Cytogenetic Analysis of 54 Cases of Myelodysplastic Syndrome M. Jotterand-Bellomo, V. Parlier, P. M. Schmidt, and Ph. Beris

ABSTRACT: Fiftyffour patients with myelodysplastic syndrome (MDS) (35 men and 19 women clge,d

34 92 years) were studied cytogenetically. Bone marraw cell culture and chromosome preparation were performed according to four different prolacols used in parallel: methotre, xate {MTX)synchronized or thymidine (TdRJ-unsynchronized techniques, and presence or absence fff 5637 conditioned medium {CM]. Some patients responded better to MTX; others had better results with TdR exposure only. Use of 5637 CM generally improved quantity and quality of metaphases. A cytogenetic result was obtained in 53 cases. 60% of the patients had a chromosome abnormality. Percentage of abnormality varied from one French-American-British (FAB) subtype to the other: 62% in refractory anemia with ringed sideroblasts (RARS. 8/13), 50% in refractory anemia (RA, 6/12). 69% in refractory anemia with excess of blasts (RAEB, 3/5), 77% in refractory anemia with excess of blasts in transformation {RAEB-T. 7/9), and 57% in chronic myelomonocytic leukemia (CMMoL. 8/14). Chromosome defects were subdivided into three categories: single, two, and complex defects. The most frequent chromosome abnormalities, either single or nne of two or complex defects were del(Sq) or monosomy 5 (13 casesl, trisomy or rearrangement af chromosome 8 [eight cases), total or partial monosomy or rearrangement of chromosome 7 {eight cases), Y loss {seven cases), and delI2Oq) (two cases). With the exception of del(5q) in macrocytic HA, this study confirms the absence of chromosome defects specific to each FAB category of MDS. Recurrent defects in MDS are relatively limited, however, in terms of chromosomes involved and type of abnormality. Consequently, these defects, mostly of deleted type, are assumed to play a specific role in the genesis of myelodysplasia.

INTRODUCTION

M y e l o d y s p l a s t i c s y n d r o m e (MDS) is a n a b n o r m a l i t y of t h e h e m a t o p o i e t i c s t e m c e l l s l e a d i n g to q u a l i t a t i v e a n d q u a n t i t a t i v e d e f e c t s of h e m a t o p o i e s i s . P r o l i f e r a t i o n a n d a b n o r m a l m a t u r a t i o n of p r e c u r s o r c e l l s g e n e r a l l y affect all l i n e a g e s , a l t h o u g h in s o m e c a s e s d y s p o i e s i s is r e s t r i c t e d to t h e e r y t h r o i d c e l l s only. B o n e m a r r o w d y s f u n c t i o n r e s u l t s i n c y t o p e n i a of v a r i a b l e n a t u r e a n d s e v e r i t y w i t h a w i d e r a n g e of c l i n i c a l m a n i f e s t a t i o n s . P e r i p h e r a l p a n c y t o p e n i a w i t h h y p e r c e l l u l a r or n o r m o c e l l u l a r b o n e m a r r o w r e p r e s e n t s o n e c h a r a c t e r i s t i c of MDS. M y e l o d y s p l a s i a p r e d o m i n a n t l y affects elderly persons. H e l m a n d M i t e l m a n [1] r e p o r t e d c l o n a l c h r o m o s o m a l a b n o r m a l i t i e s i n m o r e t h a n

From the Division de G6n6tique M6,dicale (M. I.-B., V. P.), and the Division d'H6matologie, {P. M. S.), Centre ftospitalier Universitaire Vandois, Lausanne, Switzerland, and Division d'H~matologie (Ph. B.). t l(~pital Cantonal Universitaire. Geneva, Switzerland.

Address reprint requests to: M. Jotterand-Bellomo, Division de G6ndtique M6,dicale. CHUV, 1011 Lausanne, Switzerland. Received March 2, 1989; accepted June 27, 1989.

157 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 46:157-172 (1990) 0165-4608/90/$03.50

158

M. Jotterand-Bellomo et al.

700 MDS. In most series chromosome defects were observed in one third to one half of the patients. Yunis et al. [2] reported a particular high frequency of abnormalities (79%) in their sample of 56 patients. In five recent series (314 patients), the percentage of abnormality ranged between 48 and 73 [6], a value slightly inferior to that of 1986 [3-7]. In primary acquired MDS, the most frequent abnormalities observed are del(5q), del(7q) or - 7, and + 8. Structural rearrangements such as deletions 11q, 12p, 13q, 20q, translocations (1;3), (2;11), (6;9), (11 ;21), and isochromosome 17q are less c o m m o n [8]. Single abnormalities, total or partial monosomies 5 or 7, del(12p) and t(1;7) often occur in therapy-related or secondary MDS. They may be accompanied by + 8, + 21, del or t(3p), partial or total m o n o s o m y of 17, del or t(6p), t(19q or p), t or dup(Xq13), or t(Xp11) [8]. Chromosome findings constitute a prognostic indicator in MDS [9]. Recent investigations have shown that the increased risk for developing acute leukemia is primarily associated with the presence of clones with multiple chromosome rearrangements or with a single abnormality involving partial deletion or m o n o s o m y of chromosome 7. The presence of del(5q) as sole abnormality is associated with a rather good prognosis. This article presents our results relative to the cytogenetic analysis of bone marrow cells from 54 myelodysplastic patients. Cell culture and chromosome preparation techniques were thoroughly investigated to improve the yield of bone marrow cell chromosome analysis in MDS. Chromosome findings were correlated with clinical and hematological data. Possible implications of chromosome defects in myelodysplastic transformation are discussed.

PATIENTS, MATERIALS, AND METHODS Bone Marrow Samples The cytogenetic study of bone marrow cells of patients from the Western part of Switzerland was undertaken at the end of 1985. MDS cases referred to in this article were diagnosed according to the French-American-British (FAB) classification in the University Hospitals of Lausanne (L) and Geneva (G) and in the district Hospitals of Martigny (M), Neuch~tel (N) and La-Chaux-de-Fonds (CdF). Informed consent was obtained from each patient. For living patients, clinical data were established as of October 1988.

Cell Culture and Chromosome Preparation In all cases chromosome preparation was made from bone marrow cells. Marrow samples of 0.5 to 2ml were collected in preservative-free heparin. The m o n o n u c l e a r cell fraction was isolated using Ficoll-Paque density-gradient centrifugation. Nucleated cells were cultured at 0.5 to 1.0 × 106/ml in RPMI 1640 s u p p l e m e n t e d with 20% h u m a n AB serum either with or without 10% conditioned m e d i u m (CM) derived from cultures of the h u m a n urinary bladder carcinoma cell line 5637, supplied by Dr. N. Odartchenko, Institut de Recherches Exp~rimentales sur le Cancer, Epalinges, Switzerland and Dr. M. Aapro, Laboratoire des Moelles, Oncologie, H6pital Cantonal Universitaire, Geneva, Switzerland. Cells were incubated at 37°C and u n d e r 5% CO 2 pressure for a time ranging from 48 to 72 hours. Two cytogenetic protocols were used in parallel: methotrexate (MTX) 10 -7 M for 17 hours and t h y m i d i n e (TdR) 10 _5 M for 5 hours or only TdR 10 5 M for 5 hours. Colchicine was added at 0.07 ~g/ml for 10 minutes. Hypotonic shock was performed with KC1 0.56% for 18 minutes. Cells were fixed according to two procedures, either with methanol : acetic acid 3 : 1 four to six times or according to a modification of the procedure of Islam et al. [10]. Cells were

15 767

16 197

16 283

16 297

29

34

40

31

36

RARS

RARS

RAEB-T

CMMoL

RARS

FAB

48

72

48

72

72

46,XY/47,XY, + 8 (76/62) 45% 45,X/46,XY {47/1) 98% 46,XX/46,XX,i(17q/ (24/138) 85% 4 5 , X - Y/46,XY (207/195) 51% 46,XY/47,XY, + M {1/256) 99.6%

Karyotype (no. of metaphases analyzed] a b n o r m a l cells (%)

versus unsynchronized

Incubation time (h)

of s y n c h r o n i z e d

27,3(3) 43°/,, 15{1] 100% 7(3) 95% 89(2) 50% 3(3] 100%

2(2) 3/4 1(1) 100% 17,5(2) 83°/, 30.5(2) 52% 50(3) 1/152 46,XY

MTX - T d R

23(2) 36% 11(2) 1/11 46.XY 15.7{3) 94% 39(2) 50% 19(3) 100%

T d R + 5637

M e a n n u m b e r of m e t a p h a s e s per slide ( n u m b e r of slides a n a l y z e d ) a b n o r m a l cells (%)

and 5637 CM effect assessment

MTX TdR + 5637 CM

techniques

6.3(3) 58% 5(2) 100% 19.7{3) 80°/,, 28.3(3] 54% 20{2) 100%

TdR

257

402

162

138 {151 r e p o r t e d ) 48

Total no. of metaphases analyzed

chronic myelomonocytic leukemia: RAEB-T = refractory anemia with excess of blasts in transformation.

Abbreviations: FAB - French-American-British classification; MTX = methotrexate; TdR = tbymidine; RARS = refractory anemia with ringed sideroblasts; CMMoL =

13 970

Case

Comparison

Identification no.

Table 1

160

M. J o t t e r a n d - B e l l o m o et al.

a

W ~

b

i~~¸

C

~ % W ¸

d

w

4

F i g u r e 1 Selected G-banded chromosomes from three patients of our sample: (a) refractory anemia (RA) with t(1;15](q12:p11): (b and c) chronic myelomonocytic leukemia with del[1) (p22.1p32.3]; [b) poorly banded chromosomes, (c) rather fine chromosome resolution of one metaphase selected from a GM-colony obtained by precursor cell methyh:ellulose culture; {d) RA with excess of blasts in transformation with del(7)(q22q36) and t(3:3)(q21;q26).

p r o c e s s e d on c l e a n slides from a height of 80 100 cm. After 2 - 3 days, c h r o m o s o m e s w e r e stained in G-bands (Figs. 1-4).

Comparison of Synchronized Versus Unsynchronized Techniques and 5637 Conditioned Medium Effect Assessment (Table 1) T h e m e a n n u m b e r of m e t a p h a s e s per slide was defined for five cases after one to three slides w e r e c h e c k e d e x t e n s i v e l y for each of the four different e x p e r i m e n t a l c o n d i t i o n s (MTX/TdR, TdR, + 5637 CM, - 5637 CM). So as to r e d u c e variation b e t w e e n s a m p l e s , c o n c e n t r a t i o n of cell s u s p e n s i o n was a d a p t e d from replicate to r e p l i c a t e to be relatively constant. O n e drop of cell s u s p e n s i o n was d e p o s i t e d on each slide. M o r e o v e r , to e s t i m a t e the effect of 5637 CM as w e l l as that of the M T X - T d R versus TdR p r o t o c o l on a c h r o m o s o m i c a l l y a b n o r m a l cell p r o p o r t i o n in cases w i t h an a d m i x t u r e of n o r m a l and a b n o r m a l mitoses, the five cases c h o s e n had a c h r o m o s o m e a b n o r m a l i t y in 45, 98, 85, 51, and 99.6% of the cells, r e s p e c t i v e l y .

RESULTS Of the 54 patients, 19 w e r e w o m e n a n d 35 w e r e m e n [sex ratio 1 : 1.8). Ages ranged b e t w e e n 34 and 92 years. Age and sex are s h o w n in Table 2. Patients aged less t h a n 50 years r e p r e s e n t e d 9% of the total.

161

Chromosomes in M y e l o d y s p l a s t i c S y n d r o m e

t e

r

!

1

2

3

6

7

8

13

14

19

4

5

10

11

12

15

16

17

18

2O

21

22

t 9

X

! Figure 2

M

\

Refractory anemia with ringed sideroblasts with 47,XY. + M. G-banding.

Clinical data are shown in Tables 3 and 4. Patients were classified according to FAB morphological criteria. All but one (11/15405) had de novo MDS. Case 11/15405, a 73-year-old man, had l y m p h o p l a s m a c y t o i d malignant l y m p h o m a (IgM production) diagnosed in 1984. At the beginning of 1988, the patient had p a n c y t o p e n i a (hemoglobin H 10g/dl, white blood cell count 2.5/~L, platelet count 43,000/~L). Bone marrow aspiration showed erythroid h y p e r p l a s i a with dyserythropoiesis. Iron-staining showed coarse granules r a n d o m l y distributed w i t h i n the c y t o p l a s m of erythroblasts. Diagnosis of refractory anemia (RA) secondary to lymphoproliferative s y n d r o m e was made. Cases 10/15064 and 27/13028 were considered RA-type. Actually these two patients were not anemic but had erythroid h y p e r p l a s i a and dyserythropoietic features at bone marrow aspiration. Patients were s u b d i v i d e d according to FAB classification as follows: 13 RA with ringed sideroblasts (RARS), 12 RA, 6 refractory anemia with excess of blasts (RAEB), 9 RAEB in transformation (RAEB-T) and 14 chronic m y e l o m o n o c y t i c leukemia (CMMoL). Nineteen patients were studied at the time of diagnosis. Thirteen patients received cytotoxic chemotherapy. Remaining patients received either m u l t i v i t a m i n treatment (17 patients), supportive treatment (transfusions in 9 patients), or no treatment (15 patients). Chromosome findings are shown in detail in Tables 1, 2, and 4. A cytogenetic result was obtained in 53 cases. In case 54/11186, no metaphases were available although s a m p l i n g was repeated four times (Table 2). In the 53 cases successfully analyzed, metaphases k a r y o t y p e d n u m b e r e d between 4 and 402. Chromosome resolution ranged between 350 and 650 bands. In cases 7/10979, 11/15405, and 14/13042, only four, five, and four normal metaphases were found, respectively. So a limited number of metaphases does not allow exclusion of a mosaicism in these three cases. Thirty-two cases (60%) showed a chromosome abnormality. Chromosome abnormalities were grouped according to single defects, two defects, and complex defects.

Case no.

F/69 F/87 M/75 M/76 M/60 M/71 M/64 M/34 F/66

378 408 610 741 326 142 979 278 832 064 b 405 187 7195 0425 316 541 207 971 105 106 228

092 229 237 2755

13 16 14 13

14 358 13 028 16 839

13 9705 15 564

M/70 F/92 M/58 M/63 F/77 F/71 M/67 M/63 F/41 M/64 M/73 F/81 M/66 M/58 F/40 M/64 F/70 M/49 M/60 F/85 F/63

Sex/age

13 13 13 14 15 10 10 12 13 15 15 10 12 13 13 10 13 13 15 15 15

Identification no.

RARS RARS

RARS RA-type ~ CMMoL

RA RA RAEB-T CMMoL

RARS RARS RARS RARS RARS RA RA RA RA RA-type ~: RA RAEB RAEB RAEB-T RAEB-T CMMoL CMMoL CMMoL CMMoL CMMoL CMMoL

Diagnosis (FAB}

BM BM

BM BM BM

BM BM BM BM

BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM

Material

I n i t i a l c h r o m o s o m e f i n d i n g s of 54 p a t i e n t s w i t h M D S

Normal c h r o m o s o m e s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Single c h r o m o s o m e defects Deletion of (5g} 22 23 24 25 M o n o s o m y 7 or del(7q) 26 27 28 Trisomy 8 29 30

Table 2

7(27} 8(67)

10 19[63) 26{100)

8(31} 10 1[2.8)

8

A" (%)

Mitoses analyzed

I

19 4

11

35

18

7 8 10 15 15 15 4 7 10 10 5 15 12 4 9 9 20 11 12 11 10

N

47,XY, + 8 47,XX, + 8

45,XY,- 7 46,XY, - 7, + der(1 },t(l ;7)(pl 1 ;pl 1) 45,XY,- 7

46,XX,del(5q) ° 46,XX,del(5}(qlSq33) 46,XY,del(5)(q13q33) 46,XY,del(5)(ql 5q33)

46,XY 46,XX 46,XY 46,XY 46,XX 46,XX 46,XY 46,XY 46,XX 46,XY 46,XY 46,XX 46,XY 46,XY 46,XX 46,XY 46,XX 46,XY 46,XY 46,XX 46,XX

C h r o m o s o m e findings

t-~

14 515

12 757

52

53

M/62

M/62

M/61

F/67

F/83

M/71 M/68

RAEB

CMMoL

RAEB-T

RAEB-T

RAEB

RAEB RAEB

RARS

RARS

RARS RAEB RAEB-T RAEB-T

RA RA RAIdB CMMoL

RARS RA

RARS CMMoL CMMoL CMMoL CMMoL

BM

BM

BM

BM

BM

BM BM

BM

BM

BM BM BM BM

BM BM BM BM

BM BM

BM BM BM BM BM

--12

--

--

10

19(86)

8

13(93) 12(92)

20

13

Cases in which a second chromosome analysis was performed.

Chromosome resolution not sufficient for breakpoint assessment.

3

1 1

4 2 , X Y , - 3 , - 6 , - 7 , - 1 5 , - 1 7 , - 1 8 , - 21,de1(5) (q22q23),+der(3)t{a;17)(p11;qll,q12 or q21.1), + der(6)t(6;15)(p12;q11), + der{7)t[7;21)[p11;q11} 46,XX,del(20q)(qllq13.3)(1)/46,XX,- 14, + 19, del(20q),(1)/48,XX, + 19,del(20q), + del(20q), (16)/49,XX, + 13, + 19,del(20q), + del(20q),(2} 44,X, - Y, - 5,del(8p),inv(12),del(16q),del(17q) 42,XY,-1,-3, 4,-5,-6,-7,-9,-16,-17,-19, + der(3)t(3;9)(q26 - 27;q13), + der(4)t(1;4) (q21;q31), + der(9)t(3;9)(q26 - 27;q13), + der(19)t(19;?), + 3M 4 8 , X Y , - 6 , - 20,+ 22,3p?,del(5)(q15q33),7?,8q?,l l p +, + del(16q)?,17p + , + der(6)t(6q;?}, + 2M 4 4 , X Y , - 7 , - 1 0 , - 1 3 , - 21,del(5}(q15q31), + der(7)t{7q;?), + der(10Jt{lO)(q;?), + rain 4 4 , X X , - 5 , - 7 , + 1 8 , - 1 0 , - 18,del(3p),17p + , + der(7)t(7p;?) 43,XY,-4,-5,-8, 12, 1 7 , 3 p + , 1 1 p + , + d e r ( 4 ) t [ 4 ; 8 ) [ p 1 1 ; q 1 1 ) , + M (Fig. 3)

-----

47,XY, + 8{15)/46,X, - Y, + 8(3) 46,XY,del(5)(q15q33),t{2;111{p21;q23) 47,XY, + 8,(5)/47,XY, + 8,del(5)(qllq33)(5) 46,XX,del(7)(q22q36),t(3;3)(q21;q26) (Fig. ld)

4 6 24 2

6(60/ 4(40) 138(85) 19(90/ 18 12 10 12

4 7 , X Y , + M (Fig. 2) 4 6 , X X , - 1 5 , + d e r ( 1 5 ) , t ( 1 ; 1 5 ) [ q 1 2 ; p 1 1 ) (Figs, l a and 4) 47,XY,+14 46,XX,del(20)(qllq13.3) 46,XX,i(17q)[65)/47,XX,i(17q), + i[17q) 4 6 , X Y , d e l ( 1 ) ( p 2 2 . 1 p 3 2 . 3 ) (Figs. l b and c, and 4)

1 15

3 1

256(99.6) 3(17}

45,X,-Y 45,X, Y 45,X,- Y 45,X, Y 45,X,-Y

--

195

207(51] 10 9(75) 47(98) 20

Abbreviations: MDS, myelodysplastic syndrome; M - marker chromosome; rain - minute chromosome.

11 186

9 324

51

No result 54

13 303

9 450 ~ 9 540

F/77

M/50

15 462

15 872

M/69 M/51 M/73 F/63

136 395 058 629

13 10 16 13

M/82 F/76 F/85 M/67

419 784 197 508

M/73 F/41

16 297 10 540

13 16 16 15

M/75 M/82 M/76 M/88 M/71

283 704 321 767 832

16 14 15 15 16

50

48 49

47

38 39 40 41 T w o c h r o m o s o m e defects 42 43 44 45 Complex defects 46

Y loss 31 32 33 34 35 Miscellaneous 36 37

60+

30

At d i a g n o s i s

4+

H e m o l y s i s {PNH)

13+ 20+ 2+

At d i a g n o s i s At d i a g n o s i s 2

RARS RA-type" CMMoL 46,60 ~

62+ 1+ 4/Inf 54/Traumatism

42 At d i a g n o s i s At d i a g n o s i s 46,54

RA RA RAEB-T CMMoL

RARS/ PNH RARS

Tr

5/Sepsis;RI 43/Hem 8/Henl;peritonitis 120+ 12+ 8+ 44+

4 13 7 106 6 At d i a g n o s i s 38

RAEB-T CMMoL CMMoL CMMoL CMMoL CMMoL CMMoL

15 16 17 18 19 20 21 Single c h r o m o s o m e defects Deletion (5q) 22 23 24 25 M o n o s o m y 7 or del(7q) 26 27 28 Trisomy 8 29 lnf Inf

Tr

Inf

H e m ; A N LL(M5)

Tr:Inf;ANLL[M2)

Inf Tr;Inf:Hem Tr;Inf;Bi-AL

lnf lnf Tr ANLL[M2)

Tr

Tr;Inf Tr Tr

125 + 14/CVA 90+ 50+ 50+ 93+ 32/ANLL 1/Not available 36+ 34/Sepsis 15+ 11/ANLL 29/Sepsis 12/Sepsis

116 At d i a g n o s i s 74 40 46 52 30 At d i a g n o s i s 22 30,33 10 2 12,23 8,11

RARS RARS RARS RARS RARS RA RA RA RA RA-type" RA RAEB RAEB RAEB-T

Complications

Normal chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14

S u r v i v a l Im o F c a u s e of d e a t h

Diagnosis (FAB)

Case no.

Chromosome study (mo after diagnosis)

Hydroxyurea

Idarubicine

L.D. Ara-C, Ara-C R u b i d o m y c i n e , A M S A VP-16 Ara-C, R u b i d o m y c i n e Hydroxyurea L.I). Ara-C

CHOP

I.D. Ara-C

E x p o s u r e to c a r c i n o g e n s , c h e i n o t h e r a p y for MDS

da

At d i a g n o s i s , 1 2, a n d 4

At d i a g n o s i s At d i a g n o s i s , 7 At d i a g n o s i s 6 At d i a g n o s i s At d i a g n o s i s At d i a g n o s i s

10/Undeterlnined

3/Inf 9/ H e m 1/ H e m,l nf 0,5/Inf 1/Sepsis

4/ANLL 7/Hem

12 +

22 + 29/Undetermined 4/Inf 15/Hyperleukocytosis

124 + 93 + 4/RI,BPN 1+ 4+ 39 +

48 + 26 + 26 + 3+ 22 +

m

Hem;ANLL

Inf Tr;Inf;ANLL(M4)

Tr;lnf;Hem

Tr

Tr;Hch Tr lnf Tr;ANLL(M2)

Inf;Hem -Hem Tr

Tr;Hch

Tr;lnf Inf Tr;Hem

Tr

L.D. Ara-C

L.D. Ara-C, Ara-C, rubidomycine

L.D. Ara-C

A b u s e of n o n - s t e r o i d anti-inflammatory drugs

ldarubicine Ara-C, R u b i d o m y c i n e , A M S A VP-16

Karyotyped at diagnosis in another laboratory (46,XY,N).

Dyserythropoiesis without anemia.

Abbreviations: Tr - transfusions; Inf - infection; Hem - hemorrhage; Hch ~ hemochromatosis; Bi-AL biphenotypic acute leukemia; ANLL - acute nonlymphocytic leukemia (when determined, M type was indicated); PBN - bronchopneumonia; PNt| = paroxysmal nocturnal hemoglobim~ria; CVA = cerebrovascular accident; A = abnormal; RI = renal insufficiency; Ara-C, cytarabine; MDS, myelodysplastic syndrome; CHOP cyclophosphamide; doxorubicin, vincristine, and prednisone.

RAEB

RAEB RAEB RAEB-T RAEB-T CMMoL

49 50 51 52 53

No c y t o g e n e t i c re s ult 54

RARS RAEB

RARS

C o m p l e x defects 46

47 48

4 24 At d i a g n o s i s At d i a g n o s i s

RARS RAEB RAEB-T RAEB-T

6

124 55 At d i a g n o s i s 1 4 33

48 16 20 At d i a g n o s i s 22

RARS RA RA RA RAEB CMMoL

RARS CMMoL CMMoL CMMoL CMMoL

Miscellaneous 36 37 38 39 40 41 T w o c h r o m o s o m e defects 42 43 44 45

Y loss 31 32 33 34 35

¢al

166

M. Jotterand-Bellomo et al.

Table 4

Chromosomal evolution of 6 patients with MDS Diagnosis (FAB}

Chromoson]e findings"

Identification no.

Initial karyotype

Second karyotype,

Initial analysis

13/12719 14/13042 10/15064

RAEB RAEB-T RA-type

RAEB Bi-AL ANLL(M2)

Normal (121: 100% Normal (4) Normal (10]: 100%

25/13275 29/13970 48/9450

CMMoL RARS RAEB

CMMol RARS RAEB

del{5q}(1/36):2,8% +8(7/26): 27% Complex (13/14): 93%

Abbreviations: MDS = myelodysplastic syndrome; Bi-AL

See{rod analysis Normal (201: 100% Normal {2} Aneuploidy (8/33): 25°/(, Hypodiploidy8 (7/33] 2n -- 47,XXY (1/33} del(5q){9/50): 18% +8{62/138): 45% Complex (19/20): 95%

Interval {in{}} 11 3 15

19 14 7

biphenotypic acute leukemia;

" In AN cases proportions in parentheses and percentages relate to the n u m b e r of A cells versus total number of cells. In NN cases numbers in parentheses relate to the n n m b e r of metaphases analyzed and percentages relate to the n u m b e r of N versus total n u m b e r of cells. t'tlypodiploidy: 2n - 42 {two metaphases, one with 12, 14, 18, 20, the other with 14, 20, 2l, Y), 2n 43 {one metaphase with 16, 20, 21), 2n 44 (three metaphases, the first one with - 4 , 17, the set:ond one with 12, 20, the third one with 9, 14}, and 2n 45 {one metaphase with 19).

Normal c h r o m o s o m e s (40%) and single defects (38%) were observed mare frequently than two defects or c o m p l e x defects (22%). The most frequent single defects were del(5q), m o n o s o m y 7 or del(7q), trisomy 8, and Y loss. Less frequent single defects constituted the miscellaneous category. Half of them were w e l l - k n o w n recurrent defects: t(1;15) (Figs. l a and 4), del(20q) and i(17q). The three other miscellaneous defects were trisomy 14, + M (Fig. 2) and del(1)(p22.1p32.3) (Figs. l b and c, and 4), not considered as yet as recurrent rearrangements in MDS. The M c h r o m o s o m e is difficult to interpret. Although its short arm recalls 8p, a close c o m p a r i s o n of its m o r p h o l o g y with that of c h r o m o s o m e 8 d i d not show true homology. Comparative analysis with c h r o m o s o m e s of the other pairs did not show evidence of striking homology with a particular c h r o m o s o m e arm or segment. W h e n short and poorly banded, c h r o m o s o m e I with d e l ( l p ) closely resembled der t(1;15), but high-resolution c h r o m o s o m e b a n d i n g observed in one metaphase selected from a GM-colony obtained by methylcellulose precursor cell culture clearly showed that l p is deleted between p22.1 and p32.3 (Figs. l b and c, and 4). The presence of two normal 15 c h r o m o s o m e s in this karyotype with a single normal c h r o m o s o m e 1 besides the abnormal one further supports the cytogenetic interpretation of this abnormal karyotype. Four patients showed two c h r o m o s o m e defects. In patient 45/13629 with del(7q) and t(3;3), breakpoints of t(3;3) were the same as those described in the t(3;3) observed in MDS and acute n o n l y m p h o c y t i c leukemia (ANLL) cases with thrombocytosis [1, l 1]. This patient, a 63-year-old woman, had RAEB-T. After a course of chemotherapy, she had partial remission with thrombocytosis (platelets 800 O00h, L). Complex defects affect various chromosomes. Seven of eight patients had monosomy 5, total in four patients and interstitial in three patients. In four patients chromosome 8 was either rearranged, deleted, or trisomic. Chromosome 7 was inissing, rearranged, or abnormal in five cases. In six patients a second c h r o m o s o m e analysis was performed during the course of the disease (Table 4). Two patients (13/12719 and 14/13042) retained their normal karyotype, although this conclusion is only tentative for 14/13042 because of the

167

Chromosomes in M y e l o d y s p l a s t i c S y n d r o m e

i \ 1

2

4

3

\

5

\

4V

6

7

8 ~

13

14

19

9

10

11

15

16

17

20

21

22

\

12 ~

X

18

M

Figure 3 Chronic myelomonocytic leukemia with complex defects: 43, 4. 5, 8 . - 12,- 17, 3p + , l l p +, + der(4)t(4;8)(p11;ql 1), + M. G-banding. small n u m b e r of metaphases available. Case 10/15064, transformed to ANLL, evolved to h y p o d i p l o i d y , a c h r o m o s o m e situation rarely observed in MDS. In patient 25/ 13275, a clear increase of the cell clone with del(5q) was noted (2.8-18%). Patient 29/13970 had p a r o x y s m a l nocturnal hemoglobinuria (PNH) since age 18 years. At diagnosis, c h r o m o s o m e analysis (result from another laboratory) was normal. Ten years after onset of the acquired hemolytic disease, he had macrocytosis (mean corpuscular v o l u m e 125/x 3) with marked poikilocytosis, basophilic stipplings, and a double erythrocytic population. Bone marrow studies showed numerous ringed sideroblasts and an extra c h r o m o s o m e 8 in 27% of the metaphases analyzed. One year later, ringed sideroblasts represented 80% of the erythroblastic population while trisomy 8 increased to 45%. The last patient (48/9450) had complex chromosome defects at initial diagnosis. The second analysis, performed 7 months later, did not show any change in karyotype. F o l l o w - u p of most of the patients is too short for clinical evaluation. Clinical and cytogenetic evolution will be the object of a separate study.

Figure 4 Comparison between del(1) (p22.1p32.3) and der(15)t(1;15)(ql 2;p11). (a) del(lp) with two normal chromosomes 15 (coarse resolution); (b) del(lp) with two normal chromosomes 15 (fine resolution); (c) der(15) and normal chromosome 15.

¢

o

a

•

b

a

c

168

M. Jotterand-Bellomo et al.

DISCUSSION Recent studies showed that in MDS, as well as in acute leukemia and in solid tumors, incidence of chromosome rearrangements increases with improved cytogenetic techniques. With standard methods only one third to one half of MDS showed a chromosome abnormality, whereas in the most recent studies, 4 8 - 7 3 % of the patients had a chromosome defect. In an attempt to improve the yield of our technique for both metaphase n u m b e r and quality of chromosome b a n d i n g resolution, we tested the separation of bone marrow cells on a density gradient as well as four different culture conditions: synchronization with MTX, TdR adjunction without MTX synchronization, and with or without 5637 CM. Separation of m o n o n u c l e a r cells on Ficoll-Paque gradient offers many advantages. Elimination of red blood cells (RBCs} and mature granulocytes makes chromosome preparation easier, as hypotnnic shock, fixation, and especially slide confection are not impaired by n u m e r o u s useless cells and debris. Moreover, separation of m o n o n u clear cells allows better control of culture conditions. First, interface cells can be counted so that the n u m b e r of cells per milliliter of culture m e d i u m can be determined and m a i n t a i n e d constant from one assay to the other or adapted to different culture conditions. Second, as 1.0 to 2.0 x 10 ~ cells proved sufficient for a cytogenetic assay, separated m o n o n u c l e a r cells can be used for different trials in parallel. To study the in vitro effect of 5637 CM on m o n o n u c l e a r cells from myelodysplastic marrows, either the mitotic index as Michaeli et al. [12J or the n u m b e r of metaphases suitable for accurate analysis can be considered. Despite its absolute value, mitotic index in itself may not be sufficient for cytogenetic method assessment. Actually it can be high, yet mitotic figures may be unanalyzable because of poor chromosome spread, unsatisfactory chromosome morphology, length, or b a n d i n g resolution. Therefore, we focused on the n u m b e r of metaphases of good quality obtained with and without CM. The same reasoning applied to the comparative analysis of results obtained with MTX-synchronized versus T d R - u n s y n c h r o n i z e d methods. Although our sample is of small size and m a n y variables exist, such as duration of incubation (48-72 hours), quantity of cells per milliliter of culture medium, 0.5 to 1.6 × 1 0 6 (the concentration of the cell suspension d e p e n d i n g on the total quantity of cells in the marrow sample), type of disease type (RARS, RAEB-T, CMMoL), quality of chromosome spreading, and sometimes variable morphology from slide to slide, results show that 5637 CM, as well as MTX versus TdR method, have no clear-cut effect on the n u m b e r of metaphases. In cases 29, 34, and 31, the mean n u m b e r of metaphases was clearly higher with 5637 CM, whatever the technique used, MTX or TdR. In cases 40 and 36, a higher n u m b e r of metaphases was observed without 5637 CM with MTX. However, in these two cases, metaphase n u m b e r s were not significantly different with TdR. In cases 29, 34, and 31, incubation was longer than in cases 40 and 36. Could a 24-hour incubation difference account for the negative reaction of cases 40 and 36 to 5637 CM? This is not likely. Indeed, although Michaeli et al. [12] observed a variation of the response of leukemic cells to 5637 CM between patients, they demonstrated a general increase of mitotic index in peripheral blood and bone marrow leukemic cells, already evident after 1-day culture. Despite the small size of our sample and of the relative character of our methodology, our results clearly show that in MDS use of 5637 CM may improve the yield of the cytogenetic method in terms of the quantity and consequently, as a choice is made possible, of the quality of metaphases. In contrast to Michaeli's results, however, not all cases appear to react favorably. With regard to use of MTX versus the u n s y n c h r o n i z e d technique, our results are difficult to interpret. Although C u n n i n g h a m et al. [13] demonstrated that MTX

169

Chromosomes in Myelodysplastic Syndrome

Table 5

FAB subtypes of MDS and chromosome findings

Chromosomal findings Normal Single defects del(5q) - 7/del(7q) +8 Y del(20q} del(lp) i(17q} t/1:151 +14 +M Two defects Complex defects No. of cases Abnormality (%)

FAB classification RARS 5

1 2 1

RA 6

RAEB 2

2 1

RAEB-T CMMoL 2

6

1

1 1 4

1 1 1 1 1 1 1 2 13 62

12 50

1 2 5 60

2 3 9 77

1 14 57

Total 21 4 ~

3 2 5 1 20 1 1 1 1 1 4 8 53 60

Abbreviationsas in Table 1.

synchronization does not improve bone marrow chromosome preparations, our experience brings additional evidence that, in myelodysplasia, as in other hematological disorders, as reported by Yunis et al. [2], some cases respond better to MTX, and others respond better to TdR exposure only. Although TdR was not used in the absence of MTX, comparative analysis of cytogenetic results obtained with and without MTX synchronization in a series of 90 cases of hematological disorders also showed the absence of a significant difference between both protocols [14]. To determine if 5637 CM preferentially stimulates chromosomally abnormal cells, the proportions of abnormal cells observed in replicates with and without 5637 CM were compared. Despite the small n u m b e r of cases analyzed thoroughly, results nevertheless allowed us to surmise that 5637 CM does not significantly influence the ratio of abnormal to normal cells in cases with an admixture of normal and abnormal mitoses. In case 2, a single normal metaphase was observed in the presence of 5637 CM, whereas the single normal metaphase of case 36 was observed in the trial without CM. Because the ratio of normal to abnormal cells did not appear to be influenced by 5637 CM stimulation, slides of the best quality were used to evaluate most patients. These slides were generally obtained according to the MTX/5637 CM protocol. A chromosome defect was observed in 60% of the patients at initial study (Table 5). Although it is important to outline this global percentage of abnormality and to compare it with those of other studies, the frequency of chromosome rearrangements relative to each FAB category is more specific and significant. Thus, in regard to the previously cited recent studies, our values for RARS (62%) and CMMoL (57%) are relatively high, those for RA (50%), RAEB (60%) relatively low, and the value for RAEB-T (77%) comparable to those of the other studies (Table 5). The most frequent abnormalities (single or associated with another defect or with complex defects) were del(5q) or m o n o s o m y 5 in 13 cases, trisomy 8 or rearrangement of chromosome 8 in eight cases, total or partial m o n o s o m y or rearrangement of chromosome 7 in eight cases, Y loss in seven cases, and del(2Oq) in two cases. Although Y loss appears to be an age-related chromosome defect, its biological significance in neoplastic hematological conditions, more particularly in M2 patients with

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t(8;21) and chronic myelogenous leukemia (CML) with t(9;22) where it preferentially occurs, is not clear [15-21[. Four of the seven patients with Y loss had CMMoL. Y loss is not a rare defect in CMMoL, although m o n o s o m y 7, trisomy 8, and rearrangement of 12p proved to be the most frequent defects observed in this condition (22-26]. As was the case in our sample, however, Y losses were also observed in the other FAB categories [27]. Because MDS usually occurs in persons aged more than 50 years, Y loss should not represent a surprising situation. With regard to exposure to cytotoxic substances, two cases are worthy of attention. As t(1;7) often occurred in patients exposed e n v i r o n m e n t a l l y and occupationally, patient 27/13028 with t(1;7), a blacksmith who had spent his life in a country village, was thoroughly questioned about any use or contact with toxic substances. No such exposure was found. Patient 46/15462, a 50-year-old man with RARS, showed in all metaphases an abnormal karyotype with complex defects, especially 6p rearrangement [28]. This particular cytogenetic situation was recently shown by Mecucci et al. [29] to have a relationship with m y e l o d y s p l a s i a secondary to exposure to toxic products (alkylating agents or environmental factors of occupational origin). In patient 46/ 15462, no such substances were found. Instead the patient admitted life-long abuse of nonsteroidal antiinflammatory drugs for lombalgia. Most c h r o m o s o m e defects observed in m y e l o d y s p l a s i a are reputed markers of m y e l o i d proliferative disease: e.g., partial or total m o n o s o m y 5, partial or total monosomy 7, trisomy 8, and t(2;11). In contrast, c h r o m o s o m e rearrangements specific for m y e l o i d leukemia rarely occur in MDS; e.g., abnormalities of 11q, characteristic for acute monocytic leukemia were rarely observed in CMMoL [30]. As found in other studies, our cytogenetic data show that in m y e l o d y s p l a s i a , excepting del(5q) in macrocytic RA, c h r o m o s o m e abnormalities are not specific to FAB subtypes (Tables 2 and 5). Chromosome rearrangements described in m y e l o d y s p l a s i a , however, mostly of deleted type, appear to be limited and consequently must have a specific role in the genesis of the m y e l o d y s p l a s t i c condition. Localization on 5q of very important genes in bone marrow cells differentiation [granulocyte-macrophage colony-stimulating factor (GM-CSF), CSF-1, interleukin-3 (IL-3), IL-4, IL-5, platelet-derived growth factor receptor (PDGFR)] certainly represents a decisive step in the u n d e r s t a n d i n g of the relationship between c h r o m o s o m e defects and the malignant process [31-33]. Besides the genetic specificity of c h r o m o s o m e rearrangements at the molecular level, the m o m e n t w h e n rearrangement occurs, i.e., the stage of differentiation of the bone marrow cell, is certainly decisive in the evolution of the disease. M y e l o d y s p l a s i a is considered a clonal disease originating in one stem cell through the occurrence of an initial genetic m u t a t i o n - t y p e event w h i c h probably confers on the " t r a n s f o r m e d " cell a selective proliferative advantage with regard to other stern cells [34-36]. Clonal transformed progenitor cells progressively replace polyclonal cells. The initial event, though necessary, is probably not sufficient to induce malignant cell transformation. A m o n g the subsequent steps of evolution, oncogene(s) activation, as suggested by rasgene activation studies [37-41], and c h r o m o s o m e rearrangements probably represent the most p r o m i n e n t events. The authors are indebted to Dr. N. Schmidt for assistance in preparation of the English version of the manuscript, to M. Donner for typing the tables and to R. Dimo for photographic assistance. This work was supported by Grant 350/86 from the Ligue Suisse Contre le Cancer, by the SandozStiftung, Sandoz, A.G., Basel, Switzerland, by the Ligue Vaudoise Contre le Cancer. Switzerland, and Zeiss, A. G., Switzerland. REFERENCES

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