Improved prognosis in mice with advanced myeloid leukemia following administration of GM-CSF and cytosine arabinoside

Leukemia Research Vol. 15, No. 5, pp. 321-325, 1991.

(H45 -2126/91 $3.00 + .00 Pergamon Press pie

Printed in Great Britain.

IMPROVED PROGNOSIS IN MICE WITH A D V A N C E D MYELOID L E U K E M I A FOLLOWING ADMINISTRATION OF GM-CSF AND CYTOSINE ARABINOSIDE Z I N A BEN-ISHAY, G R E G O R PRINDULL* a n d SARA SHARON

Laboratory of Experimental Hematology, Department of Anatomy and Embryology, ttebrew University-Hadassah Medical School, Jerusalem, Israel and *Department of Pediatrics, University of Gottingen, D-3400 Gottingen, F.R.G. (Received 15 June 1990. Revision accepted 29 October 1990)

Abstract--Acute myeloid leukemia (AML) was induced in C57B1mice through the i.v. innoculation of C-1498 cell line. One week later, i,e. at mid-term disease, the leukemic mice received an i.p. injection of 200 ng rmGM-CSF and 24 h later, two consecutive i.p. cytosine arabinoside (ara-C) injections at 6h intervals (2 × 200 mg/kg). The leukemic mice received 3-4 weekly courses of combined therapy and survived 4-5 weeks following leukemia induction. Control mice received araC only and survived 2-3 weeks. Moreover, leukemic mice administered both GM-CSF and ara-C had a lower marrow leukemic load than mice treated with ara-C only. From these findings, we conclude that therapy of murine AML with combined rmGM-CSF and ara-C is more effective than ara-C only. Leukemic mice treated with GM-CSF and ara-C had a longer life expectancy and a smaller leukemic load than mice administered ara-C only. Key words: Murine AML, rmGM-CSF, ara-C, CFU-C, CFU-D, L-CFU.

are subsequently killed by ara-C, a cycle-specific drug. In preliminary studies on C57BI mice with A M L induced by inoculation of acute myeloid leukemia cells of the C-1498 type, we observed that administration of rmGM-CSF results in a higher marrow leukemic load and a shorter life span than the untreated leukemic mice (unpublished observations). We therefore assumed that the C-1498 leukemic blasts become stimulated by GM-CSF. To test the hypothesis of improved eradication of growth factor stimulated leukemic blasts by subsequent administration of ara-C, we treated leukemic mice with rmGM-CSF followed by ara-C. We report here on improved prognosis of leukemic mice receiving combined GM-CSF and ara-C treatment compared to mice treated with ara-C only.

INTRODUCTION

BIOLOGICALLYengineered growth factors offer a new modality for treatment of neutropenia in cancer patients receiving X-irradiation and chemotherapy, in cases of poor or delayed engraftment following bone marrow transplantation, under conditions of immune suppression, etc. [l, 2]. Attempts at stimulation of normal myelopoiesis in acute myeloid leukemia (AML) might prove hazardous due to the simultaneous stimulation of leukemic blasts by factors such as G-CSF, GM-CSF and IL-3 [3-14]. In vitro studies of A M L cells have indicated that preexposure of leukemic blasts to G-CSF, GM-CSF or IL-3, sensitizes the cells to the killing effect of cytosine-arabinoside (ara-C) [15-17]. Occurrence of receptors for these factors on A M L blasts account for stimulation and triggering of non-cycling cells into the cell cycle [11-14]. Cycling leukemic blasts

MATERIALS AND METHODS

Correspondence to: Prof. Zina Ben-Ishay, Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School, Jerusalem, Israel. Abbreviations: A M L , acute myeloid leukemia; rmGMCSF. recombinant murine granulocyte/macrophage colony-stimulating factor: ara-C, cytosine arabinoside; CFUC, colony-forming units granulocytes/macrophages; LCFC, leukemic colony-forming cells; CFU-D, colony-forming units in peritoneal diffusion chamber cultures.

Animals and leukemia induction Male C~7 BI mice, aged 4--6weeks, were used throughout the study. AML was induced by the i.v. (the tail vein) inoculation of 105C-1498 myeloid cells. The C-1498 cells were obtained from the National Cancer Institute, Frederick Cancer Research Facility, Tumor Bank Repository, Frederick, MD, U.S.A. The disease is propagated by the intraperitoneal (i.p.) injections of ~ 106 AML cells from the peritoneal fluid of

321

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Z . BEN-ISHAY et al.

sick mice. As described (18), the i.p. induced disease consists of peritoneal solid tumors and a bone marrow load of 10-20% leukemic cells. All mice with the i.p. induced disease die within 7-9 days after cells inoculation. To induce a slower progressing disease, 105 AML cells were injected i.v. In this model, the mice do not develop peritoneal tumors while the marrow leukemic load reaches 30--40% on days 12-14, by the time of the fatal end [19, 20]. A total of 100 leukemic mice received combined rmGMCSF and ara-C treatment, 100 mice received only ara-C and 10 normal mice were used as controls.

Treatment Combined rmGM-CSF and ara-C administration. Recombinant murine GM-CSF was obtained as a gift from Dr J. J. Mermod, Glaxo Comp., Geneva, Switzerland. The specific activity of rmGM-CSF is 5.5 x 10 7 U/mg. On a dose-response curve using unseparated bone marrow cells (105/ml medium), maximal G/M colony stimulation was obtained with 200 ng rmGM-CSF. The leukemic mice received a weekly i.p. injection of 200 ng rmGM-CSF in 0.2 ml saline on days 6, 12 and 20 following leukemia inoculation. Twenty-four hours after each growth factor injection, the mice received two consecutive i.p. ara-C injections at 6 h interval (2 × 200 mg/ kg). Ara-C administration only. Two consecutive i.p. injections of ara-C (2 × 200 mg/kg) 6 h apart were given on days 7 and 13, and in the few surviving mice, on day 21. Eighty to ninety per cent of ara-C treated mice died 15-16 days after leukemia induction. Those which survived longer than that and received the third ara-C course, generally died soon after treatment. Mice in both groups were killed 2 days after the second course, and the bone marrow was obtained for study. Total and differential marrow cells counts were carried out on each specimen prior to plating in semi-solid agar cultures and in peritoneal diffusion chamber (DC) cultures. In addition, bone marrow counts and CFU-C assays were carried out four weeks after leukemia induction in a group of mice receiving three courses of combined therapy. CFU-C and L-CFC. Bone marrow cells were flushed out of femuri and single cell suspensions were prepared by repeated syringing of cells in culture medium. A total of 2 X 104-5 X 10 4 cells were plated in 3 cm diameter Petri dishes in 1 ml Iscove modified Dulbecco's medium (IMDM) (Gibco, Grand Island, N.Y.), with 20% foetal calf serum (FCS) (Gibco), 0.5% agar solution and 200 ng rmGM-CSF (Glaxo). Marrow cultures from control mice were set with each experimental group (105cells/ml medium). All cultures were continued for seven days (37°C, 5% CO2 in air and high humidity), and G/M colonies were counted by inverted microscope. Leukemic colonies were distinguished by several characteristics: generally piled up compact colonies containing uniformly sized cells with no signs of differentiation. A few whole agar cultures were processed for light microscopy (i.e. fixation with buffered formalin and staining by hematoxylin solution) and the leukemic origin of colonies was confirmed. Peritoneal DC cultures (the plasma clot diffusion chamber technique). The stem cells giving size to colonies in DC are of "young type", probably the equivalent of 12-day CFUS and CFU-Mix [21-24]. The DC technique had been described elsewhere [21, 22]. In the DC preparations for light microscopy one can readily distinguish between hematopoietic colonies (neutrophilic, megakaryocytic,

uGM-CSF + ara- c

Aoro- c

f(X)

0~)

- KX)

I

7

I

14

21

28

Do~

FIG. 1. Effect of combined administration of (A) rmGMCSF and ara-C and (B) ara-C only on survival of mice inoculated with C-1498 AML cells. Leukemic cells were inoculated on day 0. One hundred mice of group (A) received rmGM-CSF as single i.p. injections on days 6, 13 and 20 and two consecutive i.p. ara-C injections on days 7, 14 and 21. One hundred mice of group (B) were administered ara-C similarly to group (A)

occasional erythroid) and leukemic cell colonies by the morphological characteristics described above. RESULTS

Effect of combined administration o f rmG M-CS F and ara-C on survival o f mice with A M L In the present A M L model, treatment of leukemic mice was started at mid-disease, namely one week after C-1498 cell inoculation, the life expectancy of untreated mice being 12-14 days. The rationale for the late start of treatment was a better chance to assess results of different therapies in advanced A M L rather than at start of the disease. Figure 1 indicates a longer survival of mice administered combined therapy comparied to mice receiving ara-C only. In the first group, 80% of mice survived 3 weeks after leukemia induction and the remaining 20% were alive 4 weeks after disease initiation. All mice in this group received three courses of combined therapy. In contrast, 80-90% of mice treated only with ara-C died before or soon after the second course. Bone marrow assays were carried out in the few mice surviving the second ara-C course. Bone marrow differential counts These were carried out 2 days following the second course in both groups, i.e. combined therapy and ara-C only. Table 1 indicates a leukemic load of ~ 1 3 % , normal myelopoiesis of ~ 8 0 % (of which 75% were young types) and early erythroblasts (---5%) in mice receiving combined therapy. In contrast, mice receiving ara-C only had ~ 2 4 % leukemic ceils and

T r e a t m e n t of leukemia with r m G M - C S F and ara-C

323

TABLE 1. DIFFERENTIAL BONE MARROW COUNTS IN LEUKEMIC MICE, 2 DAYS FOLLOWING THE SECOND COURSE OF (A) rmGMCSF AND ara-C (B) ara-C ONLY.TOTAL CELLSPER FEMUR IN GROUP (A) WAS ~-6 X 106 AND IN GROUP ( B ) , ~4 × 106. ( C ) INDICATES DIFFERENTIAL MARROW COUNTS, 7 DAYS AFTER THE THIRD COURSE IN MICE RECEIVING COMBINED ADMINISTRATION OF r m G M - C S F AND a r a - C (20 MICE); TOTAL CELLS PER FEMUR IN THESE MICE WAS ~ 1 5 × 106

Erythroid nucleated cells

Myeloid cells

Replicative forms: myeloblasts, No. of mice Replicative Non-replicative promyeiocytes, studied forms forms myelocytes (A~ 50

5%

0

(B) 10 (C) 20

0 14%

0 2%

Non-replicative forms: metamyelocytes, granulocytes

75% (p < 0.01) 26% 51%

5% (p < 0.001) 44% 21%

Lymphocytes Leukemic ceils 2% (n.s.) 6% 2%

13% (p < 0.05) 24% 10%

TABLE 2. ASSAYS OF MARROW CFU-C, C F U - D AND L - C F U IN LEUKEMIC MICE. 2 DAYS AFTER THE SECOND COURSE OF r m G M - C S F AND a r a - C IN 50 MICE. a r a - C ONLY IN 10 MICE ( A , B ) , AND C F U - C AND L - C F U , 7 DAYS FOLLOWING THE THIRD COURSE OF COMBINED THERAPY IN 20 MICE ( C )

CFU-D per femur No. of mice studied

Treatment

(A) 50

GM-CSF + ara-C

(B) 10 (C) 20 10

ara-C GM-CSF + ara-C Normal mice (untreated)

Total nucleated cells per femur

L:CFU* per femur

Normal hematopoietic stem cells*

22000 -+ 750

240 +-- 27

10200 -+ 940

2400 +- 220

(p < 0.05)

(p < 0.001)

(n.s.)

(p < 0.005)

~4 x 106

16000 -+ 630

8000 +- 580

12800 -+ 870

9000 +- 980

~15 × 106

30600 +- 1700

1200 +- 90

~15 × 106

10000 +- 420

0

~6 X 106

CFU-C per femur

L-CFU in DC~t

not done 20000 -+ 1500

0

* Leukemic cell colonies in semi-solid agar cultures were observed by inverted microscope upon their characteristics, i.e. large, compact colonies containing piled up cells of uniform size with no signs of differentiation. t Hematopoietic colonies in DC included neutrophilic, megakaryocytic and occasional erythroid types. ~t Leukemic cell colonies in DC preparations were readily recognized by their morphological characteristics, i.e. largesize undifferentiated cells.

~ 7 0 % normal myeloid cells (of which only ~ 2 6 % were young types) while nucleated erythroid cells were absent. T a b l e 1 also indicates the m a r r o w counts in mice surviving ~ 2 8 days following r m G M - C S F and ara-C administration. A t this time, the leukemic m a r r o w load was 10% and the myelopoiesis and erythropoiesis were well represented.

Effect of rmGM-CSF and ara-C treatment on CFUC, CFU-D and L-CFU in leukemic mice Table 2 shows the data on C F U - C progenitors, C F U - D stem cells and leukemic colony-forming cells (L-CFU) in treated leukemic mice. T h e r e was a slight difference of C F U - C progenitors in the two differently treated groups, i.e. ~22000/mur with

combined therapy and ~ 16000 with ara-C only. C F U C was seen to increase in n u m b e r one week after the 3rd course of combined therapy. It is noteworthy that C F U - C values were higher in treated leukemic mice than in normal controls. Increased numbers of C F U - C with high replicative capacity occur in regenerative marrow following ara-C administration [25]. This may account for the high incidence of C F U C progenitors in both groups of treated leukemic mice receiving ara-C. Further, there was no significant difference of C F U D in the two treated groups; however, these stem cells were significantly lower in leukemic mice than in controis (10-12 x 104 vs 20 x 104 per femur). A significant finding was the lower level of L - C F U

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Z. BEN-IsHAYet al.

in agar cultures and in DC in the group of mice treated with rmGM-CSF and ara-C (~240 and ~2400/femur, respectively) than in mice treated with ara-C only (~8000 and ~9000/femur, respectively).

DISCUSSION In the present investigation we aimed to shed additional light on possible hazardous or, alternatively, beneficial effects of growth factors in the treatment of AML. Treatment of leukemic mice was started one week after cell inoculation, namely midway between disease induction and expected death. The purpose of the late start ~t~ treatment was to mimic as closely as possible the course of leukemia in humans, in which the marrow leukemic load is generally high at the time of diagnosis. In recent reports on murine B-cell leukemia and myeloid leukemia, growth factors were administered soon after cell inoculation [26, 27]. Such experimental models are apparently aimed at demonstrating possible in vivo effects of growth factors in leukemia rather than simulating more closely the human condition. In vitro studies on human leukemic cells had earlier indicated that they were highly stimulated by GMCSF, G-CSF and IL-3 due to occurence of membrane receptors for these factors [11,12]. In fact, the leukemic blasts express both receptors and growth factors functioning in an autocrine manner [28, 29]. Recent studies showed that in vitro preexposure of leukemic blasts to growth factors and subsequent exposure to cytosine arabinoside eradicate larger number of cells than exposure to ara-C only [15-17]. The present investigation is an attempt at combining growth factor and cytotoxic drug administration in murine A M L based on in vitro results of a similar schedule. The data obtained had indicated that the life expectancy of leukemic mice receiving combined treatment was higher than of mice treated with ara-C only. The former generally survived 3-4 weeks after leukemia induction and received 3-4 therapy courses, while most mice in the second group died before the third week. Furthermore, mice receiving both rmGM-CSF and ara-C had significantly lower marrow L-CFU than those receiving ara-C only. The C F U - D assay also showed lower LCFU incidence in mice which received combined treatment than in mice treated with ara-C. In addition, results of DC cultures indicated a smaller pool of C F U - D stem cells in leukemia than in controls, reflecting the reduced fraction of normal hematopoiesis in this disease. It is worth noting that the leukemic mice receiving combined therapy showed active bone marrow erythropoiesis while in

the group administered only ara-C the erythropoiesis was absent (Table 1). In a recent study, Hoelzer et al. [30] reported encouraging results in patients with myelodysplastic syndrome treated with rmGM-CSF and ara-C. In view of data reported in our animal model and based upon the report by Hoelzer et al., it is possible that administration of growth factors and ara-C in human leukemia might prove advantageous rather than hazardous as generally feared.

Acknowledgements--This study was supported by a research grant donated to the Ministry of Health, Israel, in memory of Michael Samitca. We are indebted to Dr J. J. Mermod, from the Glaxo Comp., Geneva, Switzerland, for generously providing rmGM-CSF as a gift. The authors are grateful to Mrs Yehudith Goldstein for skilled technical help.

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