Effect of procarbazine and cyclophosphamide on chromosome breakage in Fanconi anemia cells: Relevance to bone marrow transplantation

Effect of Procarbazine and Cyclophosphamide on Chromosome Breakage in F anconi Anemia Cells: Relevance to Bone Marrow Transplantation Arleen D. Auerbach,* Barbara Adler, Richard J. O'Reilly, Dahlia Kirkpatrick, and R. S. K. Chaganti

ABSTRACT: Fanconi anemia (FA) patients develop stem cell defect-based pancytopenia for which bone marrow transplantation offers the potential for correction. Recently, it has become apparent that the outcome of marrow transplantation in FA patients is poor because of the hypersensitivity of these patients to the pretransplantation conditioning regimen which includes immunosuppression with high doses of the difunctional alkylating agent cyclophosphamide. In an effort to devise a less toxic immunosuppressive regimen, we compared the clastogenic effect of cyclophosphamide with that of procarbazine in cells from FA patients and normal controls. Activation of the drugs was achieved by two alternative methods, either by injection into rats (the in vivo activation method) or by incubation with o rat-liver microsome system (the in vitro activation method). Increased sister chromatid exchange following treatment of cells with cyclaphosphamide or procarbazine was used as an indicator for the presence of activated drug metabolites in the system. Although FA cells were hypersensitive to the clastogenic effect of cyclophosphamide, they were not more sensitive than normal cells to procarbazine-induced chromosome breakage. Procarbazine may thus be a safer drug than cyclophosphamide for conditioning FA patients for bone marrow transplantation.

INTRODUCTION Fanconi anemia (FA) is an autosomal recessive disorder characterized clinically by stem cell defect-based pancytopenia, increased chromosomal instability [1], and hypersensitivity to the clastogenic effect of difunctional alkylating agents such as mitomycin C and diepoxybutane (DEB) [2]. In addition, FA patients may manifest one or more d e v e l o p m e n t a l defects [3] w h i c h tend to be highly variable, hence useless as a basis for clinical diagnosis in the absence of confirming cytogenetic evaluation. With c o n v e n t i o n a l therapy for aplastic anemia, affected i n d i v i d u al s usually die of hemorrhage, infection, or acute leukemia within 5 years of the onset of bone marrow failure [4]. A l t h o u g h bone marrow transplantation offers the potential for corFrom the Laboratory of Cancer Genetics and Cytogenetics,Department of Pathology, and the Bone Marrow Transplantation Service, Memorial Sloan-KetteringCancer Center, New York, New York. *Present address: Laboratory for InvestigativeDermatology, The RockefellerUniversity,New York, New York. Address requests for reprints to Dr. R. S. K. Chaganti, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021. Received July 5, 1982; accepted September 1, 1982. 25 © Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017

Cancer Genetics and Cytogenetics9, 25-36 (1983) 0165~4608/83/$03.00

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A.D. Auerbach et al. rection of the stem cell defect, the outcome of transplantation in these patients has been poor because of the severe and prolonged toxicity of the pretransplant conditioning regimen [5], which includes high doses of the difunctional alkylating agent cyclophosphamide, and subsequent poor tolerance of graft-versus-host (GVH)-induced dermal, enteric, and hepatic pathology. In an effort to devise a less toxic immunosuppressive regimen, we compared the clastogenic effect of cyclophosphamide with that of procarbazine, a methylhydrazine that has been widely used in chemotherapy for lymphoma [6]. Our results indicate that procarbazine may be a safer drug than cyclophosphamide for conditioning FA patients for bone marrow transplantation.

MATERIALS AND METHODS Study Population Five FA patients and three normal controls were included in these studies. In patients with either progressive pancytopenia, typical developmental abnormalities, or both, a diagnosis of FA was confirmed on the basis of increased spontaneous and DEB-induced chromosomal breakage [2]. Patient FA1 (M.D.) has been described previously [2]. Patient FA2 (D.J.) was a 21/2-year-old boy with pancytopenia; thrombocytopenia had been present since birth. His height, weight, and head circumference were less than 3rd percentile. In addition, he had microphthalmia, caf~-au-lait spots, hyperpigmentation, and flat thenar eminences. Patient FA3 (B.N.) was a 2month-old boy who weighed 5 lb, 1 oz at birth. Both forearms were shortened, and thumbs were absent. He also had a malformed ear and hypoplastic genitalia. Patient FA4 (L.G.) was a 12-year-old girl with a recent onset of pancytopenia. Her parents were second cousins, once removed. She was of small stature and had caf6-au-lait spots and hyperpigmentation. Patient FA5 (H.D.) was a 6V2-year-old boy with a recent onset of aplastic anemia. His height, weight, and head circumference were below the 3rd percentile, his thumbs were absent, and he had webbing of the toes.

Lymphocyte Cultures The culture unit in all experiments consisted of 0.4 ml of heparinized blood added to 10 ml of Eagle's minimal essential medium (MEM) (Gibco) supplemented with 15% fetal bovine serum, penicillin-streptomycin, heparin, and phytohemagglutinin (PHA) (complete blood culture medium). Metabolic activation of procarbazine or cyclophosphamide was achieved by two different methods, described in detail below. Cultures were incubated at 37°C in a 5% CO2 atmosphere at high humidity for 72 hr (in vivo activation of drugs) or 96 hr (in vitro activation of drugs). Cells were harvested, and chromosome preparations were made following standard methods.

In Vivo Activation of Drugs Sprague-Dawley rats (Charles River) weighing 250-350 g were injected intraperitoneally with an LDs0-LD10o dose of either procarbazine or cyclophosphamide. Fifteen minutes to 1 hr after the injection the rats were anesthetized and exsanguinated by cardiac puncture. The blood was allowed to clot, and the serum containing activated drug was collected. This serum, at various dilutions, was added to lymphocyte cultures for the last 48 hr of a 72-hr culture period. Serum was also collected from control rats that had not received any drug injections.

Drug-Induced Chromosome Breakage in FA

27

In Vitro Activation of Drugs In vitro activation of procarbazine, the azo derivative of procarbazine (Roche compound Ro4-8047; N-isopropyl-p-methylazoxymethyl-benzamide), and cyclophosphamide was achieved using a modification of the Ames rat-liver microsome system (S-9 mix) as described by White and Hesketh [7] for use with human lymphocytes. Procarbazine was dissolved directly in the treatment medium (Eagle's MEM containing 2.5% fetal bovine serum), and dilutions were made to give the required concentrations. Roche compound Ro4-8047 was dissolved in dimethyl sulfoxide (DMSO) and added to the treatment medium at appropriate concentrations. Cyclophosphamide was dissolved in distilled water and diluted with the treatment medium. Each treatment unit consisted of 2 ml of treatment medium containing drugs at various concentrations to which was added 0.5 ml S-9 mix (1:4 ratio) and 0.4 ml whole blood. The S-9 mix, prepared immediately before use, contained 10% S-9 rat-liver extract (Litton Bionetics) and an NADPH-generating system consisting of 0.4 M MgC12, phosphate buffer (0.2 M Na2HPO4-NaH2PO4,pH7.4), 0.1 M NADP, and 1 M glucose 6-phosphate (G-6-P). Peripheral blood lymphocytes were exposed to the drugs, in the presence of S-9 mix, for 1 hr prior to stimulation with PHA. During exposure, cells were incubated at 37°C in tightly closed glass bottles and shaken frequently to promote even distribution of activated chemical metabolites. After treatment, cells were washed three times with Eagle's MEM containing 15% fetal bovine serum, and the pelleted cells were added to complete blood culture medium.

Sister Chromatid Exchange Studies Sister chromatid exchange (SCE), which is thought to reflect DNA damage [8], was used as an indicator for the presence of activated drug metabolites. For these studies, cells were cultured in complete blood culture medium to which 20 p,M bromodeoxyuridine was added for the last 48 hr (two cell cycles) of the culture period. Slides were stained using the fluorescence plus Giemsa technique for differential staining of sister chromatids [9]. Thirty consecutive second-division cells, which appeared unbroken and showed good chromosome morphology, were examined at each drug concentration and the frequency of SCEs recorded.

Chromosome Breakage Studies Analysis of chromosome breakage was performed on 50-100 aceto-orcein-stained metaphases from each preparation. To avoid bias in cell selection, consecutive metaphases that appeared unbroken and showed sufficiently well-defined chromosome morphology were selected for study. Each cell was scored for chromosome number and for the numbers and types of structural abnormalities. Achromatic areas less than a chromatid in width were scored as gaps, and exchange configurations, translocations, and dicentric and ring chromosomes were scored as rearrangements. Gaps were excluded in the calculation of chromosome breakage frequencies, and rearrangements were scored as two breaks. RESULTS

Effects of In Vivo Activated Drugs SCE frequency was studied in two normal (control) individuals (Table 1B). Addition to the blood culture medium of serum collected from rats injected with cyclophosphamide (1:100 dilution) resulted in a fivefold increase in SCEs over the num-

A. D. Auerbach

28

et al.

normal human peripheral blood lymphoFigure 1 Induction of SCEs in second-division cytes. (A) Untreated control. (B) Treated with 1% rat serum containing activated cyclophosphamide [in vivo activation method). (C) S-9 mix control. (D] Treated with S-9 mix plus 10e5 M cyclophosphamide (in vitro activation method). Arrow indicates quadriradial configuration.

ber in untreated

controls

(Fig.

1A and B). A small

but statistically

significant

(p <

of SCEs over that in control cells (untreated and rat serum-treated) was obtained when cells were treated with serum from rats injected with procarbazine (1: 10 and 1:5 dilutions). These data indicate the presence of activated drug metabolites in the serum of injected rats. 0.01,

p < 0.005)

dose-related

increase

in the incidence

mv)ocL 00-c

dddc

I

s

do

0000

m

3 u

0

30

A . D . Auerbach et al. Lymphocytes from the four F A patients showed an increased " s p o n t a n e o u s " chromosome breakage rate. Treatment of FA cells with serum from rats to w h i c h no drug was a d m i n i s t e r e d resulted in a slight increase in c h r o m o s o m e breakage, whereas c y c l o p h o s p h a m i d e i n d u c e d a manifold increase in c h r o m a t i d breaks and exchanges in l y m p h o c y t e s from F A patients c o m p a r e d with those from n o r m a l controis (Table 1A, Fig. 2A and B). The response of FA cells was similar to that found following culture with DEB [2]. However, little d r u g - i n d u c e d c h r o m o s o m e breakage was detected after exposure of either F A or normal l y m p h o c y t e s to activated procarbazine; the increase in breakage in normal cells was greater than in F A cells (Table 1A, Fig. 2C and D).

Effect of In Vitro Activated Drugs The effect of c y c l o p h o s p h a m i d e and procarbazine, in the presence of S-9 mix, on the SCE frequency of normal cells is shown in Table 2B. S-9 mix alone d i d not affect SCEs, whereas exposure to c y c l o p h o s p h a m i d e (10 -s M) and S-9 mix y i e l d e d a fivefold increase in SCEs (Fig. 1C and D). Treatment of cells with procarbazine (10 -3 M) and S-9 mix resulted in a small but statistically significant increase in SCEs (p < 0.001) (Table 2B). FA cells were again hypersensitive to c y c l o p h o s p h a m i d e - i n d u c e d c h r o m o s o m e breakage. The small increase in breakage seen in F A cells after exposure to procarbazine and S-9 mix did not differ from that observed in similarly treated normal cells (Table 2A). In an attempt to increase the concentration of biologically active procarbazine metabolites to w h i c h cells were exposed, we used its azo derivative, Roche comp o u n d Ro4-8047. A small but statistically significant (p < 0.02) increase in SCEs was seen after treatment of normal (control) cells with 10 -3 M of the chemical, while only a slight increase in chromosome breakage was seen in both F A and normal cells (Tables 3A and B). DISCUSSION The presence of an intrinsic stem cell defect in F A patients is suggested by the results of in vitro bone marrow studies [10,11] and m a y be the basis of the increased risk for the d e v e l o p m e n t of acute n o n l y m p h o c y t i c leukemia in these patients [12]. Bone marrow transplantation offers the potential for e l i m i n a t i o n of the defective stem cells and is the treatment of choice for patients with i d i o p a t h i c aplastic anemia w h o have a suitable donor [13]. The success rate of bone marrow transplantation in n o n - F A patients with aplastic anemia has been a p p r o x i m a t e l y 50-60%; in a series of cases t r a n s p l a n t e d prior to any blood transfusions, the success rate was a p p r o x i m a t e l y 90% for non-FA patients [14]. In contrast, out of 21 patients w i t h F A that have been reported to have had marrow transplants, only 6 have survived [2,5,14-23]. Recently, it has become apparent that this poor outcome is due to the hypersensitivity of F A patients to the pretransplantation conditioning regimen, w h i c h includes i m m u n o s u p p r e s s i o n with high doses of c y c l o p h o s p h a m i d e [5,24]. The aim of these studies was to d o c u m e n t the hypersensitivity of FA patients to c y c l o p h o s p h a m i d e and to compare the sensitivity of patients and normal controls to procarbazine, an alternative i m m u n o s u p p r e s s i v e drug. Because the metabolic activation of procarbazine is c o m p l e x [25], and previous mutagenicity and cytogenetic studies have s h o w n activity m a i n l y in host-mediated [26,27] or in vivo assays [2833], we used both in vivo and in vitro systems of activation. In each of the experiments, c h r o m o s o m e breakage was used as the end point for measuring drug sensitivity, and SCE frequency was used as an indicator for the presence of activated drug metabolites in the in vitro system. The increase in SCEs found in control cells

31

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D

Figure 2 Chromosome breakage in FA and normal peripheral blood lymphocytes exposed to metabolically activated drugs (in vivo activation method). (A) Isochromatid break induced by cyclophosphamide in normal lymphocyte. (B) Multiple chromatid breaks and exchange figure induced by cyclophosphamide in FA lymphocyte. (C) Chromosome ring and fragments in normal lymphocyte treated with procarbazine. (D) Isochromatid break in FA lymphocyte treated with procarbazine.

Case

(in vitro a c t i v a t i o n of drugs)

ap < 0.001. bp < 0.0005.

Normal control

Case

0.30 0.03

0.29 0

S-9 Mix

8.27

Untreated

8.97

11.17 a

+ S-9 mix

P r o c a r b a z i n e (10 3 M)

Mean SCEs per cell

0.44 0.06

Procarbazine (10 -a M) + S-9 mix

B. Sister chromatid exchange

S-9 Mix

Untreated

Mean chromosome breaks per cell

A. Chromosome breakage

Effect of e x p o s u r e to p r o c a r b a z i n e a n d c y c l o p h o s p h a m i d e m e t a b o l i t e s o n F A a n d n o r m a l l y m p h o c y t e s

Fanconi anemia, FA4 (L. G.) Normal control

Table 2

46.5 b

Cyclophosphamide (10 5 M) + S-9 mix

1.78 0.48

Cyclophosphamide (10 5 NI) + S-9 mix

Case

Effect of exposure (in vitro activation

Normal control

Case

Fanconi Anemia, FA5 (H. D.) Normal control

Table 3

to compound of drugs)

B. Sister chromatid

11.60

Untreated

0.37 0.01

A. Chromosome

and cyclophosphamide

Untreated

Ro4-8047

Mean SCE per cell

12.05

S-9 Mix

exchange

0.38 0

S-9 Mix

breaks per cell

on FA and normal

Mean chromosome

breakage

metabolites

15.8"

Compound Ro4-8047 (10m3Ml + S-9 Mix

0.46 0.04

Compound Ro4-8047 (lOm"M) + S-9 mix

lymphocytes

w

w

34

A . D . Auerbach et al. after exposure to metabolically activated procarbazine or c y c l o p h o s p h a m i d e confirmed the presence of drug metabolites in the cultures. Similar SCE frequencies were obtained w h e n cultures were exposed to drugs activated using either the in vivo or in vitro system. Procarbazine and its azo derivative were found to be m u c h weaker inducers of SCEs than c y c l o p h o s p h a m i d e ; it was necessary to use a m u c h higher concentration of procarbazine than c y c l o p h o s p h a m i d e in order to demonstrate an effect on SCEs. Procarbazine was also a weaker clastogen than cyclophosphamide. At procarbazine concentrations that i n d u c e d significant increases in SCEs and had some clastogenic effect on normal cells, there was little increase over baseline levels of breakage in F A cells. Thus, although F A cells were h y p e r s e n s i t i v e to the clastogenic effect of c y c l o p h o s p h a m i d e , they were not more sensitive than normal cells to p r o c a r b a z i n e - i n d u c e d c h r o m o s o m e breakage. Again, data from three i n d e p e n d e n t experiments, using different systems of metabolic activation (in vivo and in vitro), were similar. The results of these studies indicate that procarbazine m a y prove to be a safer drug than c y c l o p h o s p h a m i d e for i m m u n o s u p p r e s s i o n of F A patients in preparation for bone marrow transplantation. It has previously been used successfully in conditioning regimens for marrow transplantation for severe aplastic anemia, in conjunction with a n t i t h y m o c y t e globulin and c y c l o p h o s p h a m i d e (the PAPAPA-CY regime) [16], and w i t h antithymocyte globulin and total b o d y irradiation for severe neutrophil dysfunction [34], the W i s k o t t - A l d r i c h s y n d r o m e [35], and congenital agranulocytosis [36]. The lack of hypersensitivity of F A cells to the clastogenic effect of procarbazine, c o m p a r e d to c y c l o p h o s p h a m i d e , has led the Memorial Sloan° Kettering t r a n s p l a n t a t i o n team to propose a conditioning regimen for F A transplantation that i n c l u d e s procarbazine (12.5 mg/kg/day) a d m i n i s t e r e d in three doses over 6 days, alternating with three doses of antithymocyte globulin (15 mg/kg/day) and followed by low-dose fractionated total b o d y irradiation (700 rads). In the initial clinical experience with a similar protocol, in one patient treated, toxicity was limited to m i l d acute nausea and vomiting. This course was in marked contrast to the severe mucositis, enteritis, and h e p a t i t i s observed in o w n previous experience and that reported by G l u c k m a n et al. [5] with F A patients p r e p a r e d for transplantation with c y c l o p h o s p h a m i d e . In our patient prepared for transplantation with procaro bazine, antithymocyte globulin, and irradiation (300 fads), engraftment was prompt. Unfortunately, the patient died of a s u d d e n intracerebral hemorrhage, w i t h o u t concurrent GVH disease or infection, 3 weeks after transplantation. Further studies of the effectiveness of this protocol in FA are u n d e r way.

ADDENDUM

Two additional F A patients have received transplants using this protocol. One month after transplantation they are both fully engrafted, w i t h cytogenetics showing all donor cells in the bone marrow. Toxicity, attributable to the c o n d i t i o n i n g regimen, was limited to m i l d nausea in one of the patients, and m i l d transient oral mucositis w i t h m i l d diarrhea in the other.

Supported in part by National Cancer Institute Contract CP-85665 and National Cancer Institute Grants CA 29944, CA 23766, and CA 19267; and the Charles A. Dana Foundation, Robert J. Kleberg and Helen C. Kleberg Foundation, Lila Acheson and DeWitt Wallace Fund. We thank Dr. W. E. Scott of Hoffman-LaRoche, Inc., for supplying us with procarbazine hydrochloride and its azo derivative Ro4-8047.

Drug-Induced Chromosome Breakage in FA

35

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