Cytological types of mitoses and chromosome abnormalities in acute leukemia

Leukemia ResearchVol.7, No. 2, pp. 221-236, 1983. Printedin GreatBritain.

0145-2126/8353.00 + .00 © 1983PergamonPressLtd.

CYTOLOGICAL TYPES OF MITOSES AND CHROMOSOME ABNORMALITIES IN A C U T E LEUKEMIA R. BERGER, A. BERNHE1M, M . T. DANIEL,* F. VALENSI* and G. FLANDRIN* Laboratoire de Cytog~n~tique, Institut de Recherches sur les Leuc~mies et les Maladies du Sang, H6pital Saint-Louis, Paris, France and *Laboratoire d'H~matologie, H6pital Saint-Louis, UER d'H6matologie, Paris, France (Received 5 July 1982. Revised 1 September and 2 November 1982. Accepted 20 December 1982) Abstract--In order to determine the nature of the cells in mitosis in acute leukemia, a parallel study was conducted by cytological and cytogenetic methods on the same marrow and blood samples. On direct marrow examination, erythrocyte precursors in mitosis are usually observed but ordinarily disappear following in vitro culture. In APL (M3) characterized by t(15;17) translocation, the comparison between the proportions of the different categories of cells in mitosis and of karyotypically normal and abnormal ceils suggests that erythroblasts do not belong to the leukemic clone. An analogous situation is observed in AML (M2) with t(8;21) and in monocytic leukemia (M5) with chromosome abnormalities. Erythroleukemia could be divided into two categories, one with chromosome abnormalities and persistence of erythroblast mitoses after culture, and another with no detectable chromosome abnormality and with disappearance of erythroblast mitoses following culture. Other examples of blood malignancies demonstrate the importance of the method used in determining which cell categories belong to the leukemic clone. An interpretation of the results in terms of commitment 'level' of the involved stem cells and a distinction between 'primary' and 'secondary' chromosome abnormalities is proposed. Key words: Cytology, chromosomes, mitoses, acute leukemia.

INTRODUCTION THE ANALYSIS of chromosomal abnormalities in acute leukemia (AL) has shown that certain rearrangements are specific to a given type of malignancy, such as t(15;17) translocation in acute promyelocytic leukemia (M3). The absence of clonal chromosome abnormalities in about one half of AL cases and the co-existence of normal and abnormal karyotypes in others raises the problem of ascertaining the nature of the cells with normal karyotype. The present study is based on the hypothesis that the karyotypes studied in AL do not always represent leukemic cells. While cytogenetic techniques do not permit direct morphological identification of haematopoietic cells, a correlation can be established between cytogenetic and cytologic data collected from a given specimen of marrow or blood. MATERIALS AND METHODS Seventy-six patients suffering from various blood malignancies, treated at the H6pital Saint-Louis since 1977, were chosen for this study because of the quality of the data gathered and the number of mitoses available for study. Cytological analysis was used to classify the subjects according to standard FAB nomenclature [1] as follows: 71 acute non-lymphoblastic leukemia (ANLLL with two M 1, 11 M2, nine M2 with t(8;21 ) translocation, 14 M3, three M3 variants, two M4, 15 M5a, five M5b and 10 M6; four acute lymphoblastic leukemia (ALL) with three L3 and one L2 ~vith Phi; one refractory anemia with excess blasts (RAEB) with 5q--. All but three of the patients (cases 25, 26 and 46) were studied before treatment was initiated and two of them were also studied at time of relapse (cases 54 and 56}.

Correspondence to: Dr. R. Berger. Laboratoire de Cytogenetique, lnstitut de Recherches sur les Leuc~mies et Its Maladies du Sang, H6pital Saint-Louis, 2 place du Dr. Fournier, 75475 Paris, Cedex 10. France. 221

222

R. BERGERetal.

The study was conducted using bone marrow cells examined directly (after in vitro culture with colchicine at 37°C for 1 h) and/or after culture at 37°C for 24, 48, 72 and/or 96 h, and in certain cases using cultures of nonstimulated blood kept for 24, 48 and/or 72 h. Following incubation with colchicine, the samples were divided into two parts, one for cytological study, the other for cytogeneticexamination. For karyotype determination, GTG and/or RHG banding techniques were used in all cases, and QFQ for some of the samples [15]. The number of mitoses we were able to study varied in the different specimens (see Tables 2-.6). For cytologicalexamination of the mitoses, 30,000 nucleated cells were put in suspension and placed in 'No. 2 Cytospin' cytocentrifuge (Shandon-Elliot) and centrifuged at 700 revs/min for 10 min. May-Grfl nwald-Giemsa staining and myeloperoxidaseand esterase reactions were then carried out 112]. RESULTS The clonal c h r o m o s o m e abnormalities observed are summarized in Table 1. In cases 54 and 75 various anomalies were present within the abnormal clone. In some M6 (cases 48, 50, 52) one metaphase only was a b n o r m a l without evidence of an abnormal clone. In the other cases no c h r o m o s o m e abnormalities other than those present in the stemlines were observed except probable artefactual r a n d o m losses o f chromosomes. The distribution of c h r o m o s o m e aberrations (49 out of 71 cases) is not representative of all the A N L L cases examined in our laboratory, owing to the biased selection of patients for this investigation. The results obtained by examining the cells in mitosis concurrently by cytogenetic and cytological techniques (Tables 2--6) varied with the FAB type of leukemia concerned. The difficulty in identifying the cells in mitosis on the smears was extremely variable, depending on cell type and on the extent o f morphological differences between the different cell categories concerned. The cells in mitosis are identified mainly by the appearance of the cytoplasm: basophilic strength, granulation, size and quantity of granules, presence of Auer rods, degree of hemoglobinization necessary to attribute a given mitosis to the erythroid line and by the cytochemical activity. With experience, one can come fairly close to reliable identification (Fig. 1). However, in certain cases it is difficult, even impossible, to discriminate between mitoses of cells which although morphologically similar, belong to different lines, e.g. myeloblasts vs monoblasts, young erythroblasts vs myeloblasts or megakaryoblasts. Cytochemical reactions (peroxidase, chloroacetate, esterase) while sometimes helpful, occasionally obscure all the cells by their intensity and are then of no help. In the same way, the morphological changes the cells undergo in culture makes identification impossible in certain cases. Indeed, these are the cases that present the greatest difficulty. The appearance of the chromosomes themselves can be valuable, although to a lesser extent, in identifying cells in mitosis. In general we f o u n d erythroblast chromosomes to be very dark, well contrasted and discrete whereas those of blasts (myeloblasts, monoblasts, a b n o r m a l promyelocytes) were fuzzy, difficult to discriminate, sometimes even giving images indistinguishable f r o m a nucleus with damaged interphase or a pyknotic nucleus. Rather than the nature of the cell line, what appears to be detected here is the pathological or healthy character of the cells; indeed the chromosomes o f normal granulocytic cells, while not identical to those of erythroblasts, do not display the fuzziness often found in the leukemic cells. Finally, in a few exceptional cases, the presence of a large marker chromosome, visible on the standard Giemsa-stained slide and representing the cytogenetic picture, allowed us to attribute this a b n o r m a l i t y to a given cell line (Fig. 2). It is clear then that it was easy to distinguish between mitoses of normal promyelocytes and basophilic blasts in A L L L3, between mitoses of erythroblasts and promyelocytes in A P L M3, and fairly easy to distinguish between erythroblasts and myeloblasts in M2. In fact the clearest distinction obtained was between erythroblast mitoses and that of other lines, often allowing us to draw clear conclusions.

Cytology and cytogenetics of mitoses in AL

223

TABLEl. CYTOGENETICS Blood disease APL (M3)

AML,M2,t(8;21) AMOL (M5)

Patient 1,2,5,6 3,4,7-10 } 11,12,13,15,16, 17 14 18 to 26

Various

No abnormalities t(15;17) 47,XY, + 21/47,XY, + 21, + 8 t(8;21)

27 28 29 31 32 33 34 35

47,XY, + 8,14q + ,17p + 47,XX, + 7p + ,i(Sq), 1lq--/48,XX, + 7p + ,i(8q), + i(8q),l Iq-47,XX, + 8,t(10; 11) 48,XY, +8, + 8,--15, +t(13;15) 47,XX, + ? 12 47,XX, + 8 44,X, --5,--6,1 l q - - , - - I 7,--17,--21, + 17p +, + r, + mar, + mar 46,XY,t(10;I l),t(1 ;9)

36 37 38 40 41 45 46

46,XY,t(8; 13),11q--, 13p +/46,XY,t(I 1;13), 13p + 46,XY, 11q-46,XX,t(1 ; 12) (constitutional abnormality) 47,XY, + 6,t(l 1;19) 46,XY,t(6;10;11) 47,XY, + 8 47,XXX,t(1 ;11 ) and 47,XXX (constitutional)

30,39,42--44 } Erythroleukaemias (M6)

Chromosomes (clonal abnormalities)

53 54 55 56 47-52 64 65 66 67 68 69 70 71 72 73 74 75 76 57-63 }

No abnormality Aneuploidy : 72-84 2 abnormal clones, 46,XY,--B, + D,--G various anomalies 46,XY, 1p + ,3q +,7p-46,XY,t(5; 12) No abnormality 47,XX, + 8 45,XY,--5,--7,--8,wlT, + 16 + r, + mar 48,XX, +4, +21 47,XY, + 8 Aneuploidy : 54-55 47,XX, + 8 45,XX,--7 47,XY, + 12 45,XY,--7 46,XY,t(9;22) 46,XY,t(8;22)/46,XY,t(8 ;22),t(1 ;6) 46,XY, + 18,--22,t(8; 14), + various anomalies 46,XY,t(8; 14) No abnormality

H o w e v e r , the d i s t i n c t i o n b e t w e e n p r o e r y t h r o b l a s t mitoses a n d other series was occasionally s o m e w h a t u n c e r t a i n , as was the d i s t i n c t i o n b e t w e e n m y e l o b l a s t s a n d m o n o blasts. A c o m p a r i s o n b e t w e e n the results o b t a i n e d by direct m a r r o w a n d b l o o d analyses, a n d specimens f r o m the s a m e p a t i e n t after culture for a p p r o p r i a t e intervals, gives a n idea o f d y n a m i c s of the cell p o p u l a t i o n . I n general, a p r i m a r y o r i e n t a t i o n is given by the simplification of the cytological s i t u t a t i o n of the samples in culture, where the erythroblasts a l m o s t i n v a r i a b l y d i s a p p e a r a n d o n l y one p r e d o m i n a n t cell line r e m a i n s (myeloblastic, m o n o c y t i c , p r o m y e l o c y t i c a n d so on). A q u a n t i t a t i v e e v a l u a t i o n o f the cells in mitosis at

Md Md Md Md Md Md M24 M48 M96 B72 M48 Md M48 Md M48 Md M48 Md M48 Md M48 Md B24 B48

Material*

8 17 6

44 15 39 1 il

14

1

39 50 13 26 16 48

26 6 65 31 35 53

24

32 2 37

32 15 13 48 47

2 24

Cytogenetics Normal Abnormal clonal

1 26 4 II 7 10 8 36 77 22

21 5 11 19 32 24 9 21 18 54 15 23 25

Blasts

2

13

19

27

45

8

11 14 6 17 3 7

Erythroblasts

Cytology Lymphocytes Neutrophilic precursors

Eosinophils

1

I

Questionable

*Md,M24,M48,M96: bone marrow investigated with a direct method (Md) and after in vitro culture for 24,48, or 96 h. B24,B48,B72: non-stimulated blood investigated after in vitro culture for 24,48 or 72 h. t Variant form of M3.

15t 16 t i7 t

14

13

12

11

8 9 10

I 2 3 4 5 6 7

Patient

TABLE 2. ACUTE PROMYELOCYTIC LEUKEMIA (M3). NUMBER OF MITOSES STUDIED BY CYTOGENETIC AND CYTOLOGIC METHODS

7~

4~

225

Cytology and cytogenetics of mitoses in A L TABLE 3. ACUTE MYELOBLASTIC LEUKEMIA WITH t(8;21) TRANSLOCATION. NUMBER OF MITOSES STUDIED BYCYTOGENET1CANDCYTOLOGICMETHODS

Patient

18 19 20 21 22 23 24 25 26

Material*

Md Md Md Md M48 Md M48 Md M48 Md M48 B48

Cytogenetics Normal Abnormal clonal

9 5 7 14 1 15 5 13 13 2 1

22 14 12 2 14 17 66 7 41 3 24 26

Blasts

78 21 35 1 18 9 18 23 12 21 6 9

Cytology Erythroblasts Eosinophils

50 3 16 9 4 28 9

* Md,M48: bone marrow investigated directly (Md) or after 48 h in vitro culture. B48: unstimulated blood investigated after 48 h in vitro culture.

the different stages of culture is nevertheless essential and a count was done. The results however are often disappointing and difficult to interpret because of the generally limited number of recognizable mitoses found. In acute promyelocytic leukemia (APL) the karyotypically normal mitoses observed in the samples analysed directly may represent erythroblasts (Table 2). In fact in one patient (case l l) all the mitoses studied by cytological examination represented erythroblasts, whereas chromosome analysis showed only two abnormal cells out of 46. In the specimens examined after culture almost all the mitoses seen represented promyelocytes, the majority with an abnormal karyotype. One may then conclude that in APL, erythroblasts are probably not carriers of the characteristic t(15; 17)translocation. In the M2 ANLL with t(8;21) translocation one also observes a difference in marrow examined directly and after culture (Table 3). On the whole, there is a good correlation between the proportion of karyotypically abnormal mitoses and those which represent blasts (except for case 25 in relapse). Following culture, erythroblast mitoses are no longer visible and the proportion of abnormal karyotypes increases significantly. It is interesting to note that in one subject (case 21) direct examination of the marrow revealed no abnormal karyotype (whereas the 10 mitoses examined cytologically represented nine erythroblasts and one myeloblast) and after culture of the marrow t(8;21) translocation was observed in 13 mitoses out of 16. The peculiarity of this type of ANLL is the presence of eosinophil mitoses (in two cases). One eosinophil with Auer rod was observed in several samples, suggesting that eosinophils could be carriers of the chromosome rearrangement. In monocytic leukemia (Table 4) as well as in the M1, M2 and M4 ANLLs (Table 6) where a clonal chromosome abnormality was detected, one observes a good correlation between the proportion of karyotypically abnormal mitoses and leukemic cell mitoses, with erythroblasts showing no chromosomal abnormality. However, when no chromosome abnormality is found, it is impossible to postulate the non-involvement of erythroid cells in the leukemic process. The M6 erythroleukemias (EL) (Table 5) can be divided into two groups: (1) those with no detectable chromosome abnormality (cases 47-52) and in which erythroblast mitoses are absent following in vitro culture (case 51) as in certain other forms of ANLL and (2) those which show chromosome abnormalities (cases 53-56) and in which certain erythroblasts divide even after culture (cases 54 and 56).

a a a a a a a a a a a a a a a b b b b b

27 28 29 30 31 32 33 34 35 36 37

38 39 40 41 42 43 44 45 46

Md Md Md Md Md Md Md Md Md Md Md M24 B48 B48 1348 1348 Md Md Md Md Md§ M24

Material*

11 15

15 !1

35~ 14

2

9

! 9 6 8 11

83 44 1 2 17 59 11 30

iI 5 26 10 ! 39t 10 21

40 28 27

CytoBenetics Normal Abnormal

53 23 71 56 54 7 26 30 44 19 33 14 26 11 15 7 61 33 61 28 21 18

Monoblasts or abnormal monocytes

1 ! 2 2 21 5

3

5 1

4

3

1

1 !

3

Cytology Neutrophilic precursors

1 6 1!

Erythroblasts

Questionable

* Md,M24: bone marrow studied by direct method (Md) and after 24 h in vitro culture. B48: unstimulated blood studied after 48 h in vitro culture. t Two karyotypically abnormal clones. Constitutional abnormality. § Relapse.

FAB

Patient

TABLE 4. ACUTE MONOCYT|C LEUKEMIA 0VISa AND MSb). NUM"Ea OF MITOSES STUDIED BY cY'r(X]ENETIC AND CYTOLOGIC METHODS

O~

Md Md Md Md Md M48 Md Md Md M48~; Md Md M48 Md ~t M48~; 1 5

49 28 26 16 30 38 26 I 4t 22t 9t 85t 17t 31 t 69t 44t

!

i

Cytogenetics Normal Abnormal

1 !

1

1

8 4 14

Neutrophilic precursors

3 3 3 1 4 14

4 2 5

Blasts

44 72 92 38 40 85 13 60 38

41 23 15 29 34

Erythroblasts

* Md,M48: bone marrow studied by direct method (Md) and after 48 h in vitro culture. t CIonal abnormalities. ~-Relapse. § Fanconi'sanemia.

55§ 56

52 53 54

47 48 49 50 51

Patient Material*

I

Cytology Eosinophils

I

4 I

20

Megakaryocytes Questionable

TABLE 5. ERYTHROLEUKEMIA (M6). NUMBER OF MITOSES STUDIED BYCYTOGENETIC AND CYTOLOGIC METHODS

5'

o

O

ANLL, M2

A N L L , M2 A N L L , M4 RAEB

69

70 71 72 73 74 75 76

Md Md B48 Md M48 Md Md Md B48 M24 M48 M48 Md M48 M24 B48 Md Md Md Md B48 B72 Md 9 i4 38 3

I1

19 1 17 5

10 15 10 36 50 15 19 3 4 32 6 45 29 21 124 17 11 17 10 17 6 73 36 16

1 1

Cytogenetics Normal Abnormal

6 34 5 ! 5 31 27 20 7 37 !5 11 10 10 5 9 45 12 2 16 69 9 52

Blasts

3 6 2 5 18 5

i

4 4 13

1

precursors

Neutrophilic

45 13

24

5

3 40 12

21

5

I

2 I

Cytology Erythroblasts Eosinophiis

1

(Baso:l)

Megakaryocytes

Questionable

* Md, M24, M48: bone marrow studied by direct m e t h o d (Md) or after 24 or 48 h in vitro culture. B48, B72: unstimulated blood studied after 48 or 72 h in vitroculture.

A L L , L3

ALL, L3

ALL, L3

ALL, L2

A N L L , M2 A N L L , M2

67 68

Mi MI M2 M2 M2 M4 M2 M2 M2 M2

ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL ANLL

Diagnosis Material*

57 58 59 60 61 62 63 64 65 66

Patient

TABLE 6. VARIOUS BLOOD DISORDE RS. NUMBER OF MITOSES STUDIED BY CYTOGENETIC AND CYTOLOGIC METHODS

~a

rn ~o

70

(a]-

Y~j

(c

(d)

....

(

(g)

ICh)F FIG. 1. Cytological aspects o f mitoses, a = A L L L3 (Burkitt type): mitosis o f a L3 blast cell (bone marrow, 'direct' preparation); b = A L L L3 (Burkitt type): mitosis of a promyelocyte (bone marrow, 'direct' preparation); c = A P L (M3): mitosis of an erythroblast (bone m a r r o w , 'direct' preparation); d = A P L (M3): mitoses of promyelocytes (bone marrow, culture); e = AML,t(8;21)M2: mitosis of a myeloblast with A u e r rod (bone m a r r o w , 'direct' preparation); f = AMol (M5): mitosis o f a m o n o b l a s t (bone marrow, 'direct' preparation; g = erythroleukemia (M6): mitoses of erythroblast (bone marrow, 'direct' preparation); h = erythroleukemia (M6) with predominance of proerythroblasts: mitosis with abnormally high c h r o m o s o m e n u m b e r (bone marrow, 'direct' preparation). 229

-%J

FiG. 2. Metaphase with two marker chromosomes (arrows) from blood culture, and dividing myeloblasts (bone marrow) from the same leukemic patient (M2) showing the big marker chromosome.

230

Cytology a n d cytogenetics o f mitoses in A L

231

Only four cases of ALL are included in the present study. One of these is an ALL with a t(9;22) translocation in which karyotypically normal mitoses may represent erythroblasts and granulocytic cells (case 73). Indeed, following culture of this patient's marrow for 48 h, 20 mitoses with Ph I and four normal karyotypes were observed. In Burkitt's type leukemia (L3) (cases 74-76), the proportion of normal karyotypes corresponds to that of granulocytic cell mitoses which suggests that the granulocytic cells are not carriers of the characteristic translocation. A number of discrepancies should be noted: in certain cases of APL (cases 1, 2, 6, 10, 12, 13), MI (case 57) and of M2 (cases 59 and 61) normal karyotypes were observed even though blast mitoses were visible by cytological inspection. The chromosomes of leukemic cells are fuzzy and poorly defined, and it is possible that the chromosome study is biased in so far as certain leukemic cell mitoses visible in the cytological preparations are ignored by the cytogeneticist as being impossible to analyse. That is why the chromosome preparations were examined a second time in order to determine the proportion of mitoses impossible for the cytogeneticist to study (Table 7). This demonstrates that in each case there is a variable proportion of mitoses that are dismissed in the cytogenetic examination on account of the blurred appearance of chromosomes. This may explain the absence of chromosome aberrations in certain cases.

TABLE 7. NUMBEROF MITOSESSTUDIEDBYCYTOGENETICMETHODWITH NORMAL, WELLSHAPED CHROMOSOMES AND ILL-DEFINED, FUZZY CHROMOSOMES (UNINTERPRETABLE MITOSES)

Patients

1 2 5 6 l0 12 57 59 61

Diagnosis*

M3 M3 M3 M3 M3 M3 M2 M2 M2

Material ~

Md Md Md Md Md Md Md B48 M48

Mitoses Normal shaped chromosomes

Ill-defined chromosomes

10 6 2 12 5 19 2 2 1

2 14 8 5 7 4 8 20 12

* FAB classification. "1"Md, M48, B48: Bone marrow (M) a n d blood (B) studied by direct method (Md) or after 48 h in vitro culture.

In two patients (case 70: ANLL M2 and case 72: RAEB) one monosomy 7 was found in all karyotypes and the observed mitoses represented various types of cells (blasts, granulocytic cells, erythroblasts, one basophil and one megakaryocyte). Cases 33 (M5) and 69 (M2) are unusual in that they showed two kinds of karyotypes, normal and with 8-trisomy, and that at the same time only blast cell mitoses were observed. In the category of cases without chromosome anomaly, mitoses of leukemic cells were generally observed (except for case 60), classified as normal by cytogenetic analysis. The existence of such cases shows that certain leukemias exist in which no chromosome abnormality is detectable by the usual techniques, found in types M 1, M2, M4 and M5 of our sample.

232

R. BERGEReta}.

DISCUSSION The increase in the proportion of abnormal karyotypes after culture has been reported by several authors [3, 5, 6, 8, 9, VT, 18, 21]. The concurrent study of dividing cells by cytogenetic and cytological techniques suggests at least a partial explanation for this observation. It is possible, by using a combination of both techniques on a given specimen of cultured or non-cultured cells, to identify the nature of the cells in mitosis and to compare their proportions with those of normal and abnormal karyotypes. A study of this kind has shown that erythroblasts were not carriers of the transloeation characteristic of M3 and of M2 with t(8;21) [5]. The results of the study of 75 cases of acute leukemia and one case of RAEB helps us gain a better understanding of certain aspects of the cytogenetic study of AL. There are several possible explanations for the absence of chromosome abnormalities. In certain cases, the mitoses cannot be analysed by the cytogeneticist on account of the fuzzy appearance of the chromosomes, confining the study to mitoses with well-formed chromosomes which may have a normal karyotype, thus introducing a bias into the chromosome analysis. We have verified that this could indeed happen in certain cases of APL, where examination of the mitoses rejected on first reading showed that those that could be read contained a t(15;17) translocation. Another possibility is that the chromosome study dealt with cells with no chromosome abnormalities because these cells were not directly involved in the malignant proliferation. Such is the case for erythroblasts with frequent mitoses observed on direct marrow examination while blasts are present but are not in mitosis. This situation was observed several times in the M3 and M2 types of ANLL (cases I 1 and 60). It involves the problem of the differences in proliferation dynamics in different categories of hematopoietic cells. The disappearance of erythroblast mitoses after culture of the marrow coincides with an increase in the proportion of the divisions of the leukemic blasts and consequently the probability of detecting a chromosome abnormality. This phenomenon, along with the bias in selecting mitoses for karyotype analysis, may explain why cultured cells showed chromosome abnormalities in certain ALs (cases I0, 12, 13 and 21) while direct examination of the marrow did not. The concomitant study of mitoses by cytological and cytogenetic methods showed three types of results. (1) No clonai abnormality of the karyotype was detected in certain ALs even though certain mitoses corresponded to leukemic cells. The existence of leukemias without chromosome abnormality seems to be real and the future will tell if high resolution techniques will lead to the disappearance of this category of AL, as suggested by Yunis et al. [22]. Non-clonal chromosome abnormalities were only found in few cases and they were limited to one aberrant cell. In this sense these non-clonal anomalies do not play a role in the cases presented here. (2) In other forms of AL all the haematopoietic cell lines were carriers of the clonal chromosome abnormality (case 70). The evidence for erythrocytic cell line involvement in certain ANLL has also previously been documented [7, 11, 19, 20] and recently summarized [16]. The same is true for certain RAEBs (case 72) and for chronic myeloid leukemia in which the granulocytic, monocytic, erythroid, megakaryocytic and lymphoid B lines contain the Philadelphia chromosome. (3) In cases where clonal chromosome abnormalities are present, their breakdown according to the cell line which carries them is of special interest. Erythroblasts have a normal karyotype in ANLL types M3, M2 with t(8;21), and M5 with chromosome abnormalities. Granulocytic cells are not carriers of the characteristic translocation in the ALs of type L3. These results suggest several possible interpretations. The chromosome abnormalities may be a phenomenon secondary to the leukemia and present only in certain cell lines.

Cytologyand cytogeneticsof mitosesin AL

233

The specificity of certain kinds of chromosome abnormalities for the cytological type of leukemia, e.g. t(15;17) in APL, t(8;21) in one form of M2, t(8;14), t(2;8) or t(8;22) in L3 ALL, militates against this hypothesis. Karyotype integrity of the granulocytic cells in L3 is also an important argument since the granulocytic cells do not take part in the malignant proliferation in this type of leukemia. From this we can assume that the cells whose karyotype has a clonal aberration belong to the leukemic population, whereas the cells with normal karyotype do not. For cases in which no chromosomal aberration is found, no conclusion can be drawn. Because of the relationship between phenotype and chromosome abnormality, two possibilities must be considered. Either the chromosome abnormality forces the cell in which it occurs to a particular kind of differentiation. For example, t(8;21) is compatible with granulocytic differentiation and prevents maturation of monocytes which are not found in leukemias with this translocation [5]. Eosinophils seem to possess the characteristics of leukemic cells. Or the second possibility is that the chromosome anomaly occurred in a stem cell already committed to a given type of differentiation, granulocytic in ANLL with t(8;21) and in APL. The stem cell may be more committed to differentiation in the case of APL than in AML with t(8;21) since in the latter, eosinophils also seem to be involved in the leukemic proliferation. Furthermore, since the erythroblasts generally do not belong to the leukemic clone, one can infer that the commitment to erythroid differentiation occurs earlier in the hematopoietic process than the commitment to eosinophil differentiation. This hypothesis of commitment levels accounts for the apparent karyotype integrity, in certain ANLLs, of cell lines not involved. It also accounts for observation made using variants of glucose-6phosphate dehydrogenase showing monoclonality to be limited to granulocytes and monocytes, and the non-involvement of the erythroid line in ANLL in three of five cases studied [10, 11]. The absence of certain hematopoietic lines in ANLL, such as monocytes or eosinophils may also be explained by the occurrence of regulation between different cell lines. The inhibition of normal granulocytopoiesis in ANLL explains the absence of normal granulocytic proliferation in the acute phase and the lifting of this inhibition is signalled by its resumption at time of remission. Incidentally it should be noted that during complete remission the karyotype is normal even if a chromosome abnormality was present at diagnosis. However, the regulatory mechanisms presumably set in motion by the proliferation of leukemic cells, particularly on the stem cells committed to differentiation, are poorly understood, and it is not impossible that such regulatory mechanisms exist between stem cells committed to differentiation in the course of leukemia. It is important to elucidate the concept of commitment level of the stem cell carrying the chromosome abnormality, since the discovery of differentiation antigens could in the future lead to specific therapies for different types of AL. The parallel investigation of chromosomes and the cytology of mitoses suggests that there seem to be two types of erythroleukemia [4]: one with abnormal karyotype and erythroid involvement, and another with no detectable chromosome abnormality in which the erythroblasts in culture behave as in the other varieties of ANLL; the question arises whether these ELs differ from other karyotypically normal ANLLs only in the extent of erythroid proliferation. The integrity of the erythroid line has been suggested [14] in connection with two cases of erythroleukemia which were studied concurrently by cytological and cytogenetic techniques. In recent years [2] cytogenetics has demonstrated the existence of two kinds of nonrandom chromosome aberrations in ANLL: certain types are specific for a given cytological type, such as translocations t(15;17), t(8;21) and t(8;14), while others are apparently non-specific, such as 7-monosomy or 8-trisomy. The question then arises as to whether at least some of the latter abnormalities are secondary. The finding of two kinds of karyotypes, normal and with 8-trisomy corresponding to blast mitoses (cases 33 and 69)

234

R. BERGERet aL

s u p p o r t s this hypothesis. T h e s e cases m a y be i n t e r p r e t e d as l e u k e m i a s with a n o r m a l k a r y o t y p e at the onset, f o l l o w e d by the s e c o n d a r y a p p e a r a n c e o f 8-trisomy in a l e u k e m i c cell wh i ch then p r o l i f e r a t e m o r e rapidly because o f the selective a d v a n t a g e gained by the 8-trisomy. A c c o r d i n g to this hypothesis, 8-trisomy seems to be o n l y a sign o f a m o r e a d v a n c e d stage o f e v o l u t i o n o f the l e u k e mi a, w h e r e a s the p r i m a r y event co u l d n o t be d e t e c t e d by c y t o g e n e t i c analysis. R e c e n t studies on a v i a n o n c o g e n i c viruses [13] h a v e s h o w n the i m p o r t a n c e o f D N A t r a n s p o s i t i o n s in t r a n s f o r m a t i o n . T r a n s l o c a t i o n s specific to A N L L can be c o n s i d e r e d as a n e q u i v a l e n t o f the D N A t r a n s p o s i t i o n , visible u n d e r m i c r o s c o p e . I f the m o d e l applies to h u m a n l e u k e m i a , o n e s h o u l d expect to see leukemias with no d e t e c t a b l e c h r o m o s o m e a b n o r m a l i t y , w i t h the p r i m a r y event b ei n g detectable only by the t e c h n i q u e s o f m o l e c u l a r b i o l o g y . T h u s the role o f c y t o g e n e t i c s will be to p i n p o i n t the l o c a t i o n o f c h r o m o s o m e r e a r r a n g e m e n t s in o r d e r to direct f u t u r e research o n the D N A o f certain c h r o m o s o m a l regions w h o s e r e a r r a n g e m e n t s can be d e t e r m i n a n t in the genesis o f leukemia. Acknowledgements--We sincerely acknowledge Dr. Sven-Aage Killmann for reviewing and discussing the manuscript, and Mrs. M. Le Coniat and Miss D. Vecchione for excellent technical assistance.

REFERENCES 1. BENNE'I-rJ. M., CATOVSKYD., DANIELM. T., FLANDRING., GALTOND. A. G., GRALNICKH. R. & SULTAN C. (1976)Proposals for the classification of the acute leukemias. Br. £ Haemat. 33,451. 2. BERGERR. (198 l) The chromosomes in hematology. Cancer Genet. Cytogenet. 4, 69. 3. BERGERR., BERNHEtMA., DANIELM. T., VALENSIF. • FLANDRING. (1981) Translocation t(8;21) et leuc*mie aigua granuleuse: interpr*tation des mitoses normales. C. r. hebd. S~anc. Acad. Sci., Paris 292 D, 289. 4. BERNHEIMA., BERGERR., DANIELM. T., VALENSIF. & FLANDRING. Malignant and reactive erythroblasts in erythroleukemia (M6) (in preparation). 5. BERGERR., BERNHEIMA., DANIELM. T., VALENSIF., SIGAUXF. & FLANDRING. (1982) Cytologic characterization and significance of normal karyotypes in t(8;21) acute myeloblastic leukemia. Blood 59, 171. 6. BERGERR., BERNHEIMA. & FLANDRING. (1980) Absence d'anomalie chromosomique et leuc~mie aigu~: relations avec les cellules m~dullaires normales. C. r. hebd. S~anc. Acad. Sci., Paris 290 D, 1557. 7. BLACKSTOCKA. M. & GARSONO. M. (1974) Direct evidence for involvement of erythroid cells in acute myeloblastic leukaemia. Lancet ii, 1178. 8. CARBONELLF., FLIEDNERT. M., KRAFFE. 'g"SAUERWEINK. (1979) Crecimiento de la celulas leuc6micas en -cultivo: seleccibn de clones citogen~ ticamente anormales. Sangre 24, 1057. 9. CARaONELLF., GRILLIG. & FLIEDNERT. M. (f981) Cytogenetic evidence for a clonal selection of leukemic cells in culture. Leukemia Res. 5, 395. 10. FIALKOWP. J., SINGERJ. W., ADAMSONJ. N., BERKOWR. L., FRIEDMANJ. M. JACOBSONR. J. & Mooua J. W. (1979) Acute non-lymphocytic leukemia. Expression in cells restricted to granulocytic and monocytic differentiation. New Eng/. J. Med. 301, 1. 11. FtALKOWP. J., SINGERJ. W., ADAMSONJ. W., VAIDYAK., DOW L. W., OCHSJ. & MoouR 3. W. (1981) Acute non-lymphocytic leukemia: heterogeneity of stem cell origin. Blood f7, 1068. 12. FLANDRING. & DANIELM. T. (1981) Cytochemistry in the classification of leukemia. In The Leukemic Cell (CATOVSgYD., Ed.), pp. 29-48. Churchill-Livingstone, London. 13. HAYWARDW. S., NELL B. G. & ASTalN S. M. (1981) Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature, Lond. 290, 475. 14. INOUE S., RAVINDRANATHY. & ZUELZERW. (1975) Cytogenetic analysis of erythroleukaemia in two children. Evidence of non-malignant nature of erythron. Scand. J. Haemat. 14, 129. 15. ISCN (1978) An international system of human cytogenetic nomenclature. Cytogenet. Cell Genet. 21,309. 16. KILLMANNS. A. (1976) Preleukemia: does it exist? 81oodCells 2, 81. 17. KNUUTILAS., VUOPIOP., BORGSTROMG. H. & DE LACHAPELLEA. (1980) Higher frequency of 5q-clone in bone marrow mitoses after culture than by a direct method. Scand. J. Haemat. 2,5, 358. 18. KNUUTILAS., VUOPIOP., ELON1ENE., SIMESM., KOVANENR., BORGSTROMG. H. & DE LA CHAPELLEA. (1981) Culture of hone marrow reveals more cells with chromosomal abnormalities than the direct method in patients with hematologic disorders. Blood 58, 369. 19. KROGH-JENSENM. & KILLMANNS. A. (1967) Chromosome studies in acute leukaemia. !. Evidence for chromosomal abnormalities common to erythroblasts and leukaemic white cells. Acta Med. Scand. 181, 45. 20. KaOGH-JENSENM. & KILLMANNS. A. (1971) additional evidence for chromosome abnormalities in the erythroid precursors in acute leukemia. ActaMed. Scand. 189, 97.

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21. WAGHRAYM., EQUESG,, ROWLEYJ. D., MARTIN P. & TESTA J. R. (1981) Methods of processing marrow samples may affect the frequency of detectable anenploid cells. A m . J. Hemat. 11, 409. 22. YUNlSJ. J., BLOOMFIELDC. D. & ENSRUDK. (1981) All patients with acute non-lymphocytic leukemia may have a chromosomal defect. NewEngl. J. Meal. 305,135.

NOTE FROM THE EDITOR The issue of whether other than 'ieukocytes' are directly involved in the leukemic process has for almost 20 years been a pet problem for the undersigned. The story started in 1963 when Whang et al. [1] reported that the Ph I chromosome in CML was not restricted to the granuiocytic series but also was present in erythropoietic precursors; this conclusion was reached by comparing the prevalence of Ph l-positive mitoses and differential counts of mitotic figures before and after treatment, when a fair number of erythroid mitoses again were countable. Evidence was also forthcoming that megakaryocytes probably also contained the Ph I chromosome. Krogh Jensen and Killmann then studied the problem in AML and its variants; the only selection made was that aneupioid modes and/or marker chromosomes had to be present and erythroid mitoses represented at least 10% of all mitotic figures in the direct marrow smears to allow differential counts of mitotic figures within this centennium. The results (1967, 1971, Authors' references [10] and [20]) showed that in 9 of 10 cases the frequency of erythroid mitoses (100 mitotic figures classified) clearly exceeded the prevalence of normal karyotypes, i.e. that at least some erythroid cells have a leukemic (chromosomal) phenotype. Some data also suggested direct megakaryocytic involvement. This was before the introduction of banding techniques. The scepticism created by these results, gravely and concernedly communicated by at least one prominent stem cell worker of that time to the undersigned, was eradicated by the direct iron incorporation autoradiography/karyotype studies by Biackstock and Garson (Authors' reference [7]) that proved abnormal chromosomes in erythroblasts in AML; unfortunately, only two patients were studied. More recently, Dr. Fialkow has applied his ingenious method of G-6-PD isoenzyme studies, that confirmed the involvement of a monocional pluripotential stem cell step in CML, to AML. In Authors' reference [11], two (three) children with AML are reported in whom the red cell line appeared to have the expected G-6-PD isoenzyme distribution whereas the (leukemic) blast cells only had one isoenzyme, indicating monoclonality of the blasts. But the red cells were normal. In contrast, in two elderly AML patients, not only the leukemic blasts, but also the red cells and the platelets contained a single isoenzyme, indicating a common (leukemic) ancestor cell. In the most recent information available to the undersigned, Dr. Fialkow summarized his experience in AML by stating that in four young individuals, there was no evidence, by his method, that erythropoiesis was directly involved in AML, whereas in the elderly patients, just referred to, there was direct involvement. So, here we stand: the question whether or not erythropoiesis and/or megakaryoc~opoiesis is directly 'leukemic' in AML cannot at this time be answered by a clearcut 'yes' or 'no'. It appears to vary from case to case, depending maybe, for example, on age? Treatment of blastic crisis of CML is notoriously much less rewarding than therapy of AML (or ALL). ls it because the stem cell defect goes further back in the stem cell hierarchy in CML than in AML? In AML, does it go further back in older than in younger patients, or are the results in the young just better (some will dispute this, but it is the consensus) because they tolerate aggressive therapy with its complications better? Is 'AML' 'frozen' at various developmental stages, as it is assumed in ALL (and why has the 'frozen cell' then what with a horrible clich~ is usually, almost sheepishly, referred to as a 'proliferative advantage' - - but this is another matter). To return to the subject: Dr. Berger el al. have in their above paper put forward strong (albeit indirect, but still strong) evidence that some particular abnormal chromosomal arrangements (certain translocations) are not compatible with erythroid differentiation. Does this mean that erythropoiesis then is normal, co-existent with leukemic blasts? It does not take much thinking that during development of AML, this must occur. But what when AMO or its variants, so beautifully categorized morphologically by the FAB classification (and soon to be supplemented/substituted by serological typing), is full-blown? Erythropoiesis might still be leukemic (although Dr. Fialkow's data strongly suggest that this may not be the ~ase - - how often remains to be seen); but in general, normal phenotype(s) do not prove real normality (in this case non-leukemicness), but this is an academic point probably impossible to prove or disprove but still worth keeping in mind. As far as chromosomes go, some abnormalities may be incompatible with cytologically normal erythroid differentiation. Attention is called to a case of blastic crisis in CML before and after treatment [3]. Before therapy, highly abnormal clones were present that correlated with blasts, and erythroid mitoses were scarce. After therapy, the 'worst' hyperdiploid clones were eradicated, and erythroid mitoses were much more predominant but still 46,Phi-positive. This may suggest, but does not prove, that some chromosome abnormalities are incompatible with differentiation, in the case under consideration: erythroid differentiation. The contribution by Dr. Berger et al. is important in this respect but does not cover the whole issue. More studies of the type of Drs. Blackstock and Garson and Dr. Fialkow et al. are clearly needed. As far as karyotype studies of leukemia and related diseases are concerned combinations with other techniques are clearly indicated and will at least lead to some better understanding of these diseases. What is most needed, however, is improvement of karyotype methodology; the progress made with the introduction of the banding techniques a decade ago is coming to an end, in its plain form, qualitatively speaking. Fortunately, further progress seems to be under way in some laboratories, gene mapping being a good example. That morphologically normal cells in AML may behave abnormally has been reviewed several times, most recently in 1976 [4]. SVEN-AAGEKILLMANN