Major karyotypic abnormalities in a near-tetraploid erythroleukemia

Major Karyotypic Abnormalities in a Near-Tetraploid Erythroleukemia Mirjana J. Ko ova and Avery A. Sandberg

ABSTRACT: A case of ery~hroleukemia (EL; FAB M6) is presented in which the leukemic cells af the marrow were characterized by two ranges in the chromosome number, i.e., one hypodiploid and another near-tetraploid. All characteristics of major karyotypic abnormalities (MAKA) were present, i.e., increased karyotype instability with variation of the chromosome count from metaphase to metaphase, dicentrics, acentrics, marker chromosomes, double minute chromosomes (DMs), and centromere spreading. All of these events were variably represented from metaphase to metaphase. The prognosis was poor, with survival of 1 month after the diagnosis of EL. The meaning of extreme MAKA in the classification of EL is discussed. INTRODUCTION Chromosome analysis of bone marrow cells in leukemia has been s h o w n to be a useful, if not necessary, p r o c e d u r e for diagnostic and prognostic purposes, as nonr a n d o m c h r o m o s o m e rearrangements have been found to characterize particular types of l e u k e m i a [1-9]. Even though no particular chromosome rearrangement has been found to be specific for erythroleukemia (EL), some cytogenetic events and involvement of some c h r o m o s o m e groups have been found to occur more often than others [1, 10-12]. At least three groups of EL can be distinguished on the basis of the c h r o m o s o m e findings, as p r o p o s e d by Sakurai and Sandberg [10]: (a) EL w i t h o u t c h r o m o s o m e abnormalities, (b) EL with c h r o m o s o m e abnormalities similar to those of other acute n o n l y m p h o c y t i c l e u k e m i a (ANLL) groups, and (c) EL with major karyotype abnormalities (MAKA). As p r o p o s e d by Bernheim et al. [13], in EL with c h r o m o s o m e changes, proliferating erythroblasts are a part of the leukemic clone, whereas the proliferation of the erythroblasts in cases without c h r o m o s o m e abnormalities is only a response to some u n k n o w n stimulant, with the cells not being a part of the leukemic clone. Considering the c h r o m o s o m e counts, h y p o d i p l o i d , p s e n d o d i p l o i d , and h y p e r d i p loid cases have been reported. High p o l y p l o i d y or near-tetraploidy, however, has been rarely described [11, 14]. Attempts have been m a d e recently to characterize EL cytogenetically [11, 13]. Undoubtedly, as large series of EL cases are studied, particularly with refined and a d v a n c e d cytogenetic techniques, resolution of this group of leukemias into circumscribed entities will become apparent. We report a case of EL with p r e d o m i n a n t l y near-tetraploid cells and with a host of c h r o m o s o m e changes, some of w h i c h have been previously described in EL. From the Roswell Park Memorial Institute, Buffalo,NY. Address requests far reprints to Dr. Avery A. Sandberg, Department of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263. Received June 15, 1984; accepted September 5, 1984.

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Cancer Genetics and Cytogenetics 17, 143-150 (1985) 0165-4608/85/$03.30

144

M.J. Kocova and A. A. Sandberg

CASE REPORT

A 50-year-old white male (C.D.) was admitted to another hospital in May 1983 with a history of dizziness, malaise, fatigue, and epistaxis. Severe anemia was found at that time. The patient had a hemoglobin of 6 g/dl, a WBC of 3000/mm 3, and a platelet count of 41,000/mm 3. Bone marrow examination revealed normoblastic h y p o p l a sia with features suggestive of a m y e l o d y s p l a s t i c syndrome. Serum vitamin B12 and folate levels were normal. Treatment w i t h vitamins led to no response. The patient was transfused several times prior to discharge from the hospital. He c o n t i n u e d to have nose bleeds, significant transfusion requirements, and his clinical condition deteriorated slowly. He was admitted to Roswell Park Memorial Institute in September 1983. On a d m i s s i o n his hemoglobin was 10 g/dl, WBC 2900/mm 3, and the platelet count 21,000/mm 3. Bone marrow examination revealed hypocellularity, with some h y p e r c e l l u l a r areas, with evidence of an erythroleukemia (FAB M6), including increased megaloblastic erythroid precursors (39%-57%). The erythroid cells showed abnormal mitoses and numerous vacuolated forms. The myeloblasts were also increased (29%-37%), but no Auer rods were present. The blasts were periodic acid-Schiff (PAS) positive. Treatment with A d r i a m y c i n and cytosine arabinoside (Ara-C) was performed, but no remission was achieved. The patient also received several blood and leukocyte transfusions. Two weeks after the beginning of therapy, a febrile episode was d o c u m e n t e d and P s e u d o m o n a s was cultured from the blood. Spreading cellulitis d e v e l o p e d over the right side of the neck, requiring drainage. Antibiotics were given at that time. The course of the disease was c o m p l i c a t e d by the d e v e l o p m e n t of acute renal failure. The patient expired 1 month after the diagnosis of l e u k e m i a was established and 4 months after the onset of symptoms. MATERIALS AND METHODS

Bone marrow cells obtained before c h e m o t h e r a p y were cultured for 24 hr in RPMI m e d i u m s u p p l e m e n t e d w i t h 17% fetal calf serum. Methotrexate synchronization was a c c o m p l i s h e d in some cultures according to previously described m e t h o d s [15, 16]. Colcemid was a d d e d to the n o n s y n c h r o n i z e d cultures for the last 2 hr of incubation, and for the last 15 m i n to the s y n c h r o n i z e d culture at a final concentration of 0.015 ~xg/ml, Chromosome preparations were m a d e with a conventional airdrying method. G-banding was achieved by staining the slides with Wright's stain according to the m e t h o d of Yunis [15] and C-banding with b a r i u m h y d r o x i d e and Giemsa staining [1]. Both n o n s y n c h r o n i z e d and s y n c h r o n i z e d metaphases were analyzed; a higher mitotic i n d e x and more elongated chromosomes were observed in the s y n c h r o n i z e d cultures than in the u n s y n c h r o n i z e d ones. The c h r o m o s o m e s were classified according to the International Nomenclature. RESULTS

A total of 71 r a n d o m l y chosen G- or C-banded metaphases were analyzed, w i t h the cells showing extremely variable chromosome counts (Fig. 1). Two ranges in the c h r o m o s o m e count were revealed, i.e., one h y p o d i p l o i d with a m o d e of 43 and another h y p o t e t r a p l o i d with a m o d e of 86-89 chromosomes (54% of all metaphases). Seven metaphases contained more than 95 chromosomes. No normal karyotypes were observed. The chromosome rearrangements were variable from metaphase to metaphase. Nine chromosome rearrangements with different frequencies were identified in most of the metaphases (Table 1; Fig. 2). Th~ following rearrangements were identified: 9 q + (9q34); 6 q - (6q15); an isochromo-

145

MAKA in Erythroleukemia

1

6 G) 5

~4 w nw 2

, ,,, [J,

Z I

I I I I I 28 36 37 4.t 42

I

43 44 45 4G47

1

I

I

I

I

I

I

I

i

53 54 57 58 60 63 69 73 8R 83 86 88

I

I

I

I

I

89 95100tOU04106

NUMBER OF CHROMOSOMES

F i g u r e 1 Histogram showing a bimodal distribution of the chromosome number in the bone marrow cells in the EL case presented.

F i g u r e 2 Tentative identification of chromosome changes in near-tetraploid cells. Some of the chromosome rearrangements are presented by two copies. The ring chromosome was derived from a chromosome #1 and is also represented by two copies. Breakpoints are discussed in the text.

r1

1

M1

M2

II

21

i(21q)

i M3

lS

di¢ 15

r2

rl

146

M . I . Kocova and A. A. Sandberg

O

m

o 8

e t

~o

i

o

Q

-

Figure 3 Two C-banded metaphases from bone marrow cells. Right metaphase: arrows point to two dicentric ring chromosomes. Left metaphase: arrows point to four monocentric ring chromosomes and one large dicentric chromosome. some for the long arm of a c h r o m o s o m e #21, i(21q); a dicentric c h r o m o s o m e #15, dic(15); three u n i d e n t i f i e d marker chromosomes; and two ring c h r o m o s o m e s of w h i c h one was identified as originating from c h r o m o s o m e #1 and the other possibly from c h r o m o s o m e #18. All of these k a r y o t y p i c events were r e p r e s e n t e d in 10 of 35 hyp o t e t r a p l o i d metaphases by two copies each and in single copy in 7 of 30 h y p o d i p loid cells. Ring c h r o m o s o m e s were present in 50 of 71 metaphases (1-5 rings) (Fig. 3). C-banding confirmed some of the ring c h r o m o s o m e s to he dicentrics (Fig. 4~. Numerically, chromosomes #2, #4, #17, and #18 were u n d e r r e p r e s e n t e d , whereas chromosomes #8, #9, #16, and #22 were overrepresented in the polyp l o i d cells. Loss of Y c h r o m o s o m e was seen in 40 metaphases. Other cytogenetic events were also observed, i.e., two pairs of DMs in 35 metaphases, acentric c h r o m o s o m e s (other than chromosome #15) in 3 metaphases, and centromere spreading in 2 metaphases (Fig. 5).

DISCUSSION

A variety of c h r o m o s o m a l abnormalities have been reported in EL, but none seems to be n o n r a n d o m l y associated with the disease. However, considering the chromosomal abnormalities, three groups of EL can be distinguished, as p r o p o s e d by Sakurai and Sandberg [10]: (a) cases with no c h r o m o s o m e abnormalities, (b) cases with chromosome changes similar to those in other ANLL groups, (c) cases with signifi-

MAKA in Erythroleukemia

147

Figure 4 Two metaphases from bone marrow cells shewing centromere spreading. Almost all chromosomes are affected.

cant karyotype instability and major karyotypic abnormalities (MAKA). Cases belonging to the first two categories have been reported often [10, 12, 13]. The third category seems to be heterogeneous in composition, inasmuch as two types of EL and MAKA appear to exist, i.e., one without polyploidization but with all characteristics of MAKA and another with MAKA and polyploidization [1, 11-14]. This can possibly be explained in two ways. Either two separate types of EL exist, one without tendency for polyploidization and another without such a tendency but with marked karyotype instability. Or, in the course of the disease, all EL cases with MAKA ultimately develop a tendency for polyploidization, with poor spreading of polyploid metaphases leading to their elimination from analysis. Among 71 metaphases analyzed in detail in our case, no normal metaphases were observed. We found two categories of metaphases, one in the near-diploid range (46% of the metaphases) and another in the near-tetraploid range (54% of the metaphases. Because the marker chromosomes (i.e., rings, dicentrics, DMs) were present in near-diploid cells and in two copies in the near-tetraploid cells, two possibilities may explain the observations: either the basic clone is near-diploid and the near-tetraploid cells are generated by polyploidization, or the basic clone is near-tetraploid and the near-diploid cells are generated by loss of chromosomes. However, because no near-diploid metaphase was found with two copies of any marker chromosome, the first possibility appears to us to be more likely, as proposed also by Gibas et al. [11]. The same authors assumed that hypotetraploid cells progress much slower through the cell cycle than do diploid or near-diploid ones, and thus, the hypotetraploid cells have the advantage of longer survival in the marrow, ultimately resulting in complete loss of the normal cells and the near-diploid

148

M.J. Kocova and A. A. Sandberg

Figure 5

Arrows point to two pairs of DMs in a bone marrow cell. One ring is also present.

leukemic cells. It is possible that if the survival of the patient is long enough, all marrow cells will be replaced by polyploid clones in the evolution of the disease. Erythroleukemia is thought to involve an abnormal proliferation of both myeloid and erythroblastic precursors, though the erythroblastic proliferation has been thought to be a physiologic response to some u n k n o w n stimulus accompanying the myeloid proliferation [13~. EL is a rare form of ANLL, constituting approximately 5% of all ANLL cases in large series, as indicated by Bernheim et al. [13]. The same authors suggested the existence of two types of leukemias with erythroblastic proliferation, i.e., ANLL with the erythroblasts as a part of the aneuploid leukemic clone {true EL), and another with erythroblastic proliferation but without chromosome abnormalities in the erythroblasts, in which erythroblastic proliferation is only a reactive p h e n o m e n o n to some u n k n o w n stimulant (reactive EL). Our case belongs to the first category.

Table 1

Chromosome rearrangements and their frequency in the case of EL studied Unidentified markers

Identified structural abnormalities

1 Copy 2 Copies Total

9q +

6q-

i(21q)

dic(15)

r(1)

r(18)

M2

M2

M3

17 20 37

10 12 22

18 15 33

9 5 14

20 25 45

10 13 23

9 10 19

7 -7

5 1 6

M A K A in E r y t h r o l e u k e m i a

149

Karyotypic instability has often been observed in EL. It was very impressive in our case and was represented by different manifestations among the cells, as well as by cell~to-cell variation, i.e., by m a n y rings (1-5 per metaphase, some of them being monocentric and some dicentric), acentric and dicentric chromosomes, DMs, and centromere spreading. Ring c h r o m o s o m e s have been described in EL more often than in other ANLL groups and seem to be a manifestation of the karyotypic instability [12]. DMs have also been described in EL and are not rare in other hematologic malignancies as a manifestation of gene amplification [17, 18]. Even though centromere spreading may be considered a p h e n o m e n o n resulting from vitamin B12 insufficiency [1], in our case, the level of serum vitamin B12 was normal, and hence, the centromere spreading could be the result of, and m a y have been more directly related to, the karyotypic instability, the m e c h a n i s m of w h i c h remains to be elucidated. Centromere spreading, as far as we know, has not been described in erythroleukemia. No specific c h r o m o s o m e rearrangements have been reported in EL, though some groups of chromosomes seem to be more often affected than others [1]. The relative increase in the n u m b e r of chromosomes #16, #1, and #3 was previously described by Sakurai and Sandberg [10] and later observed by Gibas et al. [11]. We also found a high frequency of c h r o m o s o m e #16. However, we did not find an increased number of c h r o m o s o m e s #1 a n d # 3 . The low range of chromosomes #17 and #18 has been p r e v i o u s l y described [10] and was present in our case. A variety of structural rearrangements in EL have been described [19, 20]; often, these are difficult to identify. A n isochromosome for the long arm of a c h r o m o s o m e #21 has been described [11]. Our findings support the consideration that this karyotypic change could possibly be a n o n r a n d o m c h r o m o s o m e rearrangement, at least in one subtype of EL. Also, the formation of ring chromosomes can be one of the characteristics of EL. A poor prognosis has been reported in patients with MAKA and EL [10-12]. Our case survived only several months after the onset of symptoms. The case of EL described by us is unique because of the n u m b e r of u n u s u a l cytogenetic events, and based on the c o m p r e h e n s i v e cytogenetic analysis, several considerations can be promulgated. EL is not a homogeneous disease. A n attempt at classification of EL into subgroups is s u p p o r t e d cytogenetically by the results of the analyzed cases. The near-tetraploid cases of EL with extreme M A K A probably form one subgroup. The p o l y p l o i d i z a t i o n in n e a r - d i p l o i d cases is probably a secondary event in ANLL following chromosome rearrangements. The remarkable karyotype instability can point to the diagnosis of EL at least in some of the cases. Chromosomes #16, #17, #18, and #21 appear to be n o n r a n d o m l y affected in EL.

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