(CANCER RESEARCH 48, 3435-3439, June 15, 1988) Induction of Sister Chromatid Exchanges and Chromosomal Aberrations by Busulfan in Philadelphia Chromosome-positive Chronic Myeloid Leukemia and Normal Bone Marrow1

Reinhard Becher2 and Gabriele Prescher

Innere Universitäts-und Poliklinik (Tumorforschung), Westdeutsches Tumorzentrum, Hufelandstrasse 55. 4300 Essen l. Federal Republic of Germany

ABSTRACT (5) human tumor cell lines and described a correlation between the sensitivity to chemotherapy measured by the colony-form Cytogenetic effects of busulfan in vitro were studied in normal bone ing efficiency assay and the rate of induced SCE. Furthermore, marrow (nine cases) and Philadelphia chromosome (Ph)-positive cells the clonal heterogeneity of chemotherapy resistance within a (10 cases) of patients with chronic myeloid leukemia. The frequency of single solid tumor was evidenced by different levels of induced chromosome aberrations and sister chromatid exchange (SCE) increased dose dependently. While there were no significant differences between SCE (6). normal and leukemic cells with regard to the induction of chromosome Our intention was to analyze SCE induction by therapeuti- aberrations, the frequency of SCE was significantly lower in Ph-positive cally relevant agents in leukemias. Ph-positive CML was chosen cells than in normal bone marrow. This difference was not only apparent for the study presented here because the leukemic cells can on the basis of the SCE frequency per cell, but also when the SCE easily be distinguished from normal cells by means of the Ph frequency was correlated to the relative chromosome length as shown by chromosome. Secondly, SCE and chromosome breaks in nor the SCE rate per chromosome group. Longitudinal studies of three mal bone marrow cells can function as control values. This patients who received long term busulfan treatment did not show a allowed us to assess differences between normal and neoplastic significant change in the frequency of induced SCE. It can be suggested cells of the same tissue origin, which seemed mandatory for the that the lower frequency of induced SCE in Ph-positive cells reveals less interpretation of induced SCE in leukemic cells. sensitivity of the leukemic cells to DNA damage by busulfan. Our data provide evidence for the inability of busulfan treatment to eradicate or In an earlier study we showed that short term cultures and in even reduce Ph-positive cells in chronic myeloid leukemia. Evaluation of vitro treatment with mitomycin C of Ph-positive leukemic cells cell proliferation by sister chromatid differentiation shows longer cell from patients with CML are possible and may yield reproduc cycle times for the Ph-positive cells. Busulfan affected the cell cycle ible results (7). Busulfan has been the most frequently used duration of leukemia and normal cells very little. drug in the routine treatment of the chronic phase of CML for many years. In order to assess its clinical relevance in the treatment of CML, we subsequently investigated the cytoge INTRODUCTION netic effects of busulfan on normal and Ph-positive cells. In SCE3, which occurs spontaneously in proliferating cells, has addition we extended this study to the evaluation of structural been shown to be a highly sensitive parameter for the monitor chromosome aberrations. ing of mutagenic and/or carcinogenic effects (1). Structural chromosome aberrations are also known to be indicators of MATERIALS AND METHODS genetic damage in exposed cells. In contrast to SCE, which occurs physiologically, structural chromosome aberrations such Patients. Normal bone marrow was obtained from healthy bone as chromosome breaks are rare under normal conditions. Since marrow donors who had made marrow available for their siblings for they may lead to random DNA loss in dividing cells, they can bone marrow transplantation (Table 1). Only patients with Ph-positive be lethal. Compared to chromosome aberrations SCE is re CML were considered for the study of leukemic cells. The study was garded as the more sensitive assay (1). performed with cell material derived from untreated patients (six cases, Nos. la, 2a, 3a, 6, 7, and 8; Table 2) at the time of diagnosis or from As chromosome aberrations and SCE mark end points of patients pretreated with busulfan or hydroxyurea who had been off DNA damage, evaluation of both cytogenetic parameters pro cytotoxic treatment for at least 2 months at the time of analysis. Three vides more comprehensive information for the assessment of patients (Nos. 1, 2, 3; Table 2) were studied before and after long term DNA damage (2). busulfan treatment. DNA damaging drugs constitute the largest group of am ¡can Normal bone marrow was obtained by sternal or iliac crest puncture. cer drugs presently in clinical use. Several authors have shown In CML patients, peripheral leukemic cells were also used. The hepa- that many of these drugs can induce SCE as well as lead to rinized cell samples were immediately delivered to the cytogenetic laboratory for further processing. increased chromosome fragility (for review see Ref. 2). Al SCE Studies. Cells were cultured in McCoy's medium which was though differences have been observed in the amount of induced chromosomal aberrations and SCE, the increase of both param supplemented with 20% fetal calf serum (Boehringer, Mannheim, FRG) and gentamicin (Boehringer, Mannheim, FRG) in a concentration of eters has been shown to correlate with the amount of cell kill SO Mg/ml. Cultures were set up in dark-stained glass culture flasks (3). Therefore the level of induced cytogenetic anomalies was which were wrapped with aluminum foil in order to completely protect examined for its value in predicting response to cytotoxic them from light exposure. The concentration of BrdU (Serva, Heidel chemotherapy. Tofilon et al. (4) studied animal and Deen et al. berg, FRG) used, was 10 ¿iM/mlof culture medium. Culture time was SO h for all cases including colcemide treatment (GIBCO, Europe) in a Received 12/30/87; revised 3/15/88; accepted 3/18/88. concentration of 0.1 ^g/ml for the final 2 h. Thereafter cells were The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in treated with hypotonie KC1 solution (75 IHM) for 20 min, fixed in a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. fresh solution of methanol/glacial acetic acid (3:1), and washed three 1Supported by the Deutsche Forschungsgemeinschaft (SFB 102, A7). 2To whom requests for reprints should be addressed. times in fresh fixative. SCE staining was performed as earlier reported 3The abbreviations used are: SCE, sister chromatid exchange; Ph, Philadel in detail (8). phia; CML, chronic myeloid leukemia; BrdU. bromodeoxyuridine; PI, prolifera SCE Frequency. SCE frequency was evaluated in M 2 metaphases tion index; SCA, structural chromosome anomalies. which had incorporated BrdU during two 5-phases of cell cycle. The 3435

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. SCE AFTER BUSULFAN IN CML methodology of SCE scoring included so-called peripheral and central chromatid exchanges (9). Each SCE event was specified according to the chromosome group where it occurred. A number of 25 and if t a possible 50 M2 cells were scored for SCE frequency per case. A minimum of 10 metaphases was necessary to fulfill the criterium of evaluability (due to reduced cell growth at higher busulfan concentra tions). Only metaphases with a modal clonal number of 46 chromo somes ±twochromosomes were scored. Leukemic cells were identified by the Ph chromosome. The SCE frequency is given as the number of SCE events per diploid I cell and per chromosome of a chromosome group, taking into consid eration the relative chromosome length in a haploid set of autosomes (10). Data were corrected for sex by adding the X chromosome to the C group and the Y chromosome to the G group. Cell cycle specific patterns were evaluated in all cultures which yielded M2 metaphases. At least 100 Ml, M2, and M3 metaphases were evaluated. The proliferation index was calculated according to the following formula: BUSUIFANftlg/mll Fig. 1. The SCE frequency per metaphase in normal marrow and Ph-positive l*nMl + 2*nM2 + 3*nM3 CML (A) and the frequency of structural chromosomal anomalies (B) after in PI vitro treatment with 1, 3, and 5 ng/ml busulfan. nMl + nM2

GROUP GY ED ex Chromatid Aberrations. Chromatid aberrations were counted exclu 1.10 sively in metaphases which proliferated less than two cell cycles in Brdl ¡-containingmedium (Ml metaphases). Single chromosome gaps and breaks were considered as one event; double gaps, double breaks, 1.00 and acentrics as two; and tetraradials as four events. Chromatid aber nB MARROWNORMAL rations were recorded as the number of events per cell. •C M L Busulfan Treatment. Busulfan was kindly provided by Wellcome 0.90 (Burgwedel, FRG) as pure substance. It was dissolved in acetone and diluted in McCoy's medium. The following three busulfan concentra 0.80 tions were studied in vitro: 1.0, 3.0, and 5.0 ¿igbusulfan/ml culture medium. Busulfan was added at the start of cultures and remained there during the whole culture time. 0.70

RESULTS 0.60 SCE. The frequency of SCE in normal bone marrow (Table 1) increased dose dependently after exposure to busulfan. While 0.50 the spontaneous SCE in the nine cases studied was within a range of 3.12 to 5.20 SCE per metaphase, it increased after 1.0 0.40 Mgbusulfan to a range of 8.36-10.85 SCE per metaphase and was found to be 12.2-21.35 SCE per metaphase after exposure 0.30 to 3.0 fig busulfan. SCE was not évaluableafter 5 ug busulfan. In Ph-positive CML, 14 analyses were performed from 10 patients. SCE control values (Table 2) ranged from 1.92-4.76 0.20 SCE per metaphase and increased after 1 fig busulfan (range, 5.68-10.69) and after 3 /¿gbusulfan (range, 12.28-17.00). Re 0.10 sults were only obtained in five of 14 experiments after treat ment with 3 fig busulfan. In two cases M2 metaphases were still available after treatment with 5 fig busulfan yielding SCE rates of 21.36 and 21.96 SCE per metaphase. Spontaneous as 12345678 well as induced SCE did not significantly differ between un RELATIVE CHROMOSOME LENGTH Fig. 2. The frequency of SCE per chromosome of each chromosome group treated and pretreated patients. Mean SCE frequencies in nor corrected for sex and correlated to the relative chromosome length within a mal bone marrow and Ph-positive CML before and after treat haploid set of autosomes. ment in vitro are shown in Fig. \A. Mean values in Ph-positive CML were consistently lower than in normal marrow. Differ Cell Cycle-specific Proliferation Patterns. The proliferation ences in SCE were significant at 1 fig busulfan (Wilcoxon test, index is given for normal marrow in Table 1 and Ph-positive p < 0.05). At higher concentrations the number of évaluable CML in Table 2. From Fig. 3 it can be seen that normal bone cases was too small for meaningful statistical analysis. marrow had shorter cell cycle times before and after busulfan If the SCE frequency per chromosome of a specific chromo treatment. Differences were significant for control values and 1 some group was taken and correlated to the relative chromo Hgbusulfan but less pronounced after 3 fig busulfan/ml. The PI some length as shown in Fig. 2, a close dose-dependent increase decreased (only slightly) after busulfan exposure in normal and of SCE could again be observed. The SCE rates per chromo leukemic cells. some in Ph-positive CML were lower not only for control Structural Chromosome Anomalies. The frequency of SCA values, but also for induced SCE. per cell was evaluated in M1 metaphases only. In normal bone 3437

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ent in CML. As our study showed significantly more SCE induction in normal bone marrow, it can be suggested that the residual normal hematopoiesis is more severely damaged than Õ Õ the intended target. Based on this observation the possibility can not be ruled out, that long term busulfan treatment may I even lead to an eradication of normal stem cells and be the 2 0. Î major factor responsible for the loss of normal hemopoiesis observed during disease progression by Carbonell et al. (13). I I 1 A follow-up study in three patients analyzed before (cases la, 2a, 3a) and after (cases Ib, le, 2b, 31»)intermittent busulfan treatment during time intervals of up to 2 years yielded no evidence for the modulation of SCE induction (see Table 2). These patients showed no signs of the development of secondary chemotherapy resistance. NORMAL A close correlation of SCE induction and resistance to MARROW chemotherapy has been reported by others for nonhuman (4) CML as well as human solid tumors (5, 6). Due to the high grade of aneuploidy in these malignancies these authors used the fre quency of SCE per chromosome as basis for comparative stud ies. The length of chromosomes, however, varies greatly, e.g., a

I chromosome of the A group is several times the size of a G 5 group chromosome. Therefore we also analyzed the impact of BUSULFAN (|ig/ml) chromosome length for the SCE frequency in our pseudodiploid Fig. 3. Proliferation indices of normal bone marrow and Ph-positive CML system by locating each SCE event according to chromosome after busulfan treatment. groups. Based on these data we found a close correlation marrow (Table 1) they were found to be within a range of 0- between SCE frequency and the relative chromosome length for spontaneous SCE (11), similar to that described by Latt 0.11 SCA per metaphase (mean, 0.04 SCA) before busulfan treatment. They increased to a mean value of 0.17 SCA per 1974 (15). This correlation remained dose dependent after metaphase after 1 ¿tgbusulfan (range, 0.03-0.34 SCA) and to busulfan treatment, as shown in Fig. 2. Accordingly, the differ ences in SCE observed between normal and Ph-positive cells a mean of 0.32 SCA per metaphase after 3 ¿igbusulfan (range, 0.21-0.47 SCA per metaphase). Only one case was évaluable before and after busulfan treatment were also present on the after 5 ¿tgbusulfan with 0.67 SCA per metaphase. The corre basis of SCE per chromosome group. This finding indicates sponding data for Ph-positive CML were similar and are shown that SCE evaluations based on chromosome groups can be in Table 2. Differences between normal bone marrow and Ph- considered an alternative method of SCE evaluation. Such a positive CML as illustrated in Fig. IB were not statistically methodology would not only save time but could also be applied significant. to aneuploid tumors and possibly replace evaluations so far based on mean SCE frequencies per chromosome, which would DISCUSSION only be representative in case of a normal distribution of the length of chromosomes. Chromosomal aberrations and SCEs correlate with cell kill Besides SCE frequencies the differentiation pattern of diro by alkylating drugs, although both are based on different mech matids makes it possible to measure cell proliferation according anisms and show some quantitative variability regarding their to the frequency of Ml, M2, and M3 metaphases (16). Using spectrum of sensitivity (3). In our study with busulfan in vitro this technique we confirmed a longer cell cycle for Ph-positive the frequency of chromosome aberrations and SCE increased CML than was described after using [3H]thymidine (17, 18). dose dependently in normal and leukemic cells. Chromosome Proliferation indices decreased with increasing levels of busul aberrations varied within a large range and did not show differ fan treatment, as shown in Fig. 3. The effect of busulfan on cell ences between normal marrow and Ph-positive CML. The cycle duration in surviving normal and leukemic cells, however, evaluation of induced SCE, however, yielded significantly lower was small. It seemed less pronounced in leukemic cells than SCE in CML. Spontaneous SCE was somewhat lower, although normal cells. However, even after treatment with 3 /ig busulfan, differences were not remarkable, possibly due to a history of pretreatment of some CML patients and the relatively small the proliferation index of normal marrow did not reach the control level of Ph-positive cells. Due to the fact that the yield number of cases. Larger studies have shown significantly lower spontaneous SCE in Ph-positive CML in the chronic phase of of mitoses was already severely reduced after 3 /¿gbusulfanand disease (11). Taking into account that SCE correlates with the below the level necessary for evaluation after 5 /ig busulfan in amount of DNA damage (12), the lower frequency of induced all except one case, we conclude that the major biological effect SCE in Ph-positive cells indicates a relatively lower suscepti of busulfan is not a general delay of cell proliferation but cell bility of leukemic cells to busulfan treatment. This finding is in kill. This assumption is in agreement with Natarajan et al. (3) agreement with the clinical experience that neither long term who studied the amount of alkylating drug necessary to kill nor high dose treatment of busulfan are capable of eradicating 50% of Chinese hamster ovary cells and correlated it with the the Ph-positive clone in CML. amount of drug necessary to double the SCE frequency. Al Although cytogenetic studies mostly yield 100% Ph-positive though the relationship varied widely according to the drug cells, it is known from in vitro culture studies (13, 14) that a used, they found that doses which doubled the SCE frequency small population of normal residual hematopoiesis is still pres mostly killed more than 50% of cells. 3438

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8. Becher, R., and Sandberg, A. A. Rapid method for Giemsa staining of sister ACKNOWLEDGMENTS chromatids. Cancer Genet. Cytogenet., 7: 223-225, 1982. 9. Becher, R., and Sandberg, A. A. Sister chromatid exchange in the centromere We thank G. Zimmer for excellent technical assistance, H. Hirche and centromeric area. Hum. Genet., 63: 358-361, 1983. for statistical analysis, C. Massion for typing the manuscript, and M. 10. Paris Conference (1971): Standardization in Human Cytogenetics. Birth Weiss for reviewing the English. Defects: Original Article Series. New York: The National Foundation, p. 42 (D), V1II:7, 1972. 11. Becher, R., Zimmer, G., Prescher, G., and Schmidt, C. G. Spontaneous sister chromatid exchange in normal bone marrow and Ph-positive chronic mye- REFERENCES locytic leukemia. Cancer Res., 48: 745-750, 1988. 12. Bodell, W. J., Rupniak, H. T. R., Rasmussen, J., Morgan, W. F., and 1. Perry, It., and Evans, H. J. Cytological detection of mutagen-carcinogen Rosenblum, M. L. Reduced level of DNA cross-links and sister chromatid exposure by sister chromatid exchange. Nature (Lond.), 258:121-125,1975. exchanges in l,3-bis(2-chloroethyl)-l-nitrosourea-resistant rat brain tumor cells. Cancer Res., 44: 3763-3767, 1984. 2. Gebhart, E. Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing. Hum. Genet., 5Ä:235-254, 1981. 13. Carbone!!, F. C., Hoelzer, D., Grilli, G., Issaragrisil, S., Harriss, E. B., and Fliedner, T. M. Chronic myelocytic leukaemia: cytogenetical studies on 3. Natarajan, A. T., Simons, J. W. I. M., Vogel, E. W., and van Zeeland, A. A. haemopoietic colonies and diffusion chamber cultures. Scand. J. Haematol., Relationship between cell killing, chromosomal aberrations, sister-chromatid 30:486-491, 1983. exchanges and point mutations induced by monofunctional alkylating agents 14. Coulombel, L., Kalousek, D. K., Eaves, C. J., Gupta, C. M., and Eaves, A. in Chinese hamster cells. Mutât.Res., 128: 31-40, 1984. C. Long-term marrow culture reveals chromosomally normal hematopoietic 4. Tofilon, P. J., Basic. I., and Milas, L. Prediction of in vivo response to progenitor cells in patients with Philadelphia chromosome-positive chronic chemotherapeutic agents by the in vitro sister chromatid exchange assay. myelogenous leukemia. N. Engl. J. Med., 308:1493-1498, 1983. Cancer Res., 45: 2025-2030, 1985. 15. Lati, S. A. Localization of sister chromatid exchanges in human chromo 5. Deen, D. F., Kendall, L. E., Marton, L. J., and Tofilon, P. J. Prediction of somes. Science (Wash. DC), 185: 74-76, 1974. human tumor cell chemosensitivity using the sister chromatid exchange 16. Becher, R., and Schmidt, C. G. Sister chromatid differentiation and cell- assay. Cancer Res., 46: 1599-1602, 1986. cycle specific patterns in chronic myelocytic leukemia and normal bone 6. Tofilon, P. J., Vines, C. M., Baker, F. L., Deen, D. F., and Brock, W. A. cis- marrow. Int. J. Cancer, 29:617-620, 1982. Diamminedichloroplatinum(Il)-induced sister chromatid exchange: an indi 17. Gavosto, F. Granulopoiesis and cell kinetics in chronic myeloid leukaemia. cator of sensitivity and heterogeneity in primary human tumor cell cultures. Cell Tissue Kinet., 7: 151-163, 1974. Cancer Res., 46: 6156-6159, 1986. 18. Ogawa, M., Freid, J., Sakai, Y., Strife, A., and Clarkson, B. D. Studies of 7. Becher, R., Zimmer, G., Schmidt, C. G., and Sandberg, A. A. Sister chro cellular proliferation in human leukemia. VI. The proliferative activity, matid exchange in normal and Ph'-positive leukemic cells after mitomycin- generation time, and emergency time of neutrophilic granulocytes in chronic C treatment in vitro. Cancer Genet. Cytogenet., 12: 239-245, 1984. granulocytic leukemia. Cancer (IMiila.). 25: 1031-1049, 1970.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. Induction of Sister Chromatid Exchanges and Chromosomal Aberrations by Busulfan in Philadelphia Chromosome-positive Chronic Myeloid Leukemia and Normal Bone Marrow

Reinhard Becher and Gabriele Prescher

Cancer Res 1988;48:3435-3439.

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