Cytogenetic Effects of Bleomycin Therapy in Man1

(CANCER RESEARCH 31, 2004-2007, December 1971] Cytogenetic Effects of Bleomycin Therapy in Man1

Richard S. Bornstein, David A. Hungerford, Gail Haller, Paul F. Engstrom, and John W. Yarbro Department of Medicine, American Oncologie Hospital [R. S. B., P. F. E., J. W. Y.J, and The institute for Cancer Research/D. A. H..G. H., J. W.Y.I, Philadelphia, Pennsylvania 19111

SUMMARY bleomycin in combination with other agents without increasing the toxicity of such agents. Prominent side effects Direct, air-dried preparations of metaphase chromosomes include skin thickening, mucosal ulcÃ©ration,fever, nausea, and, from bone marrow cells from four cancer patients were made in some instances, pulmonary fibrosis, which may ultimately prior to, during, and after treatment with the antitumor agent, lead to death. bleomycin. An increase in the number of chromosomal Investigation of the in vivo cytogenetic changes during aberrations was observed following administration of bleomycin administration has not been reported previously. In bleomycin; these persisted in two of the patients the course of our clinical studies, we have investigated the in approximately 1 month after therapy. The aberrations vivo effects of bleomycin on the chromosomes of bone consisted of hypochromatic regions, gaps, and breaks, all of marrow cells of 4 patients. which were of both the chromatid and isochromatid types; not present, however, were multiradials, dicentrics, and rings. Histopathological studies of the marrow showed no MATERIALS AND METHODS abnormalities. The disparity between cytogenetic and histopathological findings remains unexplained. Bleomycin (Bristol Laboratories, Syracuse, N. Y.) was administered i.v. at a dose of 0.3 mg/kg every 12 hr for 4 days; the course was repeated once after an interval of 10 days. INTRODUCTION Bone marrow biopsy specimens were obtained from the posterior iliac crest prior to treatment, on the last day of Bleomycin is a new cytotoxic antibiotic elaborated by a treatment (prior to the last dose of bleomycin), and 1 month mutant strain of the soil bacterium, Streptomyces verticillus after treatment. The total dose given varied between 252 and (10). It was isolated in Japan during a search for antibiotics 360 mg. The patients received no other cytotoxic agents or with an antitumor spectrum similar to that of phleomycin but radiotherapy during this period nor had they any previous lacking its nephrotoxicity. The structure of bleomycin has not exposure to such modalities. Bone marrow aspirates and been fully elucidated, although it can be separated into 2 biopsies were processed and stained in the usual manner. active substances, bleomycins A and B, which can be further Direct chromosome preparations were made by the hitherto fractionated into a number of peptides containing amino acids, unpublished method devised by one of us (G. H.), which is as sulfur, and sugars, some of which are quite unusual (12). The follows. Approximately 0.2 ml of aspirated marrow was placed drug appears to act by means of single-strand scission of DNA into a vial containing 5 ml of blood culture medium (3), (8). Bleomycin inhibits the growth of a number of both colchicine was added to a final concentration of 1 jug/ml, and gram-positive and gram-negative bacteria (11); it has also the vial was incubated at 31Â°for a total of 30 min. The culture demonstrated an effect against a wide spectrum of animal was harvested by centrifugation, and the medium was replaced tumors, including both solid and ascitic varieties (11). Recent with 0.075 M KC1 with heparin (16 U.S.P. units/ml) and clinical reports have shown the efficacy of bleomycin against colchicine (1 Mg/ml) at 31Â°for an additional 30 min. The cells well-differentiated squamous cell carcinomas involving the were then fixed in 3:1 mixture of absolute methanol in glacial skin, head and neck, larynx, esophagus, penis, lung, and cervix, acetic acid. The cells were precipitated by centrifugation, and as well as against testicular neoplasms, melanoma, and lymphomas, especially Hodgkin's disease (1). the fixative was changed once during the procedure. Slides were made by the air-dry method of Rothfels and Siminovitch This drug is of particular interest, not only because of its (5) and were stained in 1% acetic orcein for 90 min. Coverslips activity in certain tumors that have heretofore been resistant were mounted with Diaphane (Will Corp., Baltimore, Md.). to treatment, but also because of its peculiar toxicity. In All 4 patients studied by us had inoperable carcinoma. contrast with the effects of other cytotoxic chemotherapeutic These included a 44-year-old white female [MB 201026 agents, depression of the hematopoietic system has only rarely (282T)] with recurrent malignant melanoma; a 57-year-old been reported. Thus, the potential exists for the use of white male [El 090413 (285T)] and a 65-year-old white male [RF 300905 (284T)], each with squamous cell carcinoma of 'This research was supported in part by USPHS Grants CA-11266, the lung; and a 68-year-old black female [BP 251202 (286T)] CA-05903, CA-12211, GM-17551, CA-06927, RR-05539, and CA-06551 and by an appropriation from the Commonwealth of with squamous cell carcinoma of the cervix. Pennsylvania. Three categories of chromosome aberration were observed, Received July 8, 1971; accepted August 18, 1971. as follows (Fig. 1); (a) hypochromatic regions, defined as

2004 CANCER RESEARCH VOL. 31

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1971 American Association for Cancer Research. Cytogenetic Effects ofBleomycin Therapy in Man poorly staining regions, which were either chromatid or clastogens, when studied either in vitro or more rarely in vivo. isochromatid; (b) achromatic regions or gaps, defined as areas In some instances, different results have been obtained, in which staining was absent, which were either chromatid or depending on the method of study. In view of these isochromatid; (c) breaks. The term "break" was applied when differences, which may be due to changes induced by the length of the discontinuity was greater than the width of short-term culture methods or the metabolic effects of the the chromatid. host on the drug in question, it must be emphasized that in Cells with 1 or 2 aberrations were found in the same vivo studies are critical. Changes observed under in vitro frequency in all samples; these were considered normal. Only conditions may not necessarily imply that similar damage will those cells with more than 2 aberrations were classified as occur in vivo. anomalous. In this category, no fewer than 8 aberrations were Antibiotics which inhibit DNA, such as streptonigrin. observed in any cell. mitomycin C, and phleomycin, produce chromosome breaks in a nonrandom fashion (2). In vitro studies with bleomycin have suggested a similar nonrandom distribution (4). We did not RESULTS feel that it was appropriate to attempt analysis of the distribution of chromosome aberrations in our series because All 4 marrow samples taken near the termination of of the small sizes of the samples. Further studies in this regard bleomycin therapy contained more mitoses with chromosomal will be necessary. aberrations than did the control samples obtained prior to A number of inherited disorders are associated with an therapy (Table 1; Chart 1). These aberrations consisted of increased incidence of spontaneous chromosome breaks. These hypochromatic regions, achromatic regions, and breaks, all of include Bloom's syndrome, Fanconi's anemia, Kostman's which were either chromatid or isochromatid (chromosome). agranulocytosis, glutathione reducÃase deficiency, ataxia A few acentric fragments were observed, although no telangiectasia, and pernicious anemia (6). An increased dicentric, ring, or multiradial chromosomes were seen. The incidence of cancer, especially leukemia, is reported in increased frequency of aberrant cells persisted in 2 of the 4 association with each of these disorders. The association of patients examined 1 month after termination of bleomycin clastogens (such as benzene). X-rays, and certain viruses with therapy, although in all 4 patients there was an apparent cancer is also well established. These agents are potentially decrease from the peak value noted immediately after carcinogenic, mutagenic, and teratogenic. Therefore, the treatment. As shown in Table 1 and Chart 1, Patients R. F. and B. P. demonstrated an increased frequency of aberrations Table 1 in the control sample. No explanation for this was present after careful review of the patients' histories with respect to Fractions represent those cells with chromosomal aberrations over total cells studied in the bone marrow samples exposure to other known mutagens. Even in these 2 cases, however, there was a higher frequency of aberrations following Posttreatment the administration of bleomycin and a return to control Patient Pretreatment Immediate 1 month frequencies in the 1-month-posttreatment study. In contradistinction to the cytogenetic findings was the lack M. B. 0/25 13/30 11/29 of histopathological abnormality in the bone marrow. The R. F. 4/29 8/30 6/29 E.I. 0/25 9/32 5/30 bone marrow appeared normal in both a qualitative and a B. P. 5/31 20/32 7/34 quantitative sense. The preparations were evaluated in a double-blind fashion. There were no changes from control in terms of overall cellularity, differential cell count, and maturation of cell lines, nor was there evidence of nuclear anomalies or change in mitotic activity. Some minor variations were noted in the samples, but these changes, when evaluated serially in relation to the administration of bleomycin, did not appear to be significant. Furthermore, there were no changes in peripheral blood count during the course of treatment.

DISCUSSION

Bleomycin is presently being evaluated in a number of centers in this country, and its therapeutic spectrum is being 050 1.00 determined. One unique feature has been the lack of bone FRACTION OF CELLS WITH ABERRATIONS marrow toxicity in all published series. Our preliminary results have confirmed this finding. However, we have now â€¢i PRETREATMENT demonstrated that bleomycin produces abnormalities of the E5S3 POSTTREATMENT (IMMEDIATE) chromosomes. The term "chromoclastogen (clastogen)" has Chart 1. Comparison of marrow samples taken before bleomycin been suggested by Shaw (7) for such agents. Most if not all therapy and prior to the 16th (last) dose. Note the elevation of cytotoxic chemotherapeutic agents have been shown to be aberration frequency in all 4 patients.

DECEMBER 1971 2005

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1971 American Association for Cancer Research. Bornstein, Hungerford, Haller, Engstrom, and Yarbro implications of drug-induced chromosome changes are clear, the Clinical Efficiency of Bleomycin in Human Cancer. Brit. Med. albeit speculative. J., 2: 643-645, 1970. 2. Gebhart, E. The Treatment of Human Chromosomes in Vitro'. The disparity between cytogenetic and histopathological data on the bone marrow remains to be understood. It is Results. In: F. Vogel and G. Rohrborn (eds.), Chemical Mutagenesis in Mammals and Man, pp. 367-382. New York: possible that the disparity reflects a dose phenomenon, e.g., Springer-Verlag, 1970. similar results can be seen following the administration of low 3. Hungerford, D. A. Leukocytes Cultured from Small Inocula of doses of irradiation in which chromosome breakage can be Whole Blood and the Preparation of Metaphase Chromosomes by seen in the absence of other significant bone marrow toxicity. Treatment with Hypotonie KC1. Stain Technol., 40: 333-338, Comparative studies on the lethal effects of irradiation and 1965. bleomycin on cultured mammalian cells have shown a similar 4. Ohama, K., and Kadotani, T. Cytologie Effects of Bleomycin on pattern, suggesting that a similar mechanism might be involved Cultured Human Leukocytes. Japan J. Human Genetics, 14: (9). 293-297, 1970. In summary, we have documented the clastogenic effects of 5. Rothfels, K. H., and Siminovitch, L. An Air-drying Technique for Flattening Chromosomes in Mammalian Cells Grown in Vitro. bleomycin in vivo by study of direct chromosome Stain Technol., 33: 73-77, 1958. preparations. Although chromosome aberrations have occurred 6. Schroeder, T. M., and Kurth, R. Spontaneous Chromosomal with significant frequency, no rearrangements were noted. Breakage and High Incidence of Leukemia in Inherited Disease. Further clarification of the disparity between bone marrow Blood, 37: 96-112, 1971. morphology and the cytogenetic findings awaits more 7. Shaw, M. W. Human Chromosome Damage by Chemical Agents. complete studies of the mechanism of action and the Ann. Rev. Med., 21: 409-432, 1970. metabolism and mode of distribution of bleomycin in man. 8. Suzuki, H., Nagai, K., Yamaki, H., Tanaka, N., and Umezawa, H. On the Mechanism of Action of Bleomycin: Scission of DNA Strands in Vitro and in Vivo. J. Antibiotics Tokyo Ser. A, 22: ACKNOWLEDGMENTS 446-448, 1969. We are grateful to Dr. R. Philip Custer, Senior Member, The Institute 9. Terasima, T., and Umezawa, H. Lethal Effect of Bleomycin on for Cancer Research, and Professor of Pathology Emeritus, School of Cultured Mammalian Cells. J. Antibiotics Tokyo Ser. A, 23: Medicine, University of Pennsylvania, for reviewing the bone marrow 300-304, 1970. preparations. We also thank Bristol Laboratories for supplying the 10. Umezawa, H. Bleomycin and Other Antitumor Antibiotics of High bleomycin. Molecular Weight. Antimicrobial Agents Chemotherapy. 1079-1085, 1965. REFERENCES 11. Umezawa, H., Ishizuka, M.. Maeda, K., and Takeuchi, T. Studies on Bleomycin. Cancer, 20: 891-895, 1967. 1. Clinical Screening Co-operative Group of the European 12. Umezawa, H., Suhara, Y., Takita, T., and Maeda, K. Purification of Organization for Research on the Treatment of Cancer. Study of Bleomycins. J. Antibiotics Tokyo Ser. A, 19: 210-215, 1966.

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1C ID Fig. 1. /4, normal metaphase from marrow of Patient R. F. B, various effects on chromosomes in metaphase from Patient M. B. Note hypochromatic regions (arrowheads), gaps (arrows), and breaks (bent arrows). C, minimally affected metaphase from Patient B. P. Arrows indicate chromatid breaks and gaps (see text). D, maximally affected metaphase from Patient B. P. Note multiple aberrations.

DECEMBER 1971 2007

Richard S. Bornstein, David A. Hungerford, Gail Haller, et al.

Cancer Res 1971;31:2004-2007.

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