisted. He has been maintained on 6-mercaptopurine. Case 9. This 3-year-old Negro male was first seen in March, 1962, because of "swelling of the abdomen" and bruising. Physical examination revealed enlarged cervical nodes, liver palpable 6 cm. below the right costal margin arch, and a
spleen down 8 cm. below the left costal margin. Widespread petechiae and ecchymoses were noted. Following the institution of 6-mercaptopurine therapy there was a marked decrease in size of the spleen and return of the white blood cells to normal levels. The thrombopenia has persisted. FINDINGS
T h e chromosome findings are summarized in T a b l e I I I . T h e Phi-chromosome was found in 25 to 90 per cent of the countable
7 16
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Fig. 5. Metaphase plate (left) and karyotype (right) from the same metaphase from peripheral blood culture of Case 6, following splenic irradiation. Arrows indicate 2 chromosome fragments. Other evidence of radiation damage are the many chromatid breaks. metaphases of Cases 1 to 4 (children with the "adult" form of chronic granulocytic leukemia) in both peripheral blood cultures and direct marrow preparations. A diploid chromosome modal n u m b e r was present in all four. An idiogram of Case 2, outlining the Phi-chromosome is shown in Fig. 1. Case 5 presented interesting findings. In addition to cells from blood culture and marrow with diploid chromosome complements (with and without the Ph 1 marker), an aneuploid Chromosomal cell line with a modal number of 47 was identified. This cell type (Fig. 2) constituted about 60 per cent of the cells cultured from peripheral blood and made up one third of the marrow metaphases. The additional chromosome was always minute, probably derived by partial deletion from the 21-22 group. In addition, one of the 21-22 group was often smaller than the others. Thus, cells with 47 chromosomes had either 1 or 2 minute elements which resembled the Phi-chromosome. However, since there is no morphologic criterion to distinguish the Phi-chromosome from other minute elements, it was considered more appropriate for the present to indicate all the abnormally small chromosomes in the aneuploid metaph.ases as "minute" chromosomes. Other aneuploid cells with modal chromosome numbers greater than 49 were
suspected, but were unable to be analyzed because of the poor quality of these aneuploid metaphases. In Cases 6 to 9, the group with the "infantile" form of chronic~ granulocytic leukemia, the Phi-chromosome was not recognized in the metaphases of repeated blood cultures and marrows. However, an aneuploid strain with a modal number of 47 chromosomes, again with the extra chromosome being a minute one, was found in 2 cases. In Case 7, although peripheral blood cultures had only diploid cells with a normal karyotype, close to 38 per cent of the marrow metaphases contained an extra minute element (Fig. 3). Case 9 had a similar aneuploid cell with 47 chromosomes making up 40 per cent of the countable peripheral blood metaphases and about 25 per cent of the total cells counted in the marrow preparations (Fig. 4). Case 6 had an abnormal chromosome complement when studied within 4 weeks following splenic irradiationl A stem-line with 2 minute markers, probably representing fragments of chromosome material made up close to 20 per cent of the countable metaphases (Fig. 5). In this case subsequent studies have revealed a normal chromosome complement. Case 8, also in the infantile group, thus
Volume 62 Number 5
far has demonstrated an apparently normal karyotype and modal number in marrow as well as blood. DISCUSSION
A great majority of the cases of chronic granulocytic leukemia studied cytogenetically have been associated with the finding of an abnormally small chromosome, probably chromosome 21 which has lost about half of its long arm, x2 now commonly referred to as the Pha-chromosome. (Fifteen adults with chronic granulocytic leukemia studied at this institution in all stages of their disease, including the terminal "blastic crisis," showed the Phi-chromosome in metaphase figures of either peripheral blood or marrow.) Five children, Cases 1 to 5, all with typical features of the disease as seen in adults, have indeed demonstrated this "marker" chromosome. In addition, one of these children, Case 5, also showed a high fraction of aneuploid cells, with a modal chromosome number of 47. Although the symptomatology of Case 5 began in infancy (10 months of age), the course and hematologic findings up to the time of our cytogenetic studies were similar to those of the older children. Because of the gradual slope in age distribution of chronic granulocytic leukemia, one woul,d expect to find occasional cases of the adult type o{ disease in infants. Among the four cases of leukemia diagnosed and studied within the first 3 years of life, Cases 6 to 9, all of whom resemble the "infantile" or subacute form of the disease, the Phi-chromosome was not found; and of these four children, 2 revealed an aneuploid stem-line with a modal number of 47, the additional chromosome being a minute one. Thus, the infantile form of chronic granulocytic leukemia was characterized by the absence of the Phi-chromosomal anomaly among the cells with chromosome number of 46. These findings, in relation to the previously mentioned reports (and our own clinical findings) of variances from the adult form of the disease in infants, suggest that the chronic granulocytic leukemia in adults is indeed different from that in infancy.
Chronic granulocytic leukemia
721
The clinical histories and hematologic studies of all the cases were considered to be typical of chronic granutocytic leukemia of either the adult or infant form. Hyperleukocytosis, with a predominance of granulocytes and an increased number of myelocytes and metamyelocytes in both peripheral blood and bone marrow; a relatively small number of blasts and immature cells; splenomegaly, generally of massive proportions; and the absence of an acute toxic onset of the disease were present in both age groups. A decreased leukocyte alkaline phosphatase, as well as an eosinophilia or basophilia, were further diagnostic evidence and were also usually demonstrable in both groups. However, in contrast to the normal or increased platelet counts in the early stages of the disease which are generally observable in the adult disease (and in the children in the older age group), the infantile group presented early with hemorrhagic manifestations, thrombopenia, and decreased or absent megakaryopoiesis. Response to chemotherapy also differed in the two group.s, tha infants responding poorly to busulfan and impressively to 6-mercaptopurine; the older children consistently had prolonged "remissions" with busulfan. While the Phi-chromosome has been present in a great majority of the cases of chronic granulocytic leukemia reported, it must be admitted that a few well studied cases 11, 12 have failed to show this feature. Therefore, fm-ther studies in this field should contribute more evidence to the actual role of this marker in the leukemic cell. The excellent correlation between the finding of the Ph 1 chromosome and chronic granulocytic leukemia in the older children and all adults studied in our laboratories leads us to assume that the Phi-chromosome does play an etiologic role in this disorder. However, since a common finding in both groups of children was the presence of similar aneuploid stemlines, it is a possibility that too much emphasis has been placed on the finding of the Phi-chromosome as the only abnormality in chronic granulocytic leukemia.
722
Reisman and TrufilIo
If one assumes t h a t the hogt conditions in vivo of patients w i t h the a d u l t form of chronic granulocytic l e u k e m i a are f a v o r a b l e for the m a i n t e n a n c e of a continuous "stemline" w i t h the P h i - c h r o m o s o m e as a m a r k e r , it m a y be t h a t in infants the situation is s o m e w h a t different. O n e of the cells which a p p e a r s to have a selective a d v a n t a g e a n d is able to p r o p a g a t e is an a n e u p l o i d stemline. One, again, can only speculate on the relationship of the genetic stability of the c h r o m o s o m e c o m p l e m e n t of a h e m a t o p o i e t i c cell to the type of leukemic process. Evidence of the relationship of a n e u p l o i d y to l e u k e m i a is the finding of a g r e a t l y increased incidence over the e x p e c t e d of acute l e u k e m i a in D o w n ' s s y n d r o m e ? s' 19 A n increase of this disorder in persons with M o n g o l i s m w h o have trisomy of c h r o m o some 21 would certainly be expected if a n e u p l o i d y caused genetic instability and, at the least, an increased susceptibility to leukemia. I n r e g a r d to the a b n o r m a l chromosome constitution f o u n d in 20 per cent of the cells in Case 6 following r a d i a t i o n t h e r a p y , the p r o b a b i l i t y of breaks o c c u r r i n g at the same points in so m a n y cells is h i g h l y remote. I t is t h o u g h t therefore t h a t this was a stemline w h i c h was able to p r o p a g a t e itself for a brief time. It has been previously d e m o n strated by Tough, et a l ? 2 t h a t changes in chromosome m o r p h o l o g y can be i n d u c e d by t h e r a p e u t i c radiation. SUMMARY
N i n e cases of chronic g r a n u l o c y t i c leuk e m i a in childhood are r e p o r t e d , a n d cytogenetic studies on p e r i p h e r a l blood cultures have been performed. I n 5 c h i l d r e n with the a d u l t f o r m of the disease the findings of the Ph z chromosome in diploid cells a r e confirmed. H o w e v e r , in the four children with the infantile form of chronic leukemia, the P h i - c h r o m o s o m e was not present. I n 2 of these children, as well as in one of the first g r o u p of children, a high degree of aneuploidy, p r i m a r i l y of 47 m o d a l n u m b e r a n d w i t h a simitar configuration, was found. T h e significance of these findings is discussed.
May 1963
We are indebted to the Department of Pediatrics of Los Angeles County General Hospital and to Drs. Jacques Nowack and Gerritt d'Ablaing for cooperation in studying their patients; to Drs. Alfred G. Knudson, Jr., Ernest Beutler, and Wolf W. Zuelzer, for their helpful criticism of this manuscript; and to Dr. Susumu Ohno, without whose guidance and assistance this study could not have been accomplished.
REFERENCES
1. Eisenberg, A. A., and Wallerstein, H." Chronic myelosis in children, J. Lab. & Clin. Med. 19: 731, 1934. 2. Cooke, J. V.: The occurrence of leukemia, Blood 9: 340, 1954. 3. Dameshek, W., and Gunz, F.: Leukemia, New York, 1958, Grune & Stratton, Inc., pp. 3637. 4. Rodgers, C. L., Donohue, W. L., and Snelling, C. E.: Leukemia in children, Canad. M. A. J. 65: 548, 1951. 5. Dale, J. H., Jr.: Leukemia in childhood, J. PEDIAT. 34: 421, 1949. 6. Cooke, J. V.: Chronic myelogenous leukemia in children, J. PEDIAT. 42: 537, 1953. 7. Barrett, O., Conrad, M., and Crosby, W. H.: Chronic granulocytic leukemia ir~ childhood, Am. J. Med. Sc. 240: 587, 1960. 8. Keith, tI. M.: Chronic myelogenous leukemia in infancy, Am. J. Dis. Child. 69: 366, 1945. 9. Poncher, H. G., Weir, H. F., and Limarzi, L. R.: Chronic myelogenous leukemia in early infancy, J. PEDIAT. 21: 73, 1942. 10. Nowell, P. C., and Hungerford, D. A.: Chromosome studies in normal and leukemic human leukocytes, J. Nat. Cancer Inst. 25: 85, 1960. 11. Nowell, P. C., and Hungerford, D. A.: Chromosome studies in human leukemia. II. Chronic granulocytic leukemia, J. Nat. Cancer Inst. 27: 1013, 1961. 12. Tough, I. M., Court Brown, W. M., Baikie, A. G., Buckton, K. E., Harnden, D. G., Jacobs, P. A., King, M. H., and McBride, J. A.: Cytogenetic studies in chronic myeloid leukemia and acute leukemia associated with Mongolism, Lancet 1: 4.11, 1961. 13. Tough, I. M., Baikie, A. G., Harnden, D. G., Court Brown, W. M., Buckton, K. E., Jacobs, P. A., and William, J. A.: Chronic myeloid leukemia: cytogenetic studies before and after splenic irradiation, Lancet 2: I15, 1962. 14. Hauschka, T. S.: The chromosomes in ontogeny and oncogeny, Cancer Reg. 21: 957, 1961. 15. Kinlough, M. A., and Robson, H. N.: Study of chromosomes in human leukemia by a direct method, Brit. M. J. 2: 1052, 1961. 16. Adams, A., Fitzgerald, P. H., and Gunz, F. W.: A new chromosome abnormality in
Volume 62 Number 5
chronic granulocytic leukemia, Brit. M. J. 2: 1474, 1961. 17. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood, Exper. Cell Res. 20" 613, 1960.
Chronic granulocylic leukemia
723
18. Krivit, W., and Good, R. A.: Simultaneous occurrence of Mongolism and leukemia: Report of a nationwide survey, A. M'. A. J. Dis. Child. 94: 289, 1957. 19. Stewart, A., Webb, J., and Hewitt, D.: A survey of childhood malignancies, Brit. M. J. 1: 1495, 1958.