Cytogenetic studies on 519 consecutive de novo acute nonlymphocytic leukemias

Cytogenetic Studies on 519 Consecutive De Novo Acute N o n l y m p h o c y t i c Leukemias Roland Berger, Georges Flandrin, Alain Bernheim, Maryvonne Le Coniat, Dani/fle Vecchione, Annie Pacot, Josette Derr6, Marie-Th6r/~se Daniel, Fran~oise Valensi, Francois Sigaux, and Maria Elena Ochoa-Noguera

Cytogenetic studies were performed in the same laboratory on 519 untreated cases of de novo acute nonlymphocytic leukemia (ANLL) between 1977 and 1985. The overall incidence of clonal chromosome abnormalities was 54.3%; higher in children (67.5%) than in adults (50.4%). The distribution of chromosome abnormalities was uneven, according to the categories of the FAB nomenclature. The highest frequency of chromosome changes was observed in ANLL-M3 and the lowest in M1 and M6. The frequency of specific chromosome abnormalities and of their associated changes were also estimated. Monosomy 7 was detected in three patients with acute megakaryocytic leukemia (M7). Six cases with two abnormal chromosomally unrelated clones were observed and six constitutional chromosome abnormalities were detected. A clearer knowledge of the incidence of various chromosomal changes in ANLL seems necessary for better differentiation between the so-called primary and secondary chromosome abnormalities and for prognostic evaluation.

ABSTRACT:

INTRODUCTION A c o n s i d e r a b l e n u m b e r of l e u k e m i c p a t i e n t s h a v e b e e n c y t o g e n e t i c a l l y i n v e s t i g a t e d s i n c e b a n d i n g t e c h n i q u e s h a v e b e c o m e a v a i l a b l e , a n d M i t e l m a n [1] c o l l e c t e d 2150 p a t i e n t s w i t h a c u t e l e u k e m i a w i t h a b n o r m a l k a r y o t y p e s . T h e e x i s t e n c e of n o n r a n d o m c h r o m o s o m e c h a n g e s a n d of a b n o r m a l i t i e s m o r e or less s p e c i f i c for a t y p e of l e u k e m i a h a s b e e n s h o w n i n t h e last 10 y e a r s [2]. T h e o v e r a l l i n c i d e n c e of c h r o m o s o m e a b n o r m a l i t i e s i n a c u t e n o n l y m p h o c y t i c l e u k e m i a (ANLL) h a s b e e n estim a t e d m a n y t i m e s [ 3 - 1 2 ] a n d t h e p r o p o r t i o n of k a r y o t y p i c a l l y n o r m a l a c u t e l e u k e m i a s h a s b e e n r e d u c e d w i t h t h e u s e of m o r e t e c h n i q u e s [13, 14]. H o w e v e r , t h e i n c i d e n c e of d e f i n e d c h r o m o s o m a l a b n o r m a l i t i e s s u c h as t r i s o m i e s , s p e c i f i c t r a n s l o c a t i o n s , or d e l e t i o n s is still less k n o w n i n a s m u c h as t h e largest p u b l i s h e d series

From the Laboratoire d'Oncologie et d'Immunoh~matologie, LOI CNRS LP 101 (R. B., A. B., M. LeC. D. V., J. D.), Unit6 INSERM U 301, Hospital St-Louis (R. B., G. F., A. B., M. LeC., D. V., J. D., M-T. D., F. V. F. S.), and Laboratoire Central d'H6matologie, Hospital St-Louis (G. F., A. P., M-T. D., F. V., F. S., M. E O-N.), Paris, France.

Address requests for reprints to Dr. R. Berger, U 301, Centre Hayem, Hopital St-Louis, 2 Place du Dr. A. Fournier, 75475 Paris Cedex 10, France. Received December 19, 1986; accepted March 5, 1987.

9 © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genet Cytogenet 29:9-21(1987) 0165-4608/87/$03.50

10

R. Berger e,t al. of chromosome studies in leukemia were provided by the International Workshops on Chromosomes in Leukemia [15, 16] based on cases collected from different centers. We report the results of cytogenetic studies on 519 de novo cases of ANLL performed in patients e x a m i n e d in one d e p a r t m e n t and in the same cytogenetic laboratory between 1977 and 1985.

MATERIALS AND METHODS

At the end of 1977 we began a systematic cytogenetic study of ANLL patients in the Department of Hematology (H6pital St-Louis, Paris). A total of 519 patients: 399 adults (218 male and 181 female; average age 50.6± years) and 120 children under 16 (61 male and 59 female; average age 6.8 ± 4.8 years) have been successfully cytogenetically s t u d i e d by b a n d i n g techniques. The patients with secondary ANLL were e x c l u d e d from the study, as well as previously treated patients unless they had also been studied at diagnosis in the laboratory. Thirty patients were studied at diagnosis and in relapse. Chromosome studies were performed on bone marrow cells, either by a direct m e t h o d and/or after culture for 24 and 48 hours, sometimes 72 hours, and/or u n s t i m u l a t e d blood cell cultures for 24, 48, and/or 72 hours. Phytohemagglutinin-stimulated blood cell cultures were also performed on the cells of 70 patients. A bone marrow study alone was performed on 270 patients and u n s t i m u l a t e d blood cell study alone on 54 patients. Both blood and bone marrow were studied in the r e m a i n i n g 195. Banding techniques, m a i n l y RHG, GTG, and G-bands using Wright's stain and, less often, CBG and QFQ b a n d i n g [17] techniques were performed. All metaphases were a n a l y z e d on pictures taken with a x 100 oil immersion objective and the c h r o m o s o m e s were classified according to the International Nomenclature [17, 18]. A total of 15-25 metaphases were e x a m i n e d in the majority of the patients; the numbers varied between 10 and 147 mitoses. A n abnormal clone was defined according to the criteria of the Fourth International Workshop on Chromosomes in Leukemia [16]: two cells with the same extra c h r o m o s o m e or same rearrangement or three cells with loss of the same chromosome. A single normal cell was defined as a normal clone. Three categories of ANLL were considered in some circumstances: NN (only normal karyotypes), A A (only abnormal karyotypes), AN (mixture of normal and abnormal metaphases). It is clear that the choice of culture techniques and the level of expertise have changed the results over such a long period of time. The ANLL were classified according to the F r e n c h - A m e r i c a n British nomenclature (FAB) [19-22] into seven groups (M1-M7), plus the unclassifiable ANLL. In the present series the cytologic classification was a p p l i e d with the new FAB r e c o m m e n d a t i o n s [21, 22]. The smears were stained using May-Gr(inew a l d - G i e m s a stain, and various cytochemical and cytoenzymologic techniques were applied. RESULTS Incidence of Chromosomal Clonal Abnormalities in ANLL

The incidence of c h r o m o s o m a l clonal abnormalities was 54.3% (282 of 519) with a highly significant difference (p ~ 0.001) between adults (50.4%; 201 of 399) and children (67.5%; 81 of 120). A m o n g the patients with chromosomal abnormalities 103 (19.8%)--74 adults and 29 c h i l d r e n - - h a d only c h r o m o s o m a l l y abnormal metaphases (AA), whereas, 179 (34.5%)--127 adults and 52 c h i l d r e n - - h a d a mixture ot c h r o m o s o m a l l y n o r m a l and abnormal metaphases (AN).

11

C y t o g e n e t i c S t u d i e s o n 519 C o n s e c u t i v e De N o v o A N L L

T h e d i s t r i b u t i o n of t h e 519 p a t i e n t s i n t h e g r o u p s of t h e FAB n o m e n c l a t u r e is s h o w n i n T a b l e 1. T h e d i s t r i b u t i o n of 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 t h e s e g r o u p s w a s u n e v e n , t h e h i g h e s t b e i n g i n M 3 a n d M 4 E O , t h e l o w e s t i n A N L L - M 1 a n d -M4 ( T a b l e 1). T h e p e r c e n t a g e of a b n o r m a l c a s e s i n t h e M 2 s u b g r o u p w a s i n t e r m e d i a t e , p a r t l y d u e to t h e r e l a t i v e l y h i g h i n c i d e n c e of M 2 w i t h a t(8;21) t r a n s l o c a t i o n i n children and young adults.

Numerical Aberrations Psendodiploidy was the most common abnormality among cytogenetically abnorm a l l e u k e m i a s , a n d h y p e r d i p l o i d y w a s m o r e f r e q u e n t t h a n h y p o d i p l o i d y (Table 2). T w o s u b g r o u p s m i g h t b e c h a r a c t e r i z e d a m o n g h y p e r d i p l o i d ANLL, o n e w i t h s i m p l e near-diploid hyperploidy and the other with hyperdiploidy in the hypertriploidt e t r a p l o i d range. T h i s last c a t e g o r y ( 7 6 - 9 5 c h r o m o s o m e s ) h o w e v e r , w a s l i m i t e d to six cases of a d u l t A N L L of v a r i o u s c y t o l o g i c t y p e s , a n i n c i d e n c e t h a t is c l e a r l y less frequent than in acute lymphoblastic leukemia.

Trisomy 8 T r i s o m y 8 w a s t h e m o s t c o m m o n n u m e r i c a l a b n o r m a l i t y (8.9% of all c a s e s of ANLL). It w a s t h e sole a b n o r m a l i t y i n 17 p a t i e n t s (3.3%) a n d a s s o c i a t e d w i t h o t h e r c h r o m o s o m e c h a n g e s i n 29 (5.6%). T h e d i s t r i b u t i o n a m o n g t h e F A B n o m e n c l a t u r e s u b g r o u p s w a s u n e v e n . T r i s o m y 8 w a s m o r e f r e q u e n t i n t h e M 2 a n d M 5 c l a s s e s , as s h o w n i n T a b l e 3.

Table 1

D i s t r i b u t i o n of 519 A N L L c a s e s i n t h e g r o u p s of t h e F A B n o m e n c l a t u r e a n d i n c i d e n c e of 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 M1

M2

M3 + M3v

M4

M4EO

M5

M6

M7

Unclassified

Total

ADULTS NN AN AA

49 16 11

52 38 22

4 23 13

27 8 4

3 10 5

42 16 8

9 4

3 4

9 8 11

198 127 74

Total

76

112

40

39

18

66

13

7

28

399

39 52 29

CHILDREN NN AN AA

11 4 3

4 16 8

1 7 3

Total

18

28

11

5 4 6

4 4 1

5 11 3

3

4

2

1

2 6 2

15

9

19

5

5

10

120

7 4 1

11 14 13

237 179 103

38 71.1

519 54.3

TOTAL NUMBER NN AN AA

60 20 14

AN + AA (%)

94 36.2

56 54 30 140 60

5 30 16

32 12 10

7 14 6

47 27 11

12 4 2

51 90.2

54 40,7

27 74

85 44.7

18 33.3

12 41.7

12

R. Berger et al.

Table 2

Table 3

N u m b e r of A N L L cases w i t h h y p e r d i p l o i d y , h y p o d i p l o i d y , a n d p s e u d . o d i p l o i d y : P e r c e n t a g e s c a l c u l a t e d o n 519 ANLL cases Hyperdiploid (%)

Hypodiploid (%)

Pseudodiploid (%)

Total (%)

Adults Children

66 (16.5) 19 (15.8)

41 (10.3) 16 (13.3)

94 (23.6) 46 (38.3)

201 (50.4) 81 (67.5)

Total

85 (16.3)

57 (11)

140 (27)

282 (54.3)

I n c i d e n c e of t r i s o m i e s 8, 9, 11, a n d 21, a n d of m o n o s o m y 7 i n t h e F A B c l a s s e s of ANLL

+ 8 only + 8 and others Total + 9 only + 11 only + 11 and others + 21 only + 21 and others - 7 only - 7 and others Total

M1

M2

2 4 6

7 4 11 1 1 2 1 1

2 1 3 1 3 4

2 2

M3 + M3v

M4

M4EO

M5

M6

4 4

2 1 3

2 2 4

3 7 10 2

3 3

1 1 1

1 2 2

1 1 1 1 1 2

M7

Unclassified

1 1

1

4 4

2

1 2 1 3

2 1 2 3

Total 17 29 46 3 2 9 3 10 7 9 16

Monosomy 7 M o n o s o m y 7 w a s f o u n d i n 16 A N L L cases (3.1%). It w a s t h e sole a b n o r m a l i t y i n s e v e n c a s e s a n d a s s o c i a t e d w i t h o t h e r c h a n g e s i n t h e n i n e o t h e r s ( T a b l e 3). I n o n e c h i l d w i t h M 7 t w o d i f f e r e n t c l o n e s w e r e f o u n d , o n e w i t h m o n o s o m y 7 as t h e sole a b n o r m a l i t y a n d t h e o t h e r c o m p l e x w i t h 50 c h r o m o s o m e s a n d w i t h o u t m o n o s o m y 7. T w o o t h e r c a s e s w i t h M 7 h a d m o n o s o m y 7 as t h e sole a b n o r m a l i t y . T h r e e pat i e n t s w i t h M1 h a d P h - p o s i t i v e A N L L a s s o c i a t e d w i t h m o n o s o m y 7.

Trisomy 21 A n a c q u i r e d t r i s o m y 21 w a s p r e s e n t i n 13 A N L L cases (2.5% of all ANLL), e i t h e r as a n i s o l a t e d f i n d i n g i n t h r e e or a s s o c i a t e d w i t h o t h e r c h a n g e s i n t e n cases ( T a b l e 3). T h e t h r e e p a t i e n t s w i t h t r i s o m y 21 as t h e sole a b n o r m a l i t y h a d d i f f e r e n t t y p e s of ANLL.

Trisomy 11 T r i s o m y 11 w a s t h e sole c h r o m o s o m e a b e r r a t i o n i n t w o A N L L cases, o n e M 2 a n d t h e o t h e r M4, a n d w a s a s s o c i a t e d w i t h o t h e r c h a n g e s i n n i n e o t h e r s . A m o n g t h i s last s u b g r o u p t w o p a t i e n t s h a d t w o c l o n e s , o n e w i t h t r i s o m y 11 as t h e sole a b n o r mality and the other with different changes.

Other Trisomies as the Sole C h r o m o s o m e Abnormality T h r e e A N L L cases, o n e M 2 a n d t w o M5, h a d t r i s o m y 9 as t h e sole a b n o r m a l i t y , a n d two, o n e M 2 a n d o n e M5, a t r i s o m y 12. O t h e r t r i s o m i e s as t h e sole a b n o r m a l i t y w e r e o b s e r v e d i n s i n g l e cases: t r i s o m y 4 i n a n A N L L - M 1 , a n d t r i s o m y 22 i n a n M4.

13

Cytogenetic Studies on 519 Consecutive De Novo ANLL

Loss of Sex Chromosomes Loss of the Y c h r o m o s o m e (19 ANLL of 218; 8.7%) was mainly associated with other c h r o m o s o m e abnormalities such as a t(8;21). Loss of Y was found as the sole abnormality in two ANLL cases, one M4 in a 78-year-old m an and one acute erythremia in a 6-year-old patient. No female patient had a loss of an X as the sole ch ro m o s o m a l change. Loss of the X was associated with a t(8;21) in four female patients and with an inv(16) in a fifth one.

Structural Chromosome Abnormalities Clonal structural c h r o m o s o m e abnormalities were present in 207 cases of ANLL (39.9%). The majority of these abnormalities are k n o w n as specific rearrangements, but some are u n i q u e and others were i n c l u d e d among various associated abnormalities giving c o m p l e x patterns of c h r o m o s o m e changes. S t r u c t u r a l Abnormalities Related with Morphologic Subtypes of ANLL

Translocation t(8;21)(q22;q22). A total of 43 ANLL cases, 26 adults and 17 children, with a t(8;21) were collected (8.3% of all ANLL) (Table 4). In one patient, not i n c l u d e d in the 43, with a typical M2, the translocation was only detected in relapse, whereas, the karyotype had been normal at diagnosis. The group of ANLL cases with t(8;21) was not h o m o g e n e o u s because the translocation was the sole abnormality in only 18 patients (41.9% of them). The most c o m m o n associated ab-

Table 4

C h r o m o s o m e findings in 43 cases of ANLL-M2 with t(8;21) Number of patients

AN AA Males 46,XY,t(8;21) 46,XY,t(8;21)/- Y,X, - Y,t(8;21) 46,X, - Y,t(8;21) Females 46,XX,t(8;21) 46,XX,t(8;21;5)(q22;q22;q31) 46,XX,t(8;21;11)(q22;q22;p15) 46,XX,t(8;21)/X, - X,t(8;21) 46,X, - X,t(8;21) Associated chromosomal abnormalities 47,XY,t(8;21), + 8 46,X,- Y,t(8;21), ÷ 8 45,X, - Y,t(8;21)/46,X, - Y,t(8;21), + 8 47,XX,t(8;21), ÷ 8,del(9)(q13q33) 46,XY,t(8;21)/46,XY,t(8;21),del(9)(q22) 46,XX,t(8;21),del(9)(q31),del(11)(p13) 46,XX,t(8;21),del(7)(q22) 46,XX,t(8;21),del(7)(q21) 44,X, - Y,t(8;21), - 12,t(7;12)(q35;q11) 46,XY,t(8;21)/46,X,- Y,t(8;21),t(1;5)(q43;q11) 46,XY,t(8;21)/XY,t(8;21),+ variable extra chromosomes 46,X,- Y,t(8;21)/X,- Y,t(8;21),with variable ploidy (54-86)

28 15 3 6 15 13 1 1 3 1

14

R. Berger et al.

erration was loss of Y chromosome (21 of 24 male patients; 87.5%), whereas, the loss of an X was less frequent in females (four of 19; 21.1%). These abnormalities. as well as others, were either present in all abnormal mitoses or associated with a clone with the translocation as the sole abnormality. The other changes i n c l u d e d a trisomy 8 in four ANLL cases; 9q with variable breakpoints in three, associated in one with del(11)(p13); 7q in two, and with other translocations in two others (Table 4). Two variant translocations were also detected: t(8;21;5)(q22;q22;q31) and t(8;21;ll)(q22;q22;p15). Finally, 40 ANLL cases with t(8;21) were classified as M2, two as M1, and one as M4. In these three u n u s u a l ANLL cases the translocation breakpoints on chromosomes # 8 and #21 were the same as in the other t(8;21) ANLL cases. One was m i n u s Y and the two others did not exhibit additional abnormalities. One patient who relapsed as M5 has been previously reported [24].

Translocation t(15;17)(q22;q12). The t(15;17) has been detected in only ANLL belonging to the M3 and M3v (M3 variant) subgroups of the FAB classification. Thus, the t(15;17) was seen in 38 of 44 usual acute promyelocytic leukemias (M3) and in all seven variant forms (M3v) i.e., in 8.7% of all ANLL. One was a variant translocation t(15;17;4)(q22;q12;q21), in a typical M3. Four of five karyotypically normal M3 cases were investigated before we knew the importance of short-term culture for the detection of the chromosome abnormality [25]. However, in another patient, we were unable to detect the specific translocation in spite of the presence of two abnormal cell populations with 47,XY, + 8/48,XY, + 8, + 21. The t(15;17) was associated with the other abnormalities in n i n e patients, three of them with trisomy 8 (Table 5). The incidence of chromosome changes associated with t(15;17), thus, was lower than in t(8;21) ANLL. Chromosome 16 Abnormalities and Acute Myelomonocytic Leukemia with Bone Marrow Eosiniphilia Of 27 cases of ANLL with bone marrow eosinophilia (M4EO), 20 ANLL with chromosomal clonal abnormalities were detected. The most frequent was the rearrangem e n t of chromosome # 1 6 (55.6% of M4EO). An inv(16)(p13q22) was found in 13 patients, the association of inv(16) and del(16)(q22) in two, and del(16) in another.

Table 5

Chromosomal findings in 51 ANLL-M3 cases Number of patients

NN AN AA Unusual karyotypes t(15;17;4)(q22;q12;q21) t(15;17),+ 8 t(15;17)/t(15;17), + 8 t(15;17)/t(15;17), - 9 t(15;17)/t(15;17),t(4;4)(q13;q35) t(15;17), - 21, + t(21;21)(p11;q11) t(15;17),t(3;18)(p23;p11) t(15;17),19q+ ,6p+ ,variable anomalies +8/+8,+21

5 30 16

15

C y t o g e n e t i c S t u d i e s on 519 C o n s e c u t i v e De N o v o A N L L

T h e k a r y o t y p e a p p e a r e d n o r m a l in a n o t h e r p a t i e n t (NN) but a t y p i c a l inv(16) was o b s e r v e d in relapse, a s s o c i a t e d w i t h a del(2)(q23). S i n g l e cells w i t h a d d i t i o n a l chrom o s o m e a b n o r m a l i t i e s w e r e p r e s e n t in four cases of A N L L at diagnosis, but c l o n a l a d d i t i o n a l c h a n g e s a s s o c i a t e d w i t h the r e a r r a n g e m e n t of o n e c h r o m o s o m e # 1 6 w e r e o n l y d e t e c t e d in four patients. O n e h a d the k a r y o t y p e 4 7 , X X , + 2 2 , o n e an inv(14) a s s o c i a t e d w i t h inv(16), a n o t h e r h a d a m i n o r p o p u l a t i o n w i t h both inv(16) and t r i s o m y 8, and the f o u r t h h a d a loss of o n e X: 4 5 , X , - X , i n v ( 1 6 ) . C h r o m o s o m e c h a n g e s o t h e r t h a n # 1 6 r e a r r a n g e m e n t s h a v e b e e n d e t e c t e d as c l o n a l a b n o r m a l i t i e s in five o t h e r patients: 47,XY,+8 in t w o 4 8 , X X , + 8 , + 2 2 in another, 46,XX,del(1)(q21) in a fourth, and 4 7 , X Y , + m a r in the last one. Finally, n o r m a l k a r y o t y p e s w e r e s e e n in s e v e n patients at diagnosis.

Chromosome 11q Abnormalities C h r o m o s o m e 11q a b n o r m a l i t i e s h a v e b e e n d e t e c t e d in 20 A N L L (3.9%), of w h i c h 15 w e r e classified as M5 a c c o r d i n g to FAB n o m e n c l a t u r e . F i v e of t h e m w e r e deletions at b a n d s 11q23 to q24, eight t r a n s l o c a t i o n s i n v o l v i n g v a r i o u s c h r o m o s o m e s and 11q23 or 11q24, a n d t w o t r a n s l o c a t i o n s t(10;11)(p13;q14). T h r e e cases of A N L L w i t h r e a r r a n g e m e n t s at 11q23 or q24 w e r e classified as M4, t w o of t h e m in 1-yearold c h i l d r e n , a n d one w i t h a t(1;11)(q22;q22) was classified as M2. O n l y one M5 patient h a d the t ( 9 ; l l ) ( p 2 1 ; q 2 3 ) , and three M5 had t(11;11)(q23;q25). T h r e e M5 had c o m p l e x t r a n s l o c a t i o n s , a n d the 11q r e a r r a n g e m e n t was a s s o c i a t e d w i t h o t h e r abn o r m a l i t i e s in s e v e n M5 A N L L (Table 6).

Table 6

C h r o m o s o m e 11q r e a r r a n g e m e n t s in A N L L

Patient number

Age (yr)/Sex

FAB

AN/AA

2149 1965 1256

26/M 27/F 1/M

M5 M5a M5a

AN AN AA

1307

74/M

M5a

AA

1069

75/M

M5a

AA

2957 3O97 2051 3404 3914 3177 3406 1002 3090 4133 1297 2313 573 2090 1091

3/F 1/F 0.3/F 1/F 26/F 71/M 33/F 9/F 2/F 3/F 0.4/F 1/M 16/F 1.5/F 66/M

M2 M4 M5b M4 M5b M4 M5a M5a M5b M5 M5a M5a M5a M5 M5a

AA AA AA AA AA AA AN AN AN AN AN AA AN AN AA

Karyotype 46,XY,del(11)(q24) 47,XX, - 8, + 7p + ,t(7;8)(p11;q11), + i(8q),del(11)(q24) 46,XY,der(8)dup(8)(qllq24), ins(10)(p12p15),ins(11)(q23q25), del(10)(p12),del(11)(q23q25) 44,X, - Y, - 5, - C, - 17, - 21 ,del(11)(q22),17p +, + r, + mar 1,+mar 2 46,XY,t(8;13)(q24;q12),del(11)(q23q24),13p + / 46,XY,t(11;13)(p12;q12),13p + 46,XX,t(1;ll)(q22;q25) 46,XX,t(1;ll)(q21;q23) 47,XXX,t(1;11)(q21;q24).Consitutional 47,XXX 46,XX,t(1;3;11)(q22;q21;q23) 46,XX,t(1;6;11)(p36;q21;q23) 46,XY,t(5;11)(q14;q23),de|(14)(q21q31) 46,XX,t(9;11)(p22;q23) 46,XX,t(11;ll)(q23;q25) 46,XX,t(11;11)(q23;q25) 46,XX,t(11;11)(q23;q25) 46,XX,t(11;17)(q23;q23) 47,XY, + 6,t(11;19)(q23:p13) 47,XX, + 8,t(10;11)(p13;q14) 46,XX,t(1;9)(q11;q34),t(10;11)(p14;q14) 46,XY,t(6;10;11)(p22;q14;q14)

16

R. Berger et al.

5q and 7q Chromosome Abnormalities Deletions of the tong arms of chromosomes # 5 and #7 are k n o w n to be among the chromosome abnormalities in ANLL with complex changes [28]. A 5 q - chromosome has been detected in five such cases of ANLL. Only one patient with M2 had a del(5)(q14q23) as the sole abnormality and another with M5 had a del(7)(q34) as the sole change. A 7 q - chromosome was associated with t(8;21) in two M2 ANLL and with an inv(16) in one M4EO.

Chromosome 12 Short Arm (12p) Abnormalities A deletion or translocation involving the short arm of chromosome #12 was detected in eight patients (1.5%), of which seven have been recently published [26]. The 12p abnormality was the sole change in four patients and was associated with an increased n u m b e r of bone marrow basophils [27].

Philadelphia Chromosome and ANLL Three adult patients had a Ph-positive ANLL, with a c o m m o n t(9;22)(q34;q11) associated with m o n o s o m y 7. The three cases of ANLL were classified as M1.

Other Structural Chromosome Changes Various other structural changes were detected either as isolated changes or associated with other abnormalities. Such changes involved all the autosomes. Only one M2 with an increased n u m b e r of basophils in the bone marrow had the t(6;9)(p23;q34), associated with variation from cell to cell, i n c l u d i n g some metao phases with the translocation and del(12)(p12). A t(3;3)(q21;q27) was found to be associated with a 14p + chromosome in a patient with M1 and a platelet count of 100 × 109/L. Three patients had a 2 0 q - chromosome, two of them associated with other changes. A n acquired Robertsonian translocation was detected in six patients, was associated with other abnormalities in five and was the sole change in one MI: 4 6 , X X , - 1 3 , +t(13;15)(p11;q11). Two patients, one with M2 and the other with M5, had the same t(3;5)(q24;q32). Chromosomal structural changes other than specific translocation as the sole abnormality are s h o w n in Table 7.

Complex Chromosome Abnormalities An association of more than four chromosome changes, often associated with karyotypic variation from cell to cell, was detected in 58 patients (11.2% of all ANLL cases). These cases are not reported here in detail because most have been recently published [28]. FAB classification showed that 15 were M2, ten were M1, and 17 were unclassifiable. These ANLL cases occurred in all age groups, 12 were in patients u n d e r 16 years of age.

ANLL with Two Unrelated Clonal Chromosomal Changes Two unrelated clonal chromosomal abnormalities were detected in five ANLL cases. One of the clones was 47, + 8 in two patients, and 47, + 11 in two others. In three ANLL, a single n u m e r i c a l abnormality was juxtaposed to more complex changes in another clone (Table 8). The existence of one clone with a single chromosome abnormality in four of these ANLL cases is noteworthy because it could have resulted from a secondary event.

17

C y t o g e n e t i c S t u d i e s o n 519 C o n s e c u t i v e De N o v o A N L L

Table 7

S t r u c t u r a l a b n o r m a l i t i e s as t h e sole c h r o m o s o m e c h a n g e i n A N L L (specific r e a r r a n g e m e n t s e x c l u d e d )

Patient number

Age (yr)/Sex

FAB a

AN/AA

Karyotype

279 4403 3817 2601 2632 3773 4011 2148 968 1660 2687 2862 87 3579 2352 1604 4296 3808 718 1286 1122 1096 4227 4743 4739 1162 2811

47/F 25/F 2/M 1/M 4/M 61/F 23/M 86/F 31/M 7/F 16/M 32/F 12/M 48/F 1.3/M 19/M 81/M 64/M 18/M 72/M 46/F 41/F 14/M 59/M 12/M 69/M 59/M

M4EO M2 U M5b M4 U M2 M4 U M2 M5 M2 M4 M2 M6 M4 M5b M4 M4 M1 M2 M2 M2 M2 M1 M1 M2

AN AN AN AN AA AN AN AA AN AN AA AA AN AA AA AN AN AA AA AN AA AN AN AN AA AN AN

46,XX,del(1 )(q21) 46,XX,t(1;6)(q21;q27) 46,XX,inv(2)(p21q11) 46,XY,t(2;7)(p21;p22) 46,XY,t(2;9)(p21;q34) 46,XX,t(2;?)(p25;?) 46,XY,del(2)(p21) 46,XX,del(3)(q13) 46,XY,t(4;8)(q35;q11) 46,XX,t(3;5)(q24;q32) 46,XY,t(3;5)(q24;q32) 46,XX,t(4;19)(q31;p11) 46,XY,t(4;?)(q35,?) 46,XX,del(5)(q14;q23) 46,XY,t(5;12)(q13;q24) 46,XY,t(6;13)(p13;q11) 46,XY,del(7)(q34) 46,XY,t(7;11)(p15;p15) 46,XY,t(8;17)(q21;p13) 46,XY,del(9)(q12q31) 46,XX,del(9)(q12q32) 46,XX,t(9;?)(q34;?) 46,XY,dup(12)(q24) 46,XY,t(13;?)(q34;?) 46,XY,t(14;17)(q32;q12) 46,XY,del(20)(q11) 46,XY,i(22q)

°u, unclassifiable in the FAB nomenclature.

Constitutional Chromosome Abnormalities in Patients with ANLL A c o n s t i t u t i o n a l c h r o m o s o m e a b n o r m a l i t y w a s d e t e c t e d i n six c h i l d r e n (1.2% of all ANLL). T h r e e h a d t r i s o m y 21, t w o h a d t h e k a r y o t y p e 47,XXX, a n d o n e w a s a m a t e r n a l l y i n h e r i t e d t(1;12)(p33;q13). In a s e v e n t h p a t i e n t , t r i s o m y 13 r e s u l t i n g f r o m a n u n b a l a n c e d r o b ( 1 3 ; 1 5 ) w a s f o u n d i n all m e t a p h a s e s a n d it w a s i m p o s s i b l e to d i s c o v e r if t h e t r a n s l o c a t i o n w a s c o n s t i t u t i o n a l a n d p r e d i s p o s i n g to m i t o t i c n o n d i s junction.

Table 8

ANLL cases with two chromosomally unrelated abnormal clones

Patient number

Age (yr)/Sex

FAB

Karyotypes

3931 1622 2683

50/F 80/M 74/F

M2 M4 M1

2226

68/M

M6

1833

2/M

M7

47,XX, + 8/47,XX, + 10 46,XY/47,XY, + 8/47,XY, + 11 46,XX/47,XX, + 11/ 46,XX,del(10)(q11),t(4;12)(q13;13) 46,XY,mar 1,min/76,XY,mar 2,mar 3.Variation from cell to cell. 46,XY/45,XY, - 5/50,XY, + 8, + 14, + 19, + 20

Number of metaphases 8/10 2/6/16 23/17/4 10/12 2/17/12

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R. Berger et al.

DISCUSSION The overall incidence of clonal chromosomal abnormalities in untreated de novo ANLL has varied from about 50%, with the usual b a n d i n g techniques, to more than 90%, with the use of the high-resolution techniques [13, 14]. The incidence of different types of chromosomal abnormalities is more difficult to estimate because the relatively small n u m b e r of cases, and because the series collected in collaborative workshops come from various origins. The present series of 519 de novo ANLL was established from 1977 to 1985, and consisted of only untreated patients who attended the same center. Two kinds of possible biases might be expected, thus, due to the recruitment of the patients and to the technical failures of cytogenetic study. Unfortunately, these two biases were impossible to investigate. The causes of technical failures were m a i n l y an insufficient n u m b e r of cells and the absence of analyzable mitoses. The overall incidence of clonal chromosome abnormalities was 54.3%, less than the percentage of other investigators using high-resolution b a n d i n g techniques [13, 14, 29-31]. However, the first i m p r o v e m e n t in the detection of chromosome abnormalities was the use of short-term bone marrow culture [25], which allowed detection of some translocations, such as t(15;17) and t(8;21) more easily than the direct method [32-24]. The overall incidence of chromosomal abnormalities were significantly increased in children u n d e r 16 (67.5%), compared with adults (50.4%). The difference could be due to the high incidence of some forms of ANLL with specific changes in children, for example the t(8;21) associated with 16 of 22 M2 cases in children (72.7%), whereas, the incidence was 23.2%, (26 of 112) M2 cases in adults. The distribution of chromosome abnormalities among the categories of the FAB nomenclature was uneven. The lowest incidence of detected abnormalities was in M1 and M4, and the highest incidence was in M3. The differences could be due to technical reasons and to the existence of undetected subtle changes that might be more frequent in M1 and M4 than in M2. The use of improved techniques will reduce the n u m b e r of ANLL with normal karyotypes in the future. However, the existence of ANLL without any detectable cytogenetic change remains probable. As expected, the present study confirmed the relationship between some specific chromosome abnormalities such as t(15;17) and M3-M3v, t(8;21) and M2, inv(16) and M4EO, and rearrangements at bands 11q23-q24 and M5. It also showed that some specific rearrangements may occur in a small n u m b e r of ANLL classified in other than the usual categories of the FAB classification. This was not true for the rearrangements of chromosome #16 and M4EO. However, one t(8;21) ANLL was categorized as M4. These findings, also reported in the Fourth International Workshop on Chromosomes in Leukemia [16] showed that the classification in an FAB class depends not only on the n u m b e r of blasts or distribution in hematopoietic cell lines, but also on more specific cytologic features that may fit better with specific chromosome abnormalities. The present series also provided an estimation of the incidence of c o m m o n and specific abnormalities in ANLL (summarized in Table 9, in which the percentage of abnormalities was calculated among both all ANLL cases and among ANLL cases with chromosomal abnormalities). Two u n u s u a l translocations were detected t(10;11)(p13-14;q14) in two M5 and t(3;5)(q24;q34) one M1 case and one M4 case. On the contrary, the t(9;11)(p22;q23) and t(6,9)(p23;34) were found only once. Chromosomal changes in malignant cells have been classified into two categories, primary and secondary abnormalities. The primary changes are m a i n l y specific translocations or inversions related to specific forms of proliferations as, for example, t(15;17) and acute promyelocytic leukemia. Recent molecular studies have shown that not only these

19

Cytogenetic Studies on 519 Consecutive De Novo ANLL

Table 9

Incidence of c o m m o n and specific abnormalities in 519 ANLL cases

Chromosome abnormality + 8 only - 7 only + 21 only +11 only + 9 only - Y only 5 and others t(8;21) t(15;17) del(16q) or/and inv(16) 11q23-q24 abnormalities 12p abnormalities 5 q - and 7 q t(9;22) t(6;9) Others

Adults 15 5 3 1 2 1 4 26 33 12 7 6 6 3 1 73

Children 2 2 1 1 1 1 17 10 3 8 2 1

32

Percentage of all ANLL

Percentage of chromosomally abnormal ANLL

17 7 3 2 3 2 5 43 43 15 15

3.3 1.3 0.6 0.4 0.6 0.4 1.0 8.3 8.3 2.9 2.9

6.0 2.5 1.0 0.7 1.0 0.7 1.8 15.2 15.2 5.3 5.3

8 7 3 1 105

1.5 1.3 0.6 0.2 20.2

2.8 2.5 1.0 0.4 44.3

Total

ch ro m o s o m a l abnormalities but also their m o l ecu l ar consequences were c o m m o n to similar forms of malignancies in different patients with, for example, Burkitt l y m p h o m a - l e u k e m i a and chronic granulocytic leukemia. The concept of secondary c h r o m o s o m e changes in based on the existence of clonal c h r o m o s o m e evolution on the one hand [35], and on the possibility of c h r o m o s o m e changes additional to specific translocations on the other. These changes may be nonrandom, such as trisomy 8, or appear random if they are rare. In ANLL, trisomies or c h r o m o s o m e losses may be associated with specific changes. An example is the high i n ci d en ce of the Y c h r o m o s o m e loss in males with t(8;21). The cells with 45,X,-Y,t(8;21) may coexist in the same patient with cells having 46,XY,t(8;21), suggesting that the Y loss was a secondary event that occurred during the proliferation of the original abnormal clone with the translocation as the sole abnormality. Conversely, a specific translocation may appear secondarily, as sh o w n by the occurrence of a t(15;17} with a p h en o ty p e of M3 in the acute transformation of a Ph-positive chronic myelocytic leukemia [36]. When only one chromosomal, mainly numerical, abnormality (trisomy 8, m o n o s o m y 7, etc.) occurs in ANLL, it is impossible to know if it is a primary or secondary event. This uncertainty explains w h y u n i q u e c h r o m o s o m e changes have been distinguished from the associated abnormalities in the present work. A better u n d e r s ta n d in g of these different possibilities necessitates a better knowledge of the i n c i d e n c e of the n o n r a n d o m and apparently n o n r a n d o m chromosomal changes. Whatever the m o le c u lar consequences of ch r o m o so m al abnormalities are in leukemias the two types of abnormalities, primary and secondary, have a role in the progression of the disease since malignancy is clearly a multistep process. Supported by C.N.R.S. and by Grants INSERM No. 83003 and 832017.

20

~{. PJerger et al.

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21

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