Le.kemiu Research ~,ol 6. No 1. pp 17 26, 1982 0145-2126/82/010017-09503 00/0 Printed in Great Britain ~ 1982 Pergamon Press Lid

A C U T E M O N O C Y T I C L E U K E M I A

C H R O M O S O M E S T U D I E S

ROLAND BERGER,* ALAIN BERNHEIM,* FRAN(OIS SIGAUX,* MARIE-THI~RESE DANIEL.+ FRANt~'OISE VALENSI+ and GEORGES FLANDRINJ"

*Laboratoire de Cytogenetique, Centre G. Hayem and +Laboratoire d'Hematologie, UER d'Hematologie. H6pital Saint-Louis, Paris Cedex 10, France

IReceived 9 September 1981. Revised 10 October 1981. Accepted 17 October 19811

Abstract--Cytogenetic studies have been performed on 34 acute monocytic leukemia tM5~ patients. 24 of the a type and 10 of the b type. No chromosomal abnormalities were found in 12 cases, in spite of the fact that the mitoses concerned monocytes. Different chromosomal aberra- tions were present in the other cases. In 12 of them, an abnormality of the chromosome 11 long arm was observed (mainly in the poorly differentiated type of M5), on bands q22-q24 in nine cases and on band q14 in three cases. The chromosome I1 long arm thus appears preferentially rearranged in M5 although this is not apparent in every case. Concomitant study of the mitoses with cytological and cytogenetic techniques suggests that erythroblasts may not be involved in the M5 leukemic process,

Key words: Acute monocytic leukemia, chromosomes, chromosome l lq anomalies.

I N T R O D U C T I O N

IN 1980. we reported chromosome studies on 10 cases of acute monocytic leukemia (AMOL or M5) having an unexpectedly high incidence of chromosome 11 rearrange- ments [2]. Chromosome studies have been extended to a total of 34 cases of M5 which are now reported here.

MATERIAL AND METHODS Patients

Thirty-four consecutive previously untreated patients were studied: 10 children and 24 adults (Table 1/.

Cytology-cytochemistry

Diagnosis was based on the examination of blood and bone marrow smears, stained with May-Griinwald Giemsa and using the description of acute monocytic leukemia (M5t outlined in the FAB classification [1]. cases split into poorly differentiated (M5a) and well differentiated (M5b) monocytic leukemias.

Diagnosis was confirmed by cytochemical methods using the peroxidase and naphtol AS-D acetate esterase (NASDAI tests with and without sodium fluoride (NAF~ exposure [6].

Cytoqenic ~tud)

Chromosomes were studied either on bone marrow specimens examined by the direct technique and/or on bone marrow' or blood cells cultured in t'itro for 24. 48 and 72 h without stimulation. In some cases, blood cultures were also performed with phytohemagglutininin (Nos. 2, 4, 6. 8, 9, 12, 18. 20, 26, 33/.

G-banding IGTGI. R-banding (RHGt and sometimes C-banding (CBG) and Q-banding (QFQ) techniques were used in the analysis [13.14]. Cytogenetic studies were performed at diagnosis, prior to treatment of each

.4hhret'iation.~: ,4MOL. acute monocytic leukemia: ANLL. acute non-lymphocytic leukemia; APL. acute promyelocytic leukemia: CBG. C-bands by barium hydroxide using Giemsa: GTG. G-bands by trypsin using Giemsa: M2. acute myelocytic leukemia with maturation (FAB classificationj: M3, acute promyelocytic leuke- mia IFAB classificationl: M5. acute monocytic leukemia (FAB classificationl: M5a, poorly differentiated acute monocytic leukemia: MSh. well differentiated acute monocytic leukemia: NAF. sodium fluoride: NASA, AS-D acetate esterase: QFQ. Q-bands by fluorescence using quinacrine: RHG. R-bands by heating using Giemsa.

Correspomh,nce to: R. Berger. Laboratoire de Cytogenetique, Centre G. Hayem. UER d'Hematologie. H6pital Saint-Louis. 2 place du Dr A. Fournier. 75475 Paris Cedex 10. France.

17

18 R O L A N D BERGER e l cal.

TABLE 1. CLINICAL A N D HEMATOLOGICAL DATA

Blood Bone marrov, Patient Age WBC count Blasts blasts

NO (yrl* Sex x 10 s g mm I%1 I",d FAB+ Treatment; Follov,-up~

t 23 M iSII 86 95 a R C \ dead. i d 2 32 M 63 76 I00 a R. CA. \ CR C R rdap~c ~ m 3 3 F 42.6 67 100 a R. CA. \ M 2 6 CR, 15 in + 4 75 F 27.2 07 95 a 5 26 M 380 90 92 a dead. I d 6 4 ra F 450 74 60 a D. CA CR. ltl m + 7 67 M 36 5 91 a R. C~. VM2b CR. relapse, d 4! m 8 55 F 102 96 I(X) a R C~,, kCR dead 14d 9 27 F 4.5 23 ,',16 a R. CA. \ C R C R S m -

10 22 F 82 37 72 90 a R, CA CR. rctap~,. 22 m. dead 24 m I I 8 m M 45 95 95 a R. CA, \ C R dead. 1 ~ d 12 71 F 500 88 IO(/ a R. CA dead 2 d 13 IBm M 9,9 15 88 a R. CA. \ 'M26 CR, relapse 3 m, dead 8 m 14 66 M 157 84 100 a R, CA. VM2b 15 44 F 2.1 2 97 a R. CA. VM26 dead I m 16 53 M 120 90 IO0 a C \. VCR \ 'M26 CR. re[ap,c 3 m. dead 4 m 17 74 M 2 5 0 5 80 a R N.R. dead "~ m 18 21 m F 11,7 37 100 a R, CA. VM26 CR. 6 m * 19 12 M 34.2 60 90 a R. CA.. VCR CR. 7 m + 20 18 F 126 95 100 a R. C~. VM26 CR. relapse 3 m. dead 4 m 21 9 F 10.6 41 99 a D, C4. \ 'M26 dead 3 d 22 31 M 260 98 94 a R. C~. VCR dead 3 d 23 75 M 443 31 44 a R. CA ' 24 16 F 14.6 79 100 a R C R ~claps¢ 4 m. dead "; m 25 28 M 140 97 04 b R, CA. \ C R C R 12 m + 26 64 M 228 95 92 b D, CA. VM26 dead 13d 27 57 M 45 I 96 h R. CA. VCR CR. relapse 3 m, 5 m + 28 4 m g 236 79 100 b D CA CR relapse 6 m 28 relapse 25 2 55 ~0 b ? m + 29 17 M 40.2 81 89 b R. Ca. PR dead 4 m 30 26 M 244 94 100 b R. C,\, ~( 'R NR dead 5 m 31 7 M 31 l 86 90 b R. ('A. \ C R C R relapse 4 m. dead 5 m 32 13 m M 234 88 98 b R, CA ~ d + 33 53 F 13 18 77 b R. CA. VCR C R 6 m 34 52 M 45 0 81 b R. ( ' \ CR, lelap~e S m +

*Unless otherwise noted: m: month. %: poorly differentiated; b: differentiated. ~.R: rubidazone: CA: cytosine arabinoside: VCR: vincristin: D: daunorubicin. ~d: day: m: month: + : alive: CR: complete remission: PR: partial remission: NR: no remission.

p a t i e n t . T w o o f t h e p a t i e n t s w e r e s t u d i e d t w i c e , o n e d u r i n g r e l a p s e I N o . 281 a n d o n e a f t e r a l i rs t c , , c l e o f

ineffective treatment {No. 30). The number of mitoses analyzed per case varied from 12 to 128 {Table 2L

Concomitant chromosonle and cytologic studies

Cytologic and cytogenetic studies were performed concomitantly on the same samplcs of bone marrow and/or blood using the methods described previously [2]. Recognition of the origin of the mitotic cells on the Giemsa stained smears was based mainly on cytoplasmic features such as the degree of basophilia, granula- tions, cytochemical activity and hemoglobinisation of the erythroblastic line. {Figs. 1-41.

R E S U L T S

Cytology cytochemistry A m o n g the 34 patients, 24 belonged to the poorly differentiated group (M5a) and 10 to

the well differentiated group (M5b). With the exception of four patients, blast cells had NaF-sens i t ive N A S D A activity. Peroxidase activity was posit ive to various degrees in 12 patients, totally negative in 12, and faintly posit ive in a very few blast cells in seven cases. The peroxidase test was not performed on three patients.

Cytoqenetics (Fig. 5, 6)

The results of the cytogenetic studies are shown in Tables 2 and 3. One patient (case 3~ had a const i tut ional maternally transmitted t( l: 12) translocation. Another patient (No. 28) had an X trisomy. N o n e of the other patients had const i tut ional c h r o m o s o m e abnormality.

FIG. 1. Acute monocytic leukemia, undifferentiated type. Mitosis of a monoblast .

FIG. 2. Acute monocytic leukemia, differentiated type b. Mitosis of a monoblast .

FIG. 3. Acute monocytic leukemia. Mitosis of a late erythroblast.

FtG. 4. Acute monocytic leukemia. Mitosis of a myelocyte.

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AMOL chromosome studies

TABLE 2

23

Cylo logy: mitoses of Patient Cylogenet ics abnormal /normal Monocytes Erythr G r a n u l

N o Md M24 M48 M72 B 2 4 - B 4 8 - B 7 2 - B72+ Md M24 M48 B 2 4 - B 4 8 - Md M24 Md Md B 4 8 -

4 0 0 53 0;43 0;40 0/4 13 [

0/35 26

I 2 3 4 0'9 0 18" 5 I0 2 21 () 6 7 116 8 0 4 0 28 0 18/0

10 5 8 11 I2 13 50,1 14 15 2611 16 0 3 0 2 0 t 7 I 0 0 34~0 18 91 66/0 I c) 20 7 6 21 30 2 22 0 t 411 23. + 32-7 '0 24 27 1 25 1 15 I 19 26 0 1-7 27 0 2 0 20 28 28 relapse+ + 11-9 0 ~4-16 0 29 5 9 0 30 21 t 12 30 2nd stud.', 0 23 31 32 33 0 4 I 26 0:23 34 0 5 9

031"; 56 6/0 33

5/0 1021 8 2 54

0/32 0,14 32 4'2 0 2 2~

7

1 14 I

II

15

7

83,0 4,8 1 1 2

4...{/0 O~ 2 26

0,'35 0'6 92 2 39/0 729 30 3 4

39,2 2/0 1 16 44 3 I 0 3 7 0 i l0 0,'5 16/17 2 q 0 58 4

1 7 0 7 52 2 19 5

14/0 140 0 1 2 71 3 61 I

0 6 61 2 I 0'1 l 20 2 ] 2 6

28'0 38/0 30 21 18 21 5 28 2

0:3 33 I 0 4

197 32 3 2 I 28 8

0'9 29 4 3 3

Number of mitoses examined cytogenetically and cylologically. Md. M24. M48. M72: bone marrow by "direct' method: after 24, 48 and 72h of in vitro culture. B 2 4 - , B 4 8 - . B 7 2 - : blood culture without stimu- lalion: B72+: blood culture after PHA-stimulation.

*Balanced familial translocation. +Familial translocation (blood culture in remissionL ~.Two abnormal clones.

N o acquired c h r o m o s o m e abnormalities were found in 12 patients, five of the M5 a type and seven of the b type. C h r o m o s o m e anomalies, numerical structural or both numeral and structural were found in all the other cases. Autosomal trisomies involving chromosomes 8. 9, 21 and 12 (?) were found isolated in five cases, and associated with structural abnormalities in another six, C h r o m o s o m e 11 was involved in structural re- arrangements in 12 of the patients at bands q22-q24 in nine cases (Nos. 5, 6, 9, 11, 13, 17, 21. 23. 28i and at band q14 in three (Nos. 1. 14, 241. Two different abnormal cell lines were present in two of the cases (Nos. 23 and 28 in relapsel.

CollcomiHItl! cyloloHic alld cJlrotnosoltl~, study

Concomitant study of the cytology and chromosome aberrations in cells from the same bone marrow or blood specimen (Table 2) showed that in all the cases studied except No. 28 during relapse the majority of mitoses concerned monocytes or mono- blasts. In the latter case the respective proportions of mitoses in the bone marrow inxolving monocstes and erythroblasts was about the same. Cytogenetic studies showed tx~o aberrant clones: 47. XXX. t(l: l lt (the same as was found at diagnosisl which con- cerned the monocvtes, and 47. XXX not found in the previous study (lacking erythroblast mitoses) which is probably due to ervthroblasts. The chromosome abnormalities were

24 R O L A N D B E R G E R t 'f ~.sl.

T A B L E 3. K A R Y O T Y P E S

Patient No Bone marro',.,, and or unstimulaled blood

I 47. XY. + 8 . 9 q + . de l (141qq23- qter~, 1 7 p - often inconsistent abnormalities from cell to cell in 50". of mitoses: tn~ dup tl~ Iqtcr - q321

2 46, XY 3 46, XX. t l l : 12) i036: q13~* 4 46. XX 5 46, XY, del (11) ~q24- qterl 6 46. XX, t~l l : 17~(q23:q231 7 46, XY. - 8 . +8. - 15. + t l13:151 ( p l l : q l l l 8 46, XX 9 48, XX, + T p + . t l 7 : 8 ) ( p l l : q l l ) : - 8 . +il8q), +l(8q~, del [l ib(q241

47. XX. + 7 p + , t(7; 8) ( p l l ; q l l ) - 8 . +it8ql. del ( I l l ~q24! 10 47. XX, t ? 1 2 I 1 47, XY. +6. ill I : 19) iq24: pl3J 12 47, XX, + m a r [ = ? d e r ( 8 q 13 46. XY. d e l ( l O ) l p l 2 - p t e r ) . d e l ( l l ) ( q 2 3 - qter): derlSl 'dup~81(qll-qter , i n s q l O l ( p l 2 - pterl

ins I 11 ) (q23 - qterly 14 46. XY. t(6: IO: 11) (p22: q14:q141 I5 47. XX. +8 16 46. XY 17 44. - X , - 5 . - C . d e l I I l l l q 2 2 qterl, - 1 7 , + 1 7 p + . - r . + m a r l . - m a r 2

(the lacking C is ~ariable from cell to cell: 7. 8. or 91 18 46, XY, i l l : 9) Iql 1 : q341. t(10: I 1) Ip14: q14) 19 46. XY 20 47, XX. + 8 21 46, XX. t ( l l : I11 iq22: qterl 22 47, XY. - 2 t 23 46, XY. tI8:13ttq24:q12J, dellll~lq23:q24),13p+

46. XY. t ( l l : 13F~p12: q121. 13p+ 24 47. XX. +8, tqlO: I l l ( p 1 3 : q14~ 25 46. XY 26 46. XY 27 46, XY 28 47, XXX, t[l : 111 (q21 : q241

47. XXX, tl I : I 11 (q21 : q241 and 47, XXX 29 48. XY. 1 7 p - . + 19. + m a r [= ' . ' t l l : 131] 30 46. XY 31 47, XY. +9 32 46. XY 33 46. XX 34 46. XY

*Familial translocation.

different in the two cell lines. It is impossible to assess with certainty if this trisomy A ~s constitutional or acquired.

In patient No. 23, two cell lines with different chromosome aberrations were found: 46. XY, t(8; 13) (q24: q22), del (11) (q23: q24), 13p+ and 46, XY, t( l l; 13) (p12: q12), 13+. They have therefore a common abnormality, the chromosome 13p+. Cytologic examin- ation showed that mitoses concerned mainly monocytes (19 cells) but also granulocytes [five cells) which might participate in the leukemic process in this particular case.

DISCUSSION

Among 34 cases of M5 studied cytogenically and cytologically, 12 had no acquired chromosome abnormalities (one of them having a balanced familial translocation) and another probably a constitutional trisomy X. Autosomal trisomies occurred in five cases and structural rearrangements involving chromosome 11 in 12 cases. These involved two breakpoint regions: 1 lq22-1 lq24 (nine patients) and 1 lq14 (three patients).

Chromosome 11 abnormalities were found to be unusually frequent in an initial series of ten cases (Nos. 1, 2, 10, 14, 17, 21, 23, 24, 29, 331 we have previously reported I-3].

Other anomalies of the chromosome 11 long arm (11q23 llq24) in M5 have been reported by others [7, 8, 11, 15, 17 19]. In still other cases various anomalies or no ab- normalities have been found [for review, 8, 19]. Although the chromosome l lq abnor- mality has been reported in other types of malignant proliferations [3, 16 for review] it

AMOL chromosome studies 25

has rarely been reported in acute non-lymphocytic leukemia [3 for review. 8, 19]. In our experience of about 200 ANLL cases studied using banding techniques, we have never observed a long arm chromosome 11 abnormali ty except in M5. It seems to us that bands q23 q24 may be specifically involved in these cases.

A comparison of M5 with APL (M3) and M2 having t(8: 21) translocation shows important differences in the sense that no specific translocation is found in M5 and that the involvement of chromosome 11 was not found in every case. The fact that the majority of cases with anomalies on l lq were of the poorly differentiated type (a in FAB classificationl must be emphasized. No correlation with cytochemical data could however be established.

The concomitant chromosomal and cytological studies brought out some interesting points: in the majority of cases the mitoses observed concerned monocytes or mono- blasts. It is thus impossible to try to determine the level of differentiation of the hemato- poietic cells in which the chromosome abnormali ty appears as has been done for APL and M2 with the t(8: 21t translocation [2.4.5] . In one patient iNo. 2 8 ) i n relapse however a different chromosome abnormali ty was observed in monocytes (with l lq rearrangementl and erythroblasts suggesting (but not proving) that erythroblasts are either not or are involved differently from monoblas ts -monocytes in M5. In the cases with chromosome aberrations, the proport ions of mitoses of monocytes and of other cells on the one hand. and of cytogenetically abnormal and normal karyotypes on the other were comparable. Pooling the available data, since mitoses of cells other than monocytes were rare. it may be suggested that erythroblast cell lines seem not to be involved in the malignant proliferation.

The analysis of case No. 15 is also of interest: all the mitoses observed in the direct preparation concerned monocytes, two different karotypes being present, normal and trisomic 8. Since all the monocytes were believed to be leukemic, it may be concluded that trisomy 8 is a secondary event, the primary event being unrecognized, as in the other cases having no detectable chromosome abnormality. Other examples of secondary events leading to numerical chromosome abnormalities such as the loss of X or Y in acute myeloblastic leukemia with a t(8: 21) translocation [4, 10] are known in malignant diseases.

The fact that in some cases of M5 no chromosomal aberrations were found at the same time as the leukemic cells were dividing, can be explained in different ways. One explana- tion may be that chromosomal aberrations really to not exist. Another explanation. par t icularb ~alid for chromosome 11 anomalies lies in the insufficient resolution of the usual cytogenetic techniques used since, in a series of 24 cases, Yunis et al. [19] have recently found chromosome anomalies in each patient studied when high resolution techniques were used. A third possibility is that chromosome abnormali ty can be visual- ized only in some but not in other comparable cases. Recent studies on one of the genes inxolxed in avian leukosis in virus-induced tumors showed that transposition of a promoter may explain the transformation [9. 12]. It is now known that transpositions exist in eukarvotes. The transposition of a p romotor or a gene responsible for malignant transformation could theoretically also exist in humans. In this event it would not be surprising to observe a lack of chromosomal changes in some cases of AL, whereas in other cases the event is accompanied by visible chromosomal rearrangements. The linkage between molecular level events and cytogenetic changes is not a new fact for cytogeneticists involved in mutagenesis studies.

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