Leukeraia Research V o l 5. N o 4 5 . pp. 395 398.. 1981. 0t45-212681/(140395-04502.00/0 Printed in Greal Britain © 1981 Pergamon Press Ltd.

C Y T O G E N E T I C E V I D E N C E F O R A C L O N A L

S E L E C T I O N O F L E U K E M I C

C E L L S I N C U L T U R E

FELIX CARBONELL, GUALTIERO GRILLI and THEODOR M. FLIEDNER

Department of Clinical Physiology and Occupational Medicine, University of Ulm, Oberer Eselsberg M24. 7900 Ulm (Donau), West Germany

(Receit,ed 8 August 1980. Ret, ised 25 February 1981. Accepted 7 March 1981)

Abstraet--Cytogenetic analysis of bone marrow leukemic cells of 33 patients was performed in direct preparation and in short term liquid culture. In eight patients a clonal selection of abnor- mal clones was observed in the short term liquid culture. This finding is consistent with the hypothesis that leukemic cells with the most pronounced chromosomal abnormality have a growth advantage compared to the more "normal" cells.

INTRODUCTION

IN RECENT years, leukemic cell proliferation has been studied in in vitro culture systems by means of semi-solid or liquid suspension culture. This has allowed the study of the influence of regulatory factors, the cell differentiation potential and the interactions between normal and leukemic cells in culture [1, 7, 8, 10].

An important question is whether the cells produced are descendents of normal or of leukemic stem cells. One possible way to solve this question ks to undertake a cytogenetic analysis of cells from leukemia patients in culture [3, 4, 7, 11].

The aim of this study was to find out whether the different clones present in some leukemic patients differ in their proliferative capacity when present in vivo as compared to in vitro culture conditions. Cytogenetic analyses have been performed in leukemic patients using a direct preparation of the bone marrow and, for comparison a two-day culture in liquid medium.

MATERIAL AND METHODS

Patients. Bone marrow cells from 33 patients with various forms of leukemia were studied cytogenetically by means of a direct marrow preparation and a two-day in vitro culture system: 17 with chronic mvelocvtic leukemia (CML), 5 in the blast phase of the chronic myelocytic leukemia (CML B) and 11 with acute non- lymphocytic leukemia (ANLL).

Bone marrow direct preparation. Bone marrow suspension 0.2-0.5 ml was delivered into 10ml Hank's balanced salt solution containing colcemid at a concentration of 0.2/zg/ml. The suspension was incubated at 37:C for 2 h, then treated in hypotonic KCI (0.075 M) and fixed in methanol-acetic acid (3:1). Chromosomes were spread by an air-drying technique and stained with Giemsa.

Suspension culture system. Bone marrow ceils (5.106) were suspended in 5 ml McCoy's medium containing 20°,o fetal calf serum, 50 U/ml penicillin G and 50 #g/ml streptomycin. Duplicate cultures were performed in the presence or absence of 15°,o human placenta-conditioned medium (HPCM) as a source of colony stimulating activity [17]. After 2 days at 37°C, chromosomes were prepared for examination by the method of Moorhead [10].

Abbreriations: CML, chronic myelocytic leukemia: CML B, chronic myelocytic leukemia in blast phase; ANLL. acute non-lymphocytic leukemia; CSA, colony stimulating activity; HPCM, human placental con- ditioned medium.

395

396 FELIX CARBONELL, GUALTIERO GRILLI and THEODOR M. FLIEDNER

TABLE 1. RESULTS OF BONE MARROW CULTURES OF THE LEUKEMIC PATIENTS INVESTIGATED

Bone marrow direct Two-day bone marrow preparat ions liquid culture C M L C M L B ANLL Total

Patients with one cell clone (15)

Patients with two cell clones (18)

New clones in culture . . . . Growth of same clone 7 - - - - 11 No growth 3 - - 1 4

New clones in culture . . . . Growth advantage of 3 1 4 8 abnormal clone Same proport ion of 2 - - 1 3 clones No growth 2 4 1 7

Total 17 5 11 33

,,. CML, chronic myelocytic leukemia; C M L B . chronic myelocytic leukemia in blast phase: ANLL, acute non-lymphocyt ic leukemia.

Evaluation. At least 30 metaphases were studied from each preparation. A clone was defined by the presence of at least three metaphases with an identical karyotype.

R E S U L T S

The results of the cytogenetic studies performed on the direct preparations have led us to separate the patients into two groups (Table 1). The first group contains 15 patients who displayed only one cell clone. The second group contains 18 patients, in whom two cell clones were found in the bone marrow. Table 2 shows the results of eight of these patients. The proportions of the two cell clones in the direct method and in the two-day culture of the marrow differed markedly, a clonal selection was observed favouring the aneuploid cells over the diploid cells.

The comparison between the culture stimulated with HPCM and that without stimu- lation indicated the HPCM did not favour the appearance of new clones and did not change the proportions between the clones with different karyotypes.

TABLE 2. CYTOGENETIC FINDINGS IN LEUKEMIC PATIENTS WITH CLONAL GROWTH

Bone marrow direct Case Sex/Age No. metaphases No. (years) Diagnosis Karyotypes analysed

(%)

Bone marrow culture No. metaphases

analysed (%)

1 M 43 C M L 46, XY 1 l (25) 46, XY, Ph 1 13 (75)

2 M 76 C M L 46,XY 10 (29) 46,XY, Ph I 24 (71 )

3 M 33 C M L 46,XY,Ph I 32 (82) 47,XY,Ph l, + Ph 1 7(181

4 M 44 C M L B 46,XY,Ph 1 17 (77) 46,XY,Ph 1, - 17 + i(17q) 5 (23)

5 M 63 A N L L 46,XY 31 (97) 47,XY, + i(17q) 1 (3)

6 F 20 A N L L 46,XY 4 (8) 44,XX, + A, - B, - 3C, + D 48 (92)

7 F 59 A N L L 46,XX 24 (52) hypoploid with 22 (48) multiple aber.

8 M 38 A N L L 46,XY 34 (85) hyperploid with 6 (15)

multiple aber.

3 (10) 26 (90)

o(o) 30(100) 20(51) 19 (49) 8 (23)

27 (77) 19(53) 17 (47)

0(0) 35 (100)

1 (3) 30 (97)

30 (62) 18 (38)

Clonal selection of leukemic cells in culture 397

No new clones were observed in culture that had not been observed in the direct preparation.

DISCUSSION

The studies reported here together with the recent observations of Berger I-2] and Knuutila 1-9] suggest that cytogenetic abnormalities which are, in principle, present in patients with leukemia, can be amplified by employing bone marrow culture methods. At the present time, it remains to be studied why the leukemic cell clone gains an advantage over the normal cell clone in such a culture. We have observed a similar growth pat tern in cell culture without the addition of CSA, which, therefore, cannot be the sole reason for the triggering of leukemic precursor cells into division.

These findings are consistent with the results obtained in kinetic studies of human leukemic blast cells in diffusion chambers [5, 6] ; they suggest that cytokinetically quies- cent leukemic blast cells in the peripheral blood may re-enter the cell cycle for prolifer- ation under special conditions. In agreement with this, our results suggest that the observed increase of aneuploid mitoses in two-day culture using HPCM as a stimulatory factor may be explained by the re-entry into the cell cycle by leukemic cells, the sub- population of blast cells containing the more chromosomal aberrations. The chromoso- mal analysis of bone marrow cells by use of banding techniques indicates that cytogene- tic alterations in leukemic patients occur non-randomly, and may suggest that the lo- cation of gene may play an important role in the acquisition of a selective growth advantage by the aneuploid cell population 1,14, 16-1.

On the other hand, the relative decrease in the number of "normal" metaphases in the two-day culture may be an indication of an inhibitory factor produced by the abnormal cell and acting on the normal progenitor cells I-8, 13, 15].

It may be of interest to follow such leukemic patients for their entire life-span to explore whether the presence of absence of an in vitro stimulation of clonal development correlates with the eventual fate of the patient. It could, for instance, be speculated that pronounced in vitro inhibition of clonal expression of highly aneuploid cells could indi- cate a better prognosis because of a better control of the leukemic cell populations by cellular interactions.

Acknowledgements--This investigation was supported by grants from the European Atomic Energy Com- munity (EURATOM). The excellenttechnical assistance of Mrs. E. Kratt and Mrs. K. Neuhaus is gratefully acknowledged.

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398 FELIX CARBONELL, GUALTIERO GRILLI and THEODOR M. FLIEDNER

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