Trisomy of Chromosome 8 in Myelodysplastic Syndrome Significance of the Fluctuating Trisomy 8 Population Atsuhiro Iwabuchi, Kazuma Ohyashiki, Junko H. Ohyashiki, Isayo Sasao, Tomofumi Murakami, Atsushi Kodama, and Keisuke Toyama
ABSTRACT: Chromosome analyses were performed in five patients with myelodysplastic syndrome (MDS) who showed trisomy of chromosome 8 during the course of their disease. Four of these patients showed trisomy 8 at the diagnosis of MDS, and the remaining one had trisomy 8 when the leukenlia
phase developed. The proportion of bone marrow (BM) cells with trisomy 8 in the four patients who showed trisomy 8 at MDS diagnosis fluctuated, and this fluctuation was not related to the percentage of blasts in the BM or to progression of the disease. However, in two patients, metaphase cells with trisomy 8 disappeared when their anemic state improved, although leukothrombocytopenia was still present, suggesting that the decrease in the number of BM cells with trisomy 8 reflects hematologic features in some MDS patients. These findings indicate that trisomy 8 in our MDS patients was possibly not the primary event in the genesis of the disease, and that there may have been competition between a normal karyotype clone and a trisomy-8-positive clone. O a r results further suggest that the presence of a clone with trisomy 8 is not always a sign of disease progression or of poor prognosis in MDS patients.
INTRODUCTION
Numerical c h r o m o s o m e abnormalities, some of w h i c h sup e r i m p o s e on specific structural rearrangements, are frequently observed in h u m a n cancer cells [1]. For example, an extra c h r o m o s o m e 8 is detected in some patients with P h i l a d e l p h i a c h r o m o s o m e - p o s i t i v e chronic myelogenous l e u k e m i a in blast crisis; in these patients, the appearance of such a d d i t i o n a l c h r o m o s o m e abnormalities, including trisomy 8, is related to the progression of the disease [1]. In m y e l o d y s p l a s t i c s y n d r o m e s (MDS), however, the biologic and hematologic significance of numerical c h r o m o s o m e changes is not well understood. In a case exhibiting numerical changes in a d d i t i o n to a prototypic anomaly, one can easily recognize w h i c h is the original anomaly. By contrast, single n u m e r i c a l c h r o m o s o m e changes in hematopoietic neoplasias are frequently observed to have a clonal nature. In such cases, it is unclear w h e t h e r such single numerical abnormalities should be regarded as a d d i t i o n a l changes w h i c h are s u p e r i m p o s e d on undetectable genetic aberra-
From the Department of Internal Medicine (A. I., K. 0., J. H. 0.. I. S., T. M., K. T.); Chromosome Unit, Central Laboratory [A. K.], Tokyo Medical College, Tokyo 160, Japan. Address reprint requests to: Kazuma Ohyashiki, M.D., First Department of Internal Medicine, Tokyo Medical College, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan. Received March 31, 1992; accepted May 1, 1992. 70 Cancer Genet Cytogenet 62:70 74 (1992) 0165-4608/92/$05.00
tions or w h e t h e r they are p r i m a r y events in cancer development. In this investigation, we sequentially e x a m i n e d the association between clinical and cytogenetic findings in MDS patients with trisomy 8. Because MDS result from a clonal h e m o p a t h y of stem cells, most bone marrow (BM) cells obtained from MDS patients are c o n s i d e r e d to be derived from abnormal stem cells, in contrast to the situation in acute leukemia. Therefore, BM m e t a p h a s e cells in MDS patients, even if they have n o r m a l karyotype, could be considered to be cells derived from the MDS clone.
PATIENTS AND METHODS
A series of cytogenetic investigations was performed in 97 patients with MDS at the Tokyo M e d i c a l College Hospital, between January 1986 and January 1991 [21. The diagnosis of MDS was based on the FAB criteria [3]. For cytogenetic analysis, BM cells were short-term (48-hour) c u l t u r e d and processed as described p r e v i o u s l y [2]. C h r o m o s o m e s were stained with Hoechst 33258 and q u i n a c r i n e m u s t a r d and were identified in accordance with the ISCN [4]. During this series of investigations, we e n c o u n t e r e d five patients with MDS showing trisomy 8: four patients had this a n o m a l y at the time of MDS diagnosis [2], and the remaining one showed the a n o m a l y during the course of the study. A further cytogenetic s t u d y was performed in .~, 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10010
T r i s o m y 8 in M D S
Table 1 Patient no. 1
2
3
4
5
71
S e q u e n t i a l c y t o g e n e t i c f i n d i n g s in m y e l o d y s p l a s t i c s y n d r o m e s w i t h t r i s o m y 8 Date of examination
Diagnosis (FAB)
Percentage of BM blasts
8/11/86 9/6/87 5/17/88 10/26/89 2/22/90 10/6/90 10/5/91 4/25/89 5/9/89 7/24/89 9/16/89 9/18/89 8/2/88 8/11/88 8/17/88 9/1/88 11/30/89 5/31/90 7/3/90 9/26/90 1/21/91 2/13/91 3/26/91 4/8/91 4/26/89 6/21/89 11/2/89 12/4/89 6/10/88 7/24/91
RA RA RA RA RA RA RA RAEB RAEB RAEB Overt-M1 Overt-M1 RAEB RAEB RAEB RAEB RAEB RAEBT Overt-M1 Overt-M1 Overt-M1 Overt-M1 Overt-M1 Overt-M1 RA RA RA RA RARS RARS
4.0 3.2 2.8 3.0 4.0 0.8 0.4 4.8 5.6 3.0 83.6 5.4 4.4 1.8 2.4 4.2 17.0 30.0 18.8 41.6 69.0 36.6 27.6 1.8 2.0 2.0 3.4 1.0 2.0
Samples
No. of cells analyzed
BM BM BM BM BM BM BM BM BM BM BM PB BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM
21 2O 20 10 20 20 11 20 20 20 20 20 20 50 20 50 25 20 13 20 20 10 9 5 7 2O 20 20 20 20
No. of cells with + 8 (%) 8 16 18 10 10 0 0 0 0 0 2 2 1 2 2 0 2 2 5 1 0 1 0 0 0 2 8 1 20 0
(38) (80) (90) (100) (50) (0) (0) (0) (0) (0) (10) (10) (5) (4) (10) (0) (8) (10) (38) (10) (0) (5) (0) (0) (0) (10) (40) (5) (100) (0)
Abbreviations: RA, refractory anemia; RAEB, refractory anemia with an excess of blasts; RAEBT,refractory anemia with an excess of blasts in transformation;
Overt-M1, leukemic transformation with an M1 morphology; RARS, refractory anemia with ringed sideroblasts; BM, bone marrow (48-hour culture); PB, peripheral blood (48-hour culture without addition of mitogens).
t h e s e five patients, and BM cells w i t h t r i s o m y 8 w e r e f o u n d to fluctuate. T h e d e t a i l e d c l i n i c a l features of the patients are d e s c r i b e d b e l o w . Patient 1 Patient 1 was a 2 4 - y e a r - o l d male, w h o was i n i t i a l l y f o u n d to h a v e p a n c y t o p e n i a in May 1986. A d i a g n o s i s of refractory a n e m i a (RA) was m a d e in A u g u s t 1986. T h e p a t i e n t was treated w i t h an a n a b o l i c steroid ( f l u o x y m e s t e r o n e , 15 mg/ day) and p r e d n i s o l o n e I20 mg/day). H o w e v e r , the a n e m i a p r o g r e s s e d ( h e m o g l o b i n was 49 g/L) and he b e c a m e transfus i o n - d e p e n d e n t . F r o m July 1990, he was treated w i t h oral v i t a m i n D 3 (2/xg/day), a n d his a n e m i c state i m p r o v e d (105 g/L) w i t h o u t red cell t r a n s f u s i o n . Patient 2 P a t i e n t 2 was a 38-year-old female, w h o c o m p l a i n e d of n i g h t s w e a t s and fatigue in A u g u s t 1988, and w h o was a d m i t t e d in M a r c h 1989. T h i s p a t i e n t h a d a prior history of gastric cancer, in 1981, a n d h a d b e e n treated by subtotal g a s t r e c t o m y f o l l o w e d by c h e m o t h e r a p y ( m i t o m y c i n C 118 rag, tegafur 1640 rag, and f l u o r o u r a c i l 3500 mg). At the t i m e
of MDS d i a g n o s i s ( s e c o n d a r y MDS), her b l o o d test s h o w e d p a n c y t o p e n i a . T h e BM was n o r m o c e l l u l a r w i t h 4% blasts w i t h A u e r rods, suggesting the d i a g n o s i s of refractory anemia w i t h an excess of blasts (RAEB). T h e p a t i e n t w a s treated w i t h i n t r a m u s c u l a r c y t o s i n e a r a b i n o s i d e (30 mg) and vitam i n D 3 (2 /~g/day), but an i n c r e a s e (65%) of blasts in the b l o o d was n o t e d in S e p t e m b e r 1989, and c y t o t o x i c drugs w e r e t h e n a d m i n i s t e r e d . H o w e v e r , the p a t i e n t d e v e l o p e d d i s s e m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n w i t h acute renal failure; she d i e d on S e p t e m b e r 27, 1989. Patient 3 T h i s p a t i e n t was a 54-year-old male, w h o was f o u n d to h a v e p a n c y t o p e n i a in O c t o b e r 1987, w h e n a d i a g n o s i s of RAEB was m a d e . T h e p a t i e n t was treated w i t h r e c o m b i n a n t h u m a n g r a n u l o c y t e c o l o n y - s t i m u l a t i n g factor (G-CSF) in A u g u s t 1988 (case 3 in [5]), and t h e n w i t h f l n o x y m e s t e r o n e and v i t a m i n D 3 u n t i l A p r i l 1990. In A p r i l 1990, the percentage of blasts in the b l o o d i n c r e a s e d to 13% a n d he r e c e i v e d s u b c u t a n e o u s c y t o s i n e a r a b i n o s i d e (20 m g / w e e k ) . In July 1990, he p r o g r e s s e d into a l e u k e m i c p h a s e w i t h an M1 m o r p h o l o g y , a n d was f u r t h e r treated w i t h d a u n o r u b i c i n ,
72
A. Iwabuchi et al. Fluctuation in proportion of ceils with trisorny 8
vincristine, aclarubicin, and VP-16; however, he died of p n e u m o n i a in May, 1991. %
Patient 4
An 85-year-old male, patient 4 was found to have RA in April 1988 w h e n he was a d m i t t e d to our hospital with a diagnosis of arteriosclerotic dementia. His hematologic condition was stable until June 1991, without cytotoxic drugs.
CMML
100- - -
MDS
RARS
RA
RAEB
',total, eel
DQ
IIQ
! RAE
75-
Patient 5
e
This was a 64-year-old man, who c o m p l a i n e d of shortness of breath and general fatigue in 1987. The patient was diagnosed as having refractory a n e m i a with ringed sideroblasts (RARS) and has been treated with vitamin B~5.This therapy has resulted in the m a i n t e n a n c e of a hemoglobin level over 90 g/L, without red cell transfusion.
!
25-
CYTOGENETIC RESULTS
The cytogenetic changes in these five MDS patients are s u m m a r i z e d in Table 1.
RAEB
t
Patient 1
II_-
An initial cytogenetic study in August 1986 revealed that 8 of 21 (38%) BM metaphases were 47,XY, + 8. This proportion of metaphases with trisomy 8 was m a i n t a i n e d until February 1990. At this time, the patient's hemoglobin levels decreased to less than 50 g/L and he became transfusiond e p e n d e n t . In July 1990, vitamin D 3 was begun and his anemic c o n d i t i o n i m p r o v e d remarkably without red blood
Figure 1 Hematologic and cytogenetic changes during the course of the study in Patient 1. Bone marrow cells with trisomv 8 disappeared after oral vitamin D 3 treatment followed by improvement of anemia, but leukothrombocytopenia remained. [ Prednisolone LFluoxymesterone Vitamin D31
Figure 2 Proportion of cells with trisomy 8 in reported cases of myelodysplastic syndrome. CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; RA, refractory anemia: RAEB, refractory anemia with an excess of blasts: RARS, refractory anemia with ringed sideroblasts: AML, acute myeloid leukemia. (from Mitelman's Catalog [7]).
cell transfusion. In October 1990, no m e t a p h a s e s with + 8 were detected in the BM. The last cytogenetic study, in October 1991, revealed that all of 11 BM cells tested s h o w e d a normal male karyotype; his hematologic c o n d i t i o n has been m a i n t a i n e d until now (Fig. 1). During the course of the study, no progression of the disease was noted.
Repeated c h r o m o s o m e analyses during the MDS phase in this patient did not detect an abnormal karyotype clone. In the leukemic phase (in September 1989), a m i n o r population (10%) of metaphases with + 8 was detected; however, the proportion of metaphases with trisomy 8 did not correlate with the percentage of blasts in the BM and peripheral blood, as more than 80% of the BM cells were blast cells with an M1 morphology.
2Hemoglobin (g/L)
90" 6010-
r
AML
Patient 2
4-
120"
AML
Platelets ( × 109/L)
Patient 3
5100" BO" 60" 40" 20-
S
'~',,~,
~ ....
~ +8 BM cell, %o blasts BM cells
"1
1986
1990
f991
In this patient, a low percentage (two of 50 BM cells) of BM metaphases in the MDS phase had trisomy 8. After treatment with rhG-CSF, no particular change in the proportion of metaphases with trisomy 8 was noted, as reported previously [5]. After the leukemic phase d e v e l o p e d in July 1990, the percentages of cells with trisomy 8 was still low, and the i n c i d e n c e d i d not correlate with the percentage of blasts in BM.
Trisomy 8 in MDS Patient 4
This patient s h o w e d fluctuations in BM cells with trisomy 8: the percentage of cells with + 8 gradually increased during the course of this study (40%), but the last examination revealed the percentage of trisomy 8 cells to be 5%, although the percentage of blasts in the BM was usually less than 5% during the course. No particular changes in hematologic parameters were noted. Patient 5
At the time of MDS diagnosis (RARS, in June 1988), all of 20 BM cells e x a m i n e d had trisomy of c h r o m o s o m e 8. However, the last cytogenetic examination, in July 1991, revealed a normal male karyotype.
DISCUSSION
In humans, changes involving a d d i t i o n a l chromosomes imply the progression of neoplasias. In h u m a n hematopoietic neoplasias, trisomy of c h r o m o s o m e 8 is usually found as a result of n o n - d i s j u n c t i o n and this change is believed to be one of the signs of karyotypic progression [1]. For example, some patients with chronic myelogenous leukemia show the trisomy 8 anomaly, and the appearance of a trisomy 8positive clone as well as double P h i l a d e l p h i a c h r o m o s o m e is c o n s i d e r e d to be related to disease progression, i.e., blast crisis. In some patients with acute m y e l o i d leukemia, trisomy 8 is also detected as the sole c h r o m o s o m a l abnormality: in such patients, effective c h e m o t h e r a p y for leukemia can reduce the proportion of cells with trisomy 8, thus indicating that single n u m e r i c a l changes, i n c l u d i n g trisomy 8, could account for a p r i m a r y neoplastic aberration. The trisomy 8 a n o m a l y thus has two leukemogenic implications, one correlated with primary leukemogenesis, and the other linked to progression of the disease. M y e l o d y s p l a s t i c s y n d r o m e is a stem-cell h e m o p a t h y and, as most BM cells in MDS patients might be derived from an abnormal clone [1, 6], the proportion of cells with particular c h r o m o s o m e changes could reflect the progression of the disease. We first s p e c u l a t e d that the proportion of m e t a p h a s e cells with trisomy 8 may have been correlated with the progression of the disease or with the proportion of blasts in the BM. However, our results showed a heterogeneous pattern of frequency of ceils with trisomy 8 in our five MDS patients exhibiting this alteration, Furthermore, in each of these five patients, the proportion of BM cells with trisoiny 8 fluctuated in a m a n n e r that was not dependent on the stage of the disease. Moreover, we detected no non-related clones or further karyotypic changes in these five MDS patients. In Patient 2, trisomy 8 was first detected at the leukemic phase, at a very low percentage [10%), although blast cells accounted for more than 80% of the BM. In Patient 1, notably, no BM cells with t r i s o m y 8 were detected following effective v i t a m i n D 3 therapy. This t e n d e n c y was also noted in Patient 5. A review of the literature s h o w e d that about 20% of MDS patients with a sole trisomy 8 a n o m a l y had this change in all the m e t a p h a s e cells examined, whereas the remaining
73
patients had a mosaic karyotype in cells with t r i s o m y 8 and normal karyotypes in other cells (Fig. 2) [search performed using a Catolog p r e p a r e d by Dr. M i t e l m a n [7]). In this study, we encountered one patient (no. 5) who had t r i s o m y 8 in 100% of the m e t a p h a s e cells examined, but who, on reexamination, revealed no cells with this anomaly. As shown in Figure 2, fluctuation of the p r o p o r t i o n of cells with trisomy 8 has also been reported in some MDS patients in other studies, and no particular correlation between progression of the disease and these changes was noted [8-14]. This finding indicates that, in most MDS patients in w h o m this numerical c y t o g e n e t i c a n o m a l y occurs, it might be considered as an a d d i t i o n a l change, w h i c h is sometimes related to hematologic changes, w h e t h e r or not the patients receive treatment. This further suggests that BM cells with cytogenetically normal karyotypes might be derived from an MDS clone. To elucidate genetic alterations in h u m a n neoplasias, i n c l u d i n g MDS, further c h r o m o s o m e studies are required.
REFERENCES
1. Sandberg AA (1990): Myelodysplastic and myeloproliferative disorders, preleukemia and second leukemia. In: The Chromosomes in Human Cancer and Leukemia, 2rid ed. Elsevier Science Publishing, New York, pp. 521-624. 2. Ohyashiki K, Sasao I, Ohyashiki JH, Murakami T. Tauchi T, Iwabuchi A, Toyama K: Cytogenetic and clinical findings of myelodysplastic syndrome with a poor prognosis: an experience of 97 cases. Cancer (in press). 3. Bennett IM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1982): Proposals for the classification of the myelodysplastic syndrmnes. Br J Haematol 51:189199. 4. Harnden DG, Klinger HP (eds): An International System for Human Cytogenetic Nomenclature (1985), in Birth Defects, Original Article Series. Vol 21, No. 1, New York: Karger, 1985 (published in collaboration with Cytogenet Cell Genet). 5. Ohyashiki K, Ohyashiki JH, Toyama K, Takaku F (1989): Hematologic and cytogenetic findings of myelodysplastic syndrome treated in recombinant human granulocyte colonystinmlating factor. Jpn J Cancer Res 80:848-854. 6. Helm S, Mitelman F (1987): Myelodysplastic syndromes. In: Cancer Cytogenetics. Alan R. Liss. New York, pp. 111128. 7. Mitehnan F (1988): Catalog of Chromosomes Aberrations in Cancer, 3rd ed. Alan R. Liss, New York, pp. 345-351. 8. de la Chapelle A, Vuopio P, [cen A (1976): Trisomy 8 in the bone marrow associated with high red cell glutathione reductase. Blood 47:815-826. 9. Yamada K, Furusawa S (1976): Preferential involvement of chromosomes No. 8 and No. 21 in acute leukemia and preleukemia. Blood 47:679-686. 10. Mecucci C, Rege-Cambrin G, Michaux J-L, Tricot G, Van Den Berghe H (1986): Multiple chromosomally distinct cell populations in myelodysplastic syndromes and their possible significance in the evolution of the disease. Br J Haematol 64:699-706. 11. Benitez J, Carbonell F, Sanchez Fayos J, Heimpel H (1986): Karyotypic evolution in patients with myelodysplastic syndromes. Cancer Genet Cytogenet 16:157-167. 12. lacobs RH, Cornbleet MA. Vardiman JW, Larson RA, Le Beau MM, Rowley JD (1986): Prognostic implications of morphology
74
A. l w a b u c h i et al. and karyotype in primary myelodysplastic syndromes. Blood 67:1765-1772.
13. Hossfeld DK, We H-J, Kleeberg UR (1985): Low-dose cytarabine: chromosomal findings suggesting its cytostalic as wall as differentiating effect. Leuk Res 9:329-330.
14. Fenaux P. louet JP, Zandecki M, Lai IL, Simon M. Poller JP, Bauters F (1987): Chronic and subacute myelomonocytic: leukaemia in the adult: a report of 60 cases with special reference to prognositc factors. Br J Haematol 65:101 106.
ABSTRACT: Chromosome analyses were performed in five patients with myelodysplastic syndrome (MDS) who showed trisomy of chromosome 8 during the course of their disease. Four of these patients showed trisomy 8 at the diagnosis of MDS, and the remaining one had trisomy 8 when the leukenlia
phase developed. The proportion of bone marrow (BM) cells with trisomy 8 in the four patients who showed trisomy 8 at MDS diagnosis fluctuated, and this fluctuation was not related to the percentage of blasts in the BM or to progression of the disease. However, in two patients, metaphase cells with trisomy 8 disappeared when their anemic state improved, although leukothrombocytopenia was still present, suggesting that the decrease in the number of BM cells with trisomy 8 reflects hematologic features in some MDS patients. These findings indicate that trisomy 8 in our MDS patients was possibly not the primary event in the genesis of the disease, and that there may have been competition between a normal karyotype clone and a trisomy-8-positive clone. O a r results further suggest that the presence of a clone with trisomy 8 is not always a sign of disease progression or of poor prognosis in MDS patients.
INTRODUCTION
Numerical c h r o m o s o m e abnormalities, some of w h i c h sup e r i m p o s e on specific structural rearrangements, are frequently observed in h u m a n cancer cells [1]. For example, an extra c h r o m o s o m e 8 is detected in some patients with P h i l a d e l p h i a c h r o m o s o m e - p o s i t i v e chronic myelogenous l e u k e m i a in blast crisis; in these patients, the appearance of such a d d i t i o n a l c h r o m o s o m e abnormalities, including trisomy 8, is related to the progression of the disease [1]. In m y e l o d y s p l a s t i c s y n d r o m e s (MDS), however, the biologic and hematologic significance of numerical c h r o m o s o m e changes is not well understood. In a case exhibiting numerical changes in a d d i t i o n to a prototypic anomaly, one can easily recognize w h i c h is the original anomaly. By contrast, single n u m e r i c a l c h r o m o s o m e changes in hematopoietic neoplasias are frequently observed to have a clonal nature. In such cases, it is unclear w h e t h e r such single numerical abnormalities should be regarded as a d d i t i o n a l changes w h i c h are s u p e r i m p o s e d on undetectable genetic aberra-
From the Department of Internal Medicine (A. I., K. 0., J. H. 0.. I. S., T. M., K. T.); Chromosome Unit, Central Laboratory [A. K.], Tokyo Medical College, Tokyo 160, Japan. Address reprint requests to: Kazuma Ohyashiki, M.D., First Department of Internal Medicine, Tokyo Medical College, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan. Received March 31, 1992; accepted May 1, 1992. 70 Cancer Genet Cytogenet 62:70 74 (1992) 0165-4608/92/$05.00
tions or w h e t h e r they are p r i m a r y events in cancer development. In this investigation, we sequentially e x a m i n e d the association between clinical and cytogenetic findings in MDS patients with trisomy 8. Because MDS result from a clonal h e m o p a t h y of stem cells, most bone marrow (BM) cells obtained from MDS patients are c o n s i d e r e d to be derived from abnormal stem cells, in contrast to the situation in acute leukemia. Therefore, BM m e t a p h a s e cells in MDS patients, even if they have n o r m a l karyotype, could be considered to be cells derived from the MDS clone.
PATIENTS AND METHODS
A series of cytogenetic investigations was performed in 97 patients with MDS at the Tokyo M e d i c a l College Hospital, between January 1986 and January 1991 [21. The diagnosis of MDS was based on the FAB criteria [3]. For cytogenetic analysis, BM cells were short-term (48-hour) c u l t u r e d and processed as described p r e v i o u s l y [2]. C h r o m o s o m e s were stained with Hoechst 33258 and q u i n a c r i n e m u s t a r d and were identified in accordance with the ISCN [4]. During this series of investigations, we e n c o u n t e r e d five patients with MDS showing trisomy 8: four patients had this a n o m a l y at the time of MDS diagnosis [2], and the remaining one showed the a n o m a l y during the course of the study. A further cytogenetic s t u d y was performed in .~, 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10010
T r i s o m y 8 in M D S
Table 1 Patient no. 1
2
3
4
5
71
S e q u e n t i a l c y t o g e n e t i c f i n d i n g s in m y e l o d y s p l a s t i c s y n d r o m e s w i t h t r i s o m y 8 Date of examination
Diagnosis (FAB)
Percentage of BM blasts
8/11/86 9/6/87 5/17/88 10/26/89 2/22/90 10/6/90 10/5/91 4/25/89 5/9/89 7/24/89 9/16/89 9/18/89 8/2/88 8/11/88 8/17/88 9/1/88 11/30/89 5/31/90 7/3/90 9/26/90 1/21/91 2/13/91 3/26/91 4/8/91 4/26/89 6/21/89 11/2/89 12/4/89 6/10/88 7/24/91
RA RA RA RA RA RA RA RAEB RAEB RAEB Overt-M1 Overt-M1 RAEB RAEB RAEB RAEB RAEB RAEBT Overt-M1 Overt-M1 Overt-M1 Overt-M1 Overt-M1 Overt-M1 RA RA RA RA RARS RARS
4.0 3.2 2.8 3.0 4.0 0.8 0.4 4.8 5.6 3.0 83.6 5.4 4.4 1.8 2.4 4.2 17.0 30.0 18.8 41.6 69.0 36.6 27.6 1.8 2.0 2.0 3.4 1.0 2.0
Samples
No. of cells analyzed
BM BM BM BM BM BM BM BM BM BM BM PB BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM
21 2O 20 10 20 20 11 20 20 20 20 20 20 50 20 50 25 20 13 20 20 10 9 5 7 2O 20 20 20 20
No. of cells with + 8 (%) 8 16 18 10 10 0 0 0 0 0 2 2 1 2 2 0 2 2 5 1 0 1 0 0 0 2 8 1 20 0
(38) (80) (90) (100) (50) (0) (0) (0) (0) (0) (10) (10) (5) (4) (10) (0) (8) (10) (38) (10) (0) (5) (0) (0) (0) (10) (40) (5) (100) (0)
Abbreviations: RA, refractory anemia; RAEB, refractory anemia with an excess of blasts; RAEBT,refractory anemia with an excess of blasts in transformation;
Overt-M1, leukemic transformation with an M1 morphology; RARS, refractory anemia with ringed sideroblasts; BM, bone marrow (48-hour culture); PB, peripheral blood (48-hour culture without addition of mitogens).
t h e s e five patients, and BM cells w i t h t r i s o m y 8 w e r e f o u n d to fluctuate. T h e d e t a i l e d c l i n i c a l features of the patients are d e s c r i b e d b e l o w . Patient 1 Patient 1 was a 2 4 - y e a r - o l d male, w h o was i n i t i a l l y f o u n d to h a v e p a n c y t o p e n i a in May 1986. A d i a g n o s i s of refractory a n e m i a (RA) was m a d e in A u g u s t 1986. T h e p a t i e n t was treated w i t h an a n a b o l i c steroid ( f l u o x y m e s t e r o n e , 15 mg/ day) and p r e d n i s o l o n e I20 mg/day). H o w e v e r , the a n e m i a p r o g r e s s e d ( h e m o g l o b i n was 49 g/L) and he b e c a m e transfus i o n - d e p e n d e n t . F r o m July 1990, he was treated w i t h oral v i t a m i n D 3 (2/xg/day), a n d his a n e m i c state i m p r o v e d (105 g/L) w i t h o u t red cell t r a n s f u s i o n . Patient 2 P a t i e n t 2 was a 38-year-old female, w h o c o m p l a i n e d of n i g h t s w e a t s and fatigue in A u g u s t 1988, and w h o was a d m i t t e d in M a r c h 1989. T h i s p a t i e n t h a d a prior history of gastric cancer, in 1981, a n d h a d b e e n treated by subtotal g a s t r e c t o m y f o l l o w e d by c h e m o t h e r a p y ( m i t o m y c i n C 118 rag, tegafur 1640 rag, and f l u o r o u r a c i l 3500 mg). At the t i m e
of MDS d i a g n o s i s ( s e c o n d a r y MDS), her b l o o d test s h o w e d p a n c y t o p e n i a . T h e BM was n o r m o c e l l u l a r w i t h 4% blasts w i t h A u e r rods, suggesting the d i a g n o s i s of refractory anemia w i t h an excess of blasts (RAEB). T h e p a t i e n t w a s treated w i t h i n t r a m u s c u l a r c y t o s i n e a r a b i n o s i d e (30 mg) and vitam i n D 3 (2 /~g/day), but an i n c r e a s e (65%) of blasts in the b l o o d was n o t e d in S e p t e m b e r 1989, and c y t o t o x i c drugs w e r e t h e n a d m i n i s t e r e d . H o w e v e r , the p a t i e n t d e v e l o p e d d i s s e m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n w i t h acute renal failure; she d i e d on S e p t e m b e r 27, 1989. Patient 3 T h i s p a t i e n t was a 54-year-old male, w h o was f o u n d to h a v e p a n c y t o p e n i a in O c t o b e r 1987, w h e n a d i a g n o s i s of RAEB was m a d e . T h e p a t i e n t was treated w i t h r e c o m b i n a n t h u m a n g r a n u l o c y t e c o l o n y - s t i m u l a t i n g factor (G-CSF) in A u g u s t 1988 (case 3 in [5]), and t h e n w i t h f l n o x y m e s t e r o n e and v i t a m i n D 3 u n t i l A p r i l 1990. In A p r i l 1990, the percentage of blasts in the b l o o d i n c r e a s e d to 13% a n d he r e c e i v e d s u b c u t a n e o u s c y t o s i n e a r a b i n o s i d e (20 m g / w e e k ) . In July 1990, he p r o g r e s s e d into a l e u k e m i c p h a s e w i t h an M1 m o r p h o l o g y , a n d was f u r t h e r treated w i t h d a u n o r u b i c i n ,
72
A. Iwabuchi et al. Fluctuation in proportion of ceils with trisorny 8
vincristine, aclarubicin, and VP-16; however, he died of p n e u m o n i a in May, 1991. %
Patient 4
An 85-year-old male, patient 4 was found to have RA in April 1988 w h e n he was a d m i t t e d to our hospital with a diagnosis of arteriosclerotic dementia. His hematologic condition was stable until June 1991, without cytotoxic drugs.
CMML
100- - -
MDS
RARS
RA
RAEB
',total, eel
DQ
IIQ
! RAE
75-
Patient 5
e
This was a 64-year-old man, who c o m p l a i n e d of shortness of breath and general fatigue in 1987. The patient was diagnosed as having refractory a n e m i a with ringed sideroblasts (RARS) and has been treated with vitamin B~5.This therapy has resulted in the m a i n t e n a n c e of a hemoglobin level over 90 g/L, without red cell transfusion.
!
25-
CYTOGENETIC RESULTS
The cytogenetic changes in these five MDS patients are s u m m a r i z e d in Table 1.
RAEB
t
Patient 1
II_-
An initial cytogenetic study in August 1986 revealed that 8 of 21 (38%) BM metaphases were 47,XY, + 8. This proportion of metaphases with trisomy 8 was m a i n t a i n e d until February 1990. At this time, the patient's hemoglobin levels decreased to less than 50 g/L and he became transfusiond e p e n d e n t . In July 1990, vitamin D 3 was begun and his anemic c o n d i t i o n i m p r o v e d remarkably without red blood
Figure 1 Hematologic and cytogenetic changes during the course of the study in Patient 1. Bone marrow cells with trisomv 8 disappeared after oral vitamin D 3 treatment followed by improvement of anemia, but leukothrombocytopenia remained. [ Prednisolone LFluoxymesterone Vitamin D31
Figure 2 Proportion of cells with trisomy 8 in reported cases of myelodysplastic syndrome. CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; RA, refractory anemia: RAEB, refractory anemia with an excess of blasts: RARS, refractory anemia with ringed sideroblasts: AML, acute myeloid leukemia. (from Mitelman's Catalog [7]).
cell transfusion. In October 1990, no m e t a p h a s e s with + 8 were detected in the BM. The last cytogenetic study, in October 1991, revealed that all of 11 BM cells tested s h o w e d a normal male karyotype; his hematologic c o n d i t i o n has been m a i n t a i n e d until now (Fig. 1). During the course of the study, no progression of the disease was noted.
Repeated c h r o m o s o m e analyses during the MDS phase in this patient did not detect an abnormal karyotype clone. In the leukemic phase (in September 1989), a m i n o r population (10%) of metaphases with + 8 was detected; however, the proportion of metaphases with trisomy 8 did not correlate with the percentage of blasts in the BM and peripheral blood, as more than 80% of the BM cells were blast cells with an M1 morphology.
2Hemoglobin (g/L)
90" 6010-
r
AML
Patient 2
4-
120"
AML
Platelets ( × 109/L)
Patient 3
5100" BO" 60" 40" 20-
S
'~',,~,
~ ....
~ +8 BM cell, %o blasts BM cells
"1
1986
1990
f991
In this patient, a low percentage (two of 50 BM cells) of BM metaphases in the MDS phase had trisomy 8. After treatment with rhG-CSF, no particular change in the proportion of metaphases with trisomy 8 was noted, as reported previously [5]. After the leukemic phase d e v e l o p e d in July 1990, the percentages of cells with trisomy 8 was still low, and the i n c i d e n c e d i d not correlate with the percentage of blasts in BM.
Trisomy 8 in MDS Patient 4
This patient s h o w e d fluctuations in BM cells with trisomy 8: the percentage of cells with + 8 gradually increased during the course of this study (40%), but the last examination revealed the percentage of trisomy 8 cells to be 5%, although the percentage of blasts in the BM was usually less than 5% during the course. No particular changes in hematologic parameters were noted. Patient 5
At the time of MDS diagnosis (RARS, in June 1988), all of 20 BM cells e x a m i n e d had trisomy of c h r o m o s o m e 8. However, the last cytogenetic examination, in July 1991, revealed a normal male karyotype.
DISCUSSION
In humans, changes involving a d d i t i o n a l chromosomes imply the progression of neoplasias. In h u m a n hematopoietic neoplasias, trisomy of c h r o m o s o m e 8 is usually found as a result of n o n - d i s j u n c t i o n and this change is believed to be one of the signs of karyotypic progression [1]. For example, some patients with chronic myelogenous leukemia show the trisomy 8 anomaly, and the appearance of a trisomy 8positive clone as well as double P h i l a d e l p h i a c h r o m o s o m e is c o n s i d e r e d to be related to disease progression, i.e., blast crisis. In some patients with acute m y e l o i d leukemia, trisomy 8 is also detected as the sole c h r o m o s o m a l abnormality: in such patients, effective c h e m o t h e r a p y for leukemia can reduce the proportion of cells with trisomy 8, thus indicating that single n u m e r i c a l changes, i n c l u d i n g trisomy 8, could account for a p r i m a r y neoplastic aberration. The trisomy 8 a n o m a l y thus has two leukemogenic implications, one correlated with primary leukemogenesis, and the other linked to progression of the disease. M y e l o d y s p l a s t i c s y n d r o m e is a stem-cell h e m o p a t h y and, as most BM cells in MDS patients might be derived from an abnormal clone [1, 6], the proportion of cells with particular c h r o m o s o m e changes could reflect the progression of the disease. We first s p e c u l a t e d that the proportion of m e t a p h a s e cells with trisomy 8 may have been correlated with the progression of the disease or with the proportion of blasts in the BM. However, our results showed a heterogeneous pattern of frequency of ceils with trisomy 8 in our five MDS patients exhibiting this alteration, Furthermore, in each of these five patients, the proportion of BM cells with trisoiny 8 fluctuated in a m a n n e r that was not dependent on the stage of the disease. Moreover, we detected no non-related clones or further karyotypic changes in these five MDS patients. In Patient 2, trisomy 8 was first detected at the leukemic phase, at a very low percentage [10%), although blast cells accounted for more than 80% of the BM. In Patient 1, notably, no BM cells with t r i s o m y 8 were detected following effective v i t a m i n D 3 therapy. This t e n d e n c y was also noted in Patient 5. A review of the literature s h o w e d that about 20% of MDS patients with a sole trisomy 8 a n o m a l y had this change in all the m e t a p h a s e cells examined, whereas the remaining
73
patients had a mosaic karyotype in cells with t r i s o m y 8 and normal karyotypes in other cells (Fig. 2) [search performed using a Catolog p r e p a r e d by Dr. M i t e l m a n [7]). In this study, we encountered one patient (no. 5) who had t r i s o m y 8 in 100% of the m e t a p h a s e cells examined, but who, on reexamination, revealed no cells with this anomaly. As shown in Figure 2, fluctuation of the p r o p o r t i o n of cells with trisomy 8 has also been reported in some MDS patients in other studies, and no particular correlation between progression of the disease and these changes was noted [8-14]. This finding indicates that, in most MDS patients in w h o m this numerical c y t o g e n e t i c a n o m a l y occurs, it might be considered as an a d d i t i o n a l change, w h i c h is sometimes related to hematologic changes, w h e t h e r or not the patients receive treatment. This further suggests that BM cells with cytogenetically normal karyotypes might be derived from an MDS clone. To elucidate genetic alterations in h u m a n neoplasias, i n c l u d i n g MDS, further c h r o m o s o m e studies are required.
REFERENCES
1. Sandberg AA (1990): Myelodysplastic and myeloproliferative disorders, preleukemia and second leukemia. In: The Chromosomes in Human Cancer and Leukemia, 2rid ed. Elsevier Science Publishing, New York, pp. 521-624. 2. Ohyashiki K, Sasao I, Ohyashiki JH, Murakami T. Tauchi T, Iwabuchi A, Toyama K: Cytogenetic and clinical findings of myelodysplastic syndrome with a poor prognosis: an experience of 97 cases. Cancer (in press). 3. Bennett IM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1982): Proposals for the classification of the myelodysplastic syndrmnes. Br J Haematol 51:189199. 4. Harnden DG, Klinger HP (eds): An International System for Human Cytogenetic Nomenclature (1985), in Birth Defects, Original Article Series. Vol 21, No. 1, New York: Karger, 1985 (published in collaboration with Cytogenet Cell Genet). 5. Ohyashiki K, Ohyashiki JH, Toyama K, Takaku F (1989): Hematologic and cytogenetic findings of myelodysplastic syndrome treated in recombinant human granulocyte colonystinmlating factor. Jpn J Cancer Res 80:848-854. 6. Helm S, Mitelman F (1987): Myelodysplastic syndromes. In: Cancer Cytogenetics. Alan R. Liss. New York, pp. 111128. 7. Mitehnan F (1988): Catalog of Chromosomes Aberrations in Cancer, 3rd ed. Alan R. Liss, New York, pp. 345-351. 8. de la Chapelle A, Vuopio P, [cen A (1976): Trisomy 8 in the bone marrow associated with high red cell glutathione reductase. Blood 47:815-826. 9. Yamada K, Furusawa S (1976): Preferential involvement of chromosomes No. 8 and No. 21 in acute leukemia and preleukemia. Blood 47:679-686. 10. Mecucci C, Rege-Cambrin G, Michaux J-L, Tricot G, Van Den Berghe H (1986): Multiple chromosomally distinct cell populations in myelodysplastic syndromes and their possible significance in the evolution of the disease. Br J Haematol 64:699-706. 11. Benitez J, Carbonell F, Sanchez Fayos J, Heimpel H (1986): Karyotypic evolution in patients with myelodysplastic syndromes. Cancer Genet Cytogenet 16:157-167. 12. lacobs RH, Cornbleet MA. Vardiman JW, Larson RA, Le Beau MM, Rowley JD (1986): Prognostic implications of morphology
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A. l w a b u c h i et al. and karyotype in primary myelodysplastic syndromes. Blood 67:1765-1772.
13. Hossfeld DK, We H-J, Kleeberg UR (1985): Low-dose cytarabine: chromosomal findings suggesting its cytostalic as wall as differentiating effect. Leuk Res 9:329-330.
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