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

Clinical Case Reports

Missing Y Chromosome in Juvenile Chronic Myelogenous Leukemia*

Taru Hays , J a m e s R. H u m b e r t , D a v i d C, Peakman , John J. Hur te r , Helvise G. Morse, A r t h u r Robinson, and Charles S. Augus 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

" Juven i l e " chronic myelogenous leukemia (CML) differs from the adu l t - type , ph i -pos i t i ve CML b y its much more ma l ignan t clinical course, the usual absence of a Ph 1 chromosome, the presence of high fetal hemoglobin levels, and i ts oc- currence in children. The absence of a P h 1 chromosome also carries a poor prog- nosis in adu l t pa t i en t s wi th CML (Ezdinl i et al., 1970). On the o ther hand, adu l t male pa t i en t s wi th p h i - p o s i t i v e CML who had a missing ¥ chromosome in the i r bone mar row cells have been descr ibed; the pa r t i cu la r ben ign i ty of the i r clinical evolu t ion has been a t t r i b u t e d to the missing Y chromosome (Bauters et al., 1970; Sandberg and Hossfeld, 1970) a l though this po in t has been deba ted recen t ly (Lawler et al., 1974). No child wi th juveni le CML associa ted wi th a missing ¥ chromosome has been repor ted to date , and thus the possible beneficial effect of t h a t aneup lo idy has not been eva lua ted in t h a t condit ion. This paper is con- cerned wi th such a case, who ran a course as ma l ignan t as t h a t o rd ina r i ly observed in o ther pa t i en t s wi th P h 1 chromosome-nega t ive juveni le 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.

260 T. Hays et a l .

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 adeno- pathies were observed. Hemoglobin and hematocr i t 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, wi thout increased blast cells. The leukocyte alkaline phosphatase score was decreased in blood and bone marrow granulocytes. Fetal hemoglobin (alkali de- na tura t ion method) was markedly elevated to 53%. Analysis of gamma chains of fetal hemo- globin 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 adul t fetal hemoglobin (Schroeder et a l . , 1968). Serum vi tamin B12 level was elevated to more t han 2000 pcg/ml (normal: less t han 200 peg/ml). A chromosome prep- arat ion 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 pat ient ' s clinical course is summarized in Fig. 1. His leukocytosis f luctuated between 15000 and 134000 cells/txl; thrombocytopenia persisted despite splenectomy in November, 1974 and caused symptomat ic bleeding in the last 6 weeks of his life. Blastic crisis was evident in November, 1974 when the bone marrow contained 35O/o blasts. Trea tment consisted of a short course of busulfan in June, 1974 which temporari ly controlled the leukocytosis. In No- vember, 1974, t r ea tment with vincristine, daunorubicin and cytosine arabinoside was s tar ted

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arabinoside

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 mar row cells were cu l tu red for 24 hrs a t 37°C in McCoy's med ium supp lemen ted wi th 15~o fe ta l calf serum. Mitoses were a r res ted wi th Colcemid. Af te r centr i fugat ion, cells were suspended in 0.075 M KC1 for 20 rain. They were resuspended in Carnoy 's f ixat ive several t imes, t hen sp read on a cold wet glass slide b y flaming. Af te r 1 - -2 weeks aging a t room t empera tu re , Giemsa and Quinacr ine band ing were performed. Six to 10 sec exposure to t ryps in (0.0125 ~o concent ra t ion , t ryps in 1:250 from Difco Labora tor ies , Det ro i t , Michigan) was necessary pr ior to s ta in ing for op t ima l G-banding. 24-hour uns t imu la t e d l eukocyte cul tures were processed s imilar ly. Skin f ibroblasts and phy tohe ma gg lu t i n in (PH A ) - s t imu la t ed l eukocyte cul tures were processed according to accepted me thods (Puck et al., 1958; I lunger fo rd , 1965). De tec t ion of Y bodies was u n d e r t a k e n using the fluorescence me thods (Pearson et al., 1970), and Burr bodies in buccal smears were eva lua t ed using an accepted m e t h o d (Robinson and Puck, 1965).

B. Results. I n J a n u a r y , 1974, a t the t ime of the diagnosis, P H A - s t i m u l a t e d leukocytes revea led a no rma l 4 6 , X ¥ k a r y o t y p e (Table 1). On Augus t 15, a bone mar row chromosome p repa ra t i on wi th Giemsa and Quinacr ine band ing showed all mitoses to have 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 leukocyte cul tures revealed 4 5 , X / 4 6 , X ¥ mosaicism, wi th the m a j o r i t y of cells showing the 45 ,X ka ryo type . A skin f ibroblas t p r e p a r a t i o n revea led a 4 6 , X ¥ k a r y o t y p e in all cells ; the buccal smear a t t h a t t ime showed a p redominance of ¥ bodies and no Bur r bodies, consis tent wi th a male ka ryo type . I n November , 1974 (while the pa t i e n t was going into relapse) 45 ,X/46 ,XY mosaicism was ev ident in bone mar row cells, in the uns t imu la t ed 24-hour l eukocyte cultures, and in the P H A - s t i m u l a t e d leuko- cyte cultures. I n all 3 of these p repara t ions , mos t mitoses had the 45,X k a r y o t y p e . Bone mar row and u n s t i m u l a t e d leukocyte cul tures from cells ob ta ined the d a y

Table 1. Summary of cytogenetic studies

Material Date Karyotype Total number of metaphases

45,X 46,XY examined

Leukocytes with PHA 1 29 74 0 Bone marrow 8 15-74 25 Leukocytes with PHA 8-15-74 73 Paternal leukocytes with PHA 8-15-75 0 Fibroblasts 9 12-74 0 Bone marrow 11-19-74 11 Leukocytes without PHA 11-19-74 10 Leukocytes with PHA 11-19-74 4 Bone marrow a 2-7-75 13 Leukocytes without PHA a 2-7-75 9 Leukocytes with PHA a 2-7-75 6

10 10 0 25

13 86 20 20 20 20

2 13 8 18 3 7 0 13 0 9

i3 19

a Samples obtained the day of death.

262 T. Hays et al.

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 PHA- stimulated leukocytes revealed a normal 46,XY karyotype. Examinat ion of the banding pat tern of metaphases containing the 45,X karyotype did not reveal any abnormali ty 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 PHA- stimulated 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 PtIA- stimulated 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; Sandberg and Sakura i , 1973; Garson and Milligen, 1972). Similar ly , in an e lder ly p a t i e n t wi th p h i - n e g a t i v e CML, Hossfeld and W e n d e h o r s t (1974) r epo r t ed an unusua l ly benign clinical course as opposed to the r a p id ly ma l ignan t evolu t ion of mos t P h i - n e g a t i v e CML cases in adu l t s (Ezdinl i et al., 1970). T h e y specula ted t h a t t he missing ¥ cons t i tu t ion of bone mar row cells was p r o b a b l y responsible for t he more favorab le ou tcome in these va r i an t s of CML. Hurd l e et al. (1972) also r epo r t ed a group of pa t i en t s wi th an i so la ted loss of ¥ chromosome in bone mar row t issue dur ing chronic mye lomonocy t i c leukemia, who were fol- lowing a more chronic course t h a n mos t pa t i en t s wi th t h a t form of leukemia. Al l of these pa t ien ts , however , as well as the one of t tossfe ld and Wendehors t , were over 65 years of age. I t is impossible, in these cases, to rule out a coincidenta l age- r e l a t ed loss of the ¥ chromosome (Pierre and Hoag land , 1972; Cour t -Brown, 1967). A recent r epo r t (Lawler et al., 1974) emphasizes th is v iew and also casts serious doub t s upon the beneficial effect of a missing Y chromosome in adul t , p h i - p o s i t i v e CML : the med ian length of the chronic phase was ident ica l (27 months) amongs t the i r 8 - - Y pa t i en t s and the i r 66 X ¥ pa t ien ts . I n our 3 ~ - y e a r - o l d pa t ien t , ear ly and fu lminan t deve lopmen t of acu te b las t ic crisis was no t influenced b y the presence of the 45,X k a r y o t y p e in mye lo id tissue. A l though more cases are needed to resolve this issue, a missing Y chromosome in bone mar row ceils d id no t seem to confer a prognos t ic a d v a n t a g e to th is child wi th t he juveni le 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

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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-posi-

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Scand. J. Haemat. 9, 186--192 (1972) Hossfeld, D. K., Wendehorst, E. : 1)hi-negative chronic myelocytic leukemia with a missing

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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)

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264 T. Hays et al.

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Schroeder, W. A., Huisman, T. H. J., Shelton, J. R., Kleihauer, E. F., Dozy, A. )5., Robber- son, 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