Humangenetik 29, 259--264 (1975) © by Springer-Verlag 1975

Clinical Case Reports Missing Y Chromosome in Juvenile Chronic Myelogenous Leukemia* T a r u H a y s , J a m e s R. H u m b e r t , D a v i d C, P e a k m a n , J o h n J. H u r t e r , Helvise G. Morse, A r t h u r Robinson, a n d Charles S. A u g u s t Department of Biophysics/Genetics and Pediatrics, University of Colorado Medical Center, National Jewish Hospital, and the Oncology Center of Denver Children's Hospital, Denver, Colorado Received April 21, 1975 / May 22, 1975

Summary. A child with phi-negative juvenile chronic myelogenous leukemia (CML) is presented. The only chromosomal abnormality in hematopoietic tissues consisted of an absent ¥ chromosome. While a missing Y chromosome in adult patients with CML may be associated with a better prognosis, the clinical course in our patient was as malignant as that usually observed in other children with phi-negative juvenile CML. Introduction " J u v e n i l e " chronic m y e l o g e n o u s l e u k e m i a (CML) differs from t h e a d u l t - t y p e , p h i - p o s i t i v e CML b y its m u c h m o r e m a l i g n a n t clinical course, t h e usual a b s e n c e of a P h 1 chromosome, t h e presence of high fetal h e m o g l o b i n levels, a n d its occurrence in children. T h e absence of a P h 1 chromosome also carries a poor prognosis in a d u l t p a t i e n t s w i t h CML (Ezdinli et al., 1970). On t h e o t h e r hand, a d u l t male p a t i e n t s w i t h p h i - p o s i t i v e CML who h a d a missing ¥ c h r o m o s o m e in t h e i r bone m a r r o w cells h a v e been described; t h e p a r t i c u l a r b e n i g n i t y of t h e i r clinical e v o l u t i o n has been a t t r i b u t e d to t h e missing Y c h r o m o s o m e (Bauters et al., 1970; S a n d b e r g a n d Hossfeld, 1970) a l t h o u g h this p o i n t has been d e b a t e d r e c e n t l y (Lawler et al., 1974). No child w i t h j u v e n i l e CML associated w i t h a missing ¥ c h r o m o s o m e has been r e p o r t e d to date, a n d t h u s t h e possible beneficial effect of t h a t a n e u p l o i d y has n o t been e v a l u a t e d in t h a t condition. This p a p e r is concerned w i t h such a case, who r a n a course as m a l i g n a n t as t h a t o r d i n a r i l y o b s e r v e d in o t h e r p a t i e n t s w i t h P h 1 c h r o m o s o m e - n e g a t i v e juvenile CML. * Supported in part by the following Public Health Service Grants: Research Grants CA-15564-01, CA-12247, and CA-13419 from the National Cancer Institute, by a general Research Support Grant No. RR-05357 and by Grant No. HD-00622 from the National Institute of Child Health and Human Development, by funds provided by the House Bill No. 1578 of the State of Colorado Legislature (Sickle Cell Anemia Treatment and Research Center) and by a Biological Medical Award from the Swiss Academy of Medical Sciences. This publication is No. 626 from the Department of Biophysics and Genetics, University of Colorado Medical Center.

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Case Report This 31~-year-old white male was evaluated at Denver Children's Hospital in January, 1974 for an upper respiratory illness. Mild hepatosplenomegaly, cervical and inguinal adenopathies were observed. Hemoglobin and hematocrit were normal. Leukocyte count was 45000/~1, with a predominance of granulocytes including numerous immature granulocytic precursors. Nucleated red cell precursors were observed in the peripheral blood. The platelet count was 40000/lzl. A bone marrow aspirate was hyper-cellular, with predominance of granulocytic precursors, without increased blast cells. The leukocyte alkaline phosphatase score was decreased in blood and bone marrow granulocytes. Fetal hemoglobin (alkali den a t u r a t i o n method) was markedly elevated to 53%. Analysis of gamma chains of fetal hemoglobin for the ratio of glycine to alanine a t position of 136 revealed a ratio of 3 : 1. This ratio is similar to t h a t observed in newborn fetal hemoglobin as contrasted to the ratio of 2:3 observed in normal adult fetal hemoglobin (Schroeder et a l . , 1968). Serum vitamin B12 level was elevated to more t h a n 2000 pcg/ml (normal: less t h a n 200 peg/ml). A chromosome preparation of 1)HA-stimulated leukocytes revealed a 46,XY karyotype, with no evidence of a 1)h 1 chromosome. Bone marrow cell chromosomal analysis on admission was unsuccessful due to lack of observable mitoses. Other subsequent laboratory findings supporting the diagnosis of juvenile CML included consistently low A 2 hemoglobin levels (0--0.3%) and the presence in red blood cells of the neonatal i antigen. The patient's clinical course is summarized in Fig. 1. His leukocytosis fluctuated between 15000 and 134000 cells/txl; thrombocytopenia persisted despite splenectomy in November, 1974 and caused symptomatic bleeding in the last 6 weeks of his life. Blastic crisis was evident in November, 1974 when the bone marrow contained 35O/o blasts. T r e a t m e n t consisted of a short course of busulfan in June, 1974 which temporarily controlled the leukocytosis. I n November, 1974, t r e a t m e n t with vincristine, daunorubicin and cytosine arabinoside was started

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Missing Y Chromosome in Juvenile CML

261

in an attempt to control the acute blastic crisis. Despite a temporary improvement, drug resistance quickly developed and the child expired in February, 1975--12]/2 months after diagnosis.

Cytogenetic Studies A. Methods. Bone m a r r o w cells were c u l t u r e d for 24 hrs at 37°C in McCoy's m e d i u m s u p p l e m e n t e d w i t h 15~o fetal calf serum. Mitoses were a r r e s t e d w i t h Colcemid. A f t e r centrifugation, cells were s u s p e n d e d in 0.075 M KC1 for 20 rain. T h e y were r e s u s p e n d e d in C a r n o y ' s fixative several times, t h e n s p r e a d on a cold wet glass slide b y flaming. A f t e r 1 - - 2 weeks aging a t r o o m t e m p e r a t u r e , Giemsa a n d Quinacrine b a n d i n g were performed. Six to 10 sec exposure to t r y p s i n (0.0125 ~o c o n c e n t r a t i o n , t r y p s i n 1:250 from Difco L a b o r a t o r i e s , D e t r o i t , Michigan) was necessary prior to staining for o p t i m a l G-banding. 24-hour u n s t i m u l a t e d l e u k o c y t e cultures were processed similarly. Skin fibroblasts a n d p h y t o h e m a g g l u t i n i n ( P H A ) s t i m u l a t e d l e u k o c y t e cultures were processed according to a c c e p t e d m e t h o d s ( P u c k et al., 1958; I l u n g e r f o r d , 1965). D e t e c t i o n of Y bodies was u n d e r t a k e n using t h e fluorescence m e t h o d s (Pearson et al., 1970), a n d Burr bodies in buccal smears were e v a l u a t e d using an a c c e p t e d m e t h o d (Robinson a n d Puck, 1965). B. Results. I n J a n u a r y , 1974, a t t h e t i m e of t h e diagnosis, P H A - s t i m u l a t e d l e u k o c y t e s r e v e a l e d a n o r m a l 4 6 , X ¥ k a r y o t y p e (Table 1). On A u g u s t 15, a bone m a r r o w chromosome p r e p a r a t i o n w i t h Giemsa a n d Quinacrine b a n d i n g showed all mitoses to h a v e a 45,X k a r y o t y p e (Fig. 2). P H A - s t i m u l a t e d l e u k o c y t e cultures r e v e a l e d 4 5 , X / 4 6 , X ¥ mosaicism, w i t h t h e m a j o r i t y of cells showing t h e 4 5 , X k a r y o t y p e . A skin fibroblast p r e p a r a t i o n r e v e a l e d a 4 6 , X ¥ k a r y o t y p e in all cells ; t h e buccal s m e a r a t t h a t t i m e showed a p r e d o m i n a n c e of ¥ bodies a n d no B u r r bodies, consistent w i t h a male k a r y o t y p e . I n N o v e m b e r , 1974 (while t h e p a t i e n t was going into relapse) 4 5 , X / 4 6 , X Y mosaicism was e v i d e n t in bone m a r r o w cells, in t h e u n s t i m u l a t e d 24-hour l e u k o c y t e cultures, a n d in t h e P H A - s t i m u l a t e d leukoc y t e cultures. I n all 3 of these p r e p a r a t i o n s , m o s t mitoses h a d t h e 45,X k a r y o t y p e . B o n e m a r r o w a n d u n s t i m u l a t e d l e u k o c y t e cultures from cells o b t a i n e d t h e d a y

Table 1. Summary of cytogenetic studies Material

Leukocytes with PHA Bone marrow Leukocytes with PHA Paternal leukocytes with PHA Fibroblasts Bone marrow Leukocytes without PHA Leukocytes with PHA Bone marrow a Leukocytes without PHA a Leukocytes with PHA a

Date

1 29 74 8 15-74 8-15-74 8-15-75 9 12-74 11-19-74 11-19-74 11-19-74 2-7-75 2-7-75 2-7-75

a Samples obtained the day of death.

Karyotype 45,X

46,XY

Total number of metaphases examined

0 25 73 0 0 11 10 4 13 9 6

10 0 13 20 20 2 8 3 0 0 i3

10 25 86 20 20 13 18 7 13 9 19

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Fig. 2. Karyotype from a 24-hour bone marrow cell culture, demonstrating the missing Y chromosome, The banding pattern of remaining chromosomes appears normal. (Giemsa)

of the patient's death showed only the 45,X karyotype. The father's PHAstimulated leukocytes revealed a normal 46,XY karyotype. Examination of the banding pattern of metaphases containing the 45,X karyotype did not reveal any abnormality besides the loss of the Y chromosome.

Discussion The acquired nature of our patient's chromosomal disorder in his myeloid cells is attested by the normal karyotype of his fibroblasts and of his original PHAstimulated leukocytes, as well as by the male karyotype of his buceal smear. Subsequent 45,X/46,XY mosaieism reflects the simultaneous presence, in bone marrow tissue, of normal dividing male cells and leukemic cells; and in PtIAstimulated leukocytes, of leukemic cells growing along with normal lymphoeytes responding to PHA. In unstimulated (24-hour) leukocyte cultures, 4 5 , X / 4 6 , X Y mosaicism was consistent with leukemic cells growing with normal lymphocytes stimulated by an in vivo antigen (possibly a respiratory virus, as our patient suffered from severe viral respiratory illness at the time). This mosaieism could also have been due to premature release of normal bone marrow cells into the circulation along with the leukemic cells (Meissner et al., 1970) or to the existence of two parallel clones of leukemic cells. In adult patients with Ph 1 CML, loss of the Y chromosome seems to coincide with a considerably prolonged survival (Bauters et al., 1970; Sandberg and Hoss-

Missing ¥ Chromosome in Juvenile CML

263

feld, 1970; S a n d b e r g a n d S a k u r a i , 1973; G a r s o n a n d Milligen, 1972). Similarly, in an e l d e r l y p a t i e n t w i t h p h i - n e g a t i v e CML, Hossfeld a n d W e n d e h o r s t (1974) r e p o r t e d an u n u s u a l l y benign clinical course as o p p o s e d to t h e r a p i d l y m a l i g n a n t e v o l u t i o n o f m o s t P h i - n e g a t i v e CML cases in a d u l t s (Ezdinli et al., 1970). T h e y s p e c u l a t e d t h a t t h e missing ¥ c o n s t i t u t i o n of bone m a r r o w cells was p r o b a b l y responsible for t h e m o r e f a v o r a b l e o u t c o m e in these v a r i a n t s of CML. H u r d l e et al. (1972) also r e p o r t e d a group of p a t i e n t s w i t h a n i s o l a t e d loss of ¥ c h r o m o s o m e in bone m a r r o w tissue d u r i n g chronic m y e l o m o n o c y t i c leukemia, who were following a m o r e chronic course t h a n m o s t p a t i e n t s w i t h t h a t form of leukemia. All of these p a t i e n t s , however, as well as t h e one of t t o s s f e l d a n d W e n d e h o r s t , were over 65 y e a r s of age. I t is impossible, in these cases, t o rule o u t a coincidental ager e l a t e d loss of t h e ¥ c h r o m o s o m e (Pierre a n d H o a g l a n d , 1972; Court-Brown, 1967). A r e c e n t r e p o r t (Lawler et al., 1974) emphasizes this view a n d also casts serious d o u b t s u p o n t h e beneficial effect of a missing Y chromosome in a d u l t , p h i - p o s i t i v e CML : t h e m e d i a n l e n g t h of t h e chronic p h a s e was i d e n t i c a l (27 m o n t h s ) a m o n g s t t h e i r 8 - - Y p a t i e n t s a n d t h e i r 66 X ¥ p a t i e n t s . I n our 3 ~ - y e a r - o l d p a t i e n t , e a r l y a n d f u l m i n a n t d e v e l o p m e n t of a c u t e b l a s t i c crisis was n o t influenced b y t h e presence of t h e 45,X k a r y o t y p e in m y e l o i d tissue. A l t h o u g h m o r e cases are n e e d e d to resolve this issue, a missing Y c h r o m o s o m e in bone m a r r o w ceils d i d n o t seem t o confer a p r o g n o s t i c a d v a n t a g e t o this child w i t h t h e j u v e n i l e t y p e of CML.

Acknowledgements. We gratefully acknowledge the determination of glycine/alanine ratios in position 136 of fetal hemoglobin gamma globin chains by Dr. T. H. J. Huisman at the Medical College of Georgia and Dr. W. A. Schroeder at the California Institute of Technology. We also thank Dr. B. L. Rich for his referral of this patient to the Oncology Center of Denver Children's Hospital. References Bauters, F., Croquette, M. F., Delmas-l~[arsalet, Y., Deminati, M., Goudemand, M. : Une forme partieuli~re de leuc@mie my61oide chez l'homme: 6volution prolong6e et pr@sence du chromosome 1)hiladelphie avec perte du chromosome Y dans les cellules my6loidcs (£ propos de trois observations). Nouv. Rev. franc. H@mat. 10, 697--708 (1970) Court-Brown, W. M. : Human population cytogenetics. Amsterdam: North-Holland 1967 Ezdinli, E. Z., Sokal, J. E., Crosswhite, L., Sandberg, A. A. : 1)hiladelphia-chromosome-positive and -negative chronic myelocytic leukemia. Ann. intern. Med. 72, 175--182 (1970) Garson, O. M., Milligan, W. J. : The 45, XO, Ph 1 subgroup of chronic granulocytic leukaemia. Scand. J. Haemat. 9, 186--192 (1972) Hossfeld, D. K., Wendehorst, E. : 1)hi-negative chronic myelocytic leukemia with a missing Y chromosome. Acta haemat. (Basel) 52, 232--237 (1974) tIungerford, D. A. : Lenkocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonie KC1. Stain Technol. 40, 333--338 (1965) Hurdle, A. D. F., Garson, O. M., Buist, D. G. 1). : Clinical and cytogenetic studies in chronic myelomonocytic leukaemia. :Brit. J. Haemat. 22, 773--782 (1972) Lawler, S. D., Lobb, D. S., Wiltshaw, E.: 1)hiladelphia-chromosome positive bone-marrow cells sharing loss of the ¥ in males with chronic myeloid leukaemia. Brit. J. Haemat. 27, 247--252 (1974) Meisner, L. F., Inhorn, S.L., Chuprevich, T. W.: Cytogenetic analysis as a diagnostic aid in leukemia. Amer. J. clin. Path. 60, 435--444 (1973) Pearson, 1). L., Bobrow, M., Vosa, C. G. : Technique for identifying Y chromosomes in human interphase nuclei. Nature (Lond.) 226, 78--80 (1970)

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Pierre, R.V., Hoagland, H.C.: Age-associated aneuploidy: Loss of Y chromosome from human bone marrow cells with aging. Cancer (Philad.) ~0, 889--894 (1972) Puck, T. T., Ciecinra, S. J., Robinson, A. : Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. exp. Med. 108, 945--956 (1958) Robinson, A., Puck, T. T. : Sex chromatin in newborns: Presumptive evidence for external factors in human nondisjunction. Science 148, 83--85 (1965) Sandberg, A. A., I-Iossfeld, D. K. : Chromosomal abnormalities in human neoplasia. Ann. Rev. Med. 21, 379--408 (1970) Sandberg, A.A., Sakurai, M.: The missing ¥ chromosome and human leukaemia. Lancet 1978 I, 375 Schroeder, W. A., Huisman, T. H. J., Shelton, J. R., Kleihauer, E. F., Dozy, A. )5., Robberson, B. : Evidence for multiple structural genes for the ? chain of human fetal hemoglobin. Proe. nat. Acad. Sci. (Wash.) 60, 537--544 (1968) Taru Hays, M.D. Oncology Center Denver Children's Hospital Denver, Co. 80220, USA