Cell differentiation and therapeutic effect of low doses of cytosine arabinoside in human myeloid leukemia

Leukemta Re.~eorch Vol. 8. No 5. pp, 783-790. 1984. Printed in Great Britain

0145-2126/8453.00 + 0.00 ;,~, 1984 Pergamon P r ~ Lid

CELL DIFFERENTIATION AND THERAPEUTIC EFFECT OF LOW DOSES OF CYTOSINE ARABINOSIDE IN HUMAN MYELOID LEUKEMIA RITA MICHALEWICZ, JOSEPH LOTEM* and LEO SACHS* Hematology Unit, Medical Center of Tel Aviv, lchilov Hospital, Tel Aviv 64239 and the *Department of Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

(Received 18 November 1983. Accepted in revised form 15 March 1984) Abstract--Bone marrow cells from 2 patients over 60 years of age with acute myeloblastic (AML) or monoblastic (AMoL) leukemia were cultured in the presence of a low dose of cytosine arabinoside. In the cells from the AML patient this treatment induced differentiation to metamyelocytes and a decrease in the number of blasts, so that there was an I I-fold increase in the ratio of differentiated myeloid cells to blasts. In the patient with A M o l there was differentiation to monocytes and macrophages and only a 3-fold increase in the ratio of differentiated mydoid cells to blasts. In the latter patient actinomycin D was a more potent inducer of differentiation than cytosine arabinoside, daunomycin was similar to cytosine arabinoside and adriamycin showed the lowest response. Four courses of low dose treatment with cytosine arabinoside produced remission in the patient with AML and in another patient with AMoL whose cells were not tested in culture. No remission was induced by this low dose treatment in the patient with AMoL whose cells showed only a small decrease in blast cells in culture with cytosine arabinoside, it is suggested that prescreening for effective compounds in patients with myeloid leukemias and the use of low dose therapy can be of help in obtaining remission without serious side effects. This could be eslxcially useful in patients where there may be severe toxic effects after high dose chemotherapy.

Key words: Induction of differentiation, low dose therapy, pre-screening in culture, human myeloid leukemia, cytosine arabinoside.

INTRODUCTION EXPERIMENTS with human and mouse myeioid leukemic cells in culture have shown that some clones can be induced to differentiate to mature macrophages or granulocytes by the normal macrophage and granulocyte differentiation-inducing protein which we now call MGI-2. These clones, as well as clones that are not inducible by MGI-2, can be induced to partially or completely differentiate to mature macrophages or ganuiocytes by low doses of certain compounds presently used in cancer chemotherapy ]reviewed in 4, 15, 19, 20, 22]. As a result of these experiments we have previously suggested that it may be possible to introduce a form of therapy based on induction of differentiation. This would include pre-screening in culture to select for the most effective compounds, and using these compounds for a low-dose chemotherapy protocol aimed at inducing cell differentiation [15, 18-21]. Different myeloid leukemic clones respond differently in culture and in vivo to MGI-2 and other compounds [6, 12, 13, 16, 18, 20-22] and such differences can also be expected to occur in leukemic cells from different patients [15, 18, 20, 21]. Based on these suggestions, encouraging clinical results were obtained with the use of low dose cytosine arabinoside treatment of some patients with refractory anemia with excess blasts [1, 3] and acute myeloid leukemia [3, 10]. Other trials with low dose cytosine arabinoside did not show an improvement in patients with refractory anemia [5] or myeloid leukemia [9]. Cytosine arabinoside was chosen for low-dose chemotherapy Abbreviattons: AML, acute myeloblastic leukemia; AMoL, acute monoblastic leukemia; PMN, polymorphonuclear cells; MGI-2, macrophage and granulocyte differentiation-inducing protein. Correspondence to: Prof. Leo Sachs, Department of Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. 783

784

RITA MICHALEv,I( 7, JOSEPH LOFFM and LEo Sa(Hs

w i t h o u t t e s t i n g its d i f f e r e n t i a t i o n - i n d u c i n g a b i l i t y in cells o f t h e s e p a t i e n t s . It is t h e r e f o r e p o s s i b l e t h a t cells o f the n o n - r e s p o n d i n g p a t i e n t s m i g h t a l s o h a v e b e e n n o n - r e s p o n s i v e to c y t o s i n e a r a b i n o s i d e in c u l t u r e , b u t m a y h a v e r e s p o n d e d to o t h e r c o m p o u n d s . We now present data for 2 patients with myeloid leukemia (acute myeloblastic and a c u t e m o n o b l a s t i c l e u k e m i a ) w h o s e cells w e r e tested f o r d i f f e r e n t i a t i o n in c u l t u r e a n d a t h i r d p a t i e n t w i t h a c u t e m o n o c y t i c l e u k e m i a w h o s e cells w e r e n o t c u l t u r e d . T h e 3 p a t i e n t s w e r e t r e a t e d w i t h a low d o s e o f c y t o s i n e a r a b i n o s i d e a n d the results s h o w that 2 o u t o f t h e s e 3 p a t i e n t s e n t e r e d r e m i s s i o n w h e r e a s t h e r e was n o i m p r o v e m e n t in the t h i r d p a t i e n t . T h e t h i r d p a t i e n t ' s cells a l s o o n l y s h o w e d a s m a l l e f f e c t o f c y t o s i n e a r a b i n o s i d e in c u l t u r e . It is h o p e d t h a t l a r g e r scale trials i n c l u d i n g p r e - s c r e e n i n g o f the p a t i e n t ' s cells will be c a r r i e d o u t to o b t a i n a b r o a d e r e v a l u a t i o n o f this t y p e o f t r e a t m e n t . PATIENTS

AND METHODS

Patients Patient MS : A 75-year-old woman was admitted to hospital because of high fever and leukocytosis lasting t~o weeks. Examination of the peripheral blood showed anemia (Hb 8 g/dl), leukocytosis (WBC 42 × 10'/I with 15% polymorphonuclear cells, 51% monocytes, 10% lymphocytes and 24% a-naphthyl-acetate esterase positive blasts) and thrombocytopenia (platelets 6 x I0'/I). The bone marrow showed an acute monoblastic leukemia (AMoL) with 90% ct-naphthyl-acetate-esterase positive monoblasts. Patient LR : A 73-year-old woman with a one year history of untreated refractory anemia with a slight excess of blasts was admitted to hospital with symptoms of severe weakness and echymosis. Examination of peripheral blood showed anemia (Hb 7.5 g/dl), leukocytosis (WBC I I x 10'/I with 48~70 polymorphonuclear cells, 35% monocytes, 15'/0 lymphocytes and 2% Q-naphthyl-acetate-esterase positive blasts) and thrombocytopenia (platelets 7 x 10'/I). The bone marrow showed acute monoblastic leukemia with 25070 a-naphthyl-acetateesterase positive blasts. Patient KE : A 65-year-old man was admitted to hospital because of pancytopenia. Examination of his peripheral blood showed anemia (Hb 9.3 g/dl), leukopenia (WBC 3.3 x 10'/1 with 40~0 polymorphonuclear cells, 6~, monocytes and 54% lymphocytes) and thrombocytopenia (platelets 20 × 10'/I). The bone marrow showed acute myeloblastic leukemia (AML) with 30% naphthol AS-o-chloracetate-esterase positive blast cells. Cell cultures Bone marrow was aspirated in heparin, diluted to 8 ml with phosphate-buffered saline pH 7.4 and separated by FicolI-Hypaque density centrifugation [2] for 30 min at 400 g at room temperature. The cells at the interface were collected and washed twice in 10 ml RPMI 1640 medium [Grand Island Biological Co. (GIBCO), Grand Island, New York] with 20% fetal calf serum (GIBCO). The cells were seeded at 1 × 10' cells per 35 mm tissue culture Petri dish (Falcon Plastics, Oxnard, CA) in 2 ml culture medium containing RPMI 1640. 20% fetal call serum and 10~0 human leukocyte conditioned medium. The human leukocyte conditioned medium was obtained 48 hours after seeding normal human peripheral blood leukocytes at 5 x 10' cells per ml in RPMI 1640 with 20~0 fetal calf serum, 0.05 ml PHA-M (Difco Lab.. Detroit, MI) per ml and 5 x I0-~ M mercaptoethanol. The bone marrow cells were incubated for 4 days at 37°C and were then counted and analysed for cell morphology and the production of lysozyme. The materials tested for induction of differentiation were: cytosine arabinoside (Sigma Chemical Co., St. Louis, MI), actinomycin D (Cal Biochem. San Diego, CA), adriamycin (Farmitalia Carlo Erba, Milan, Italy), and daunomycin (Specia, Paris) and the concentrations tested were like those previously used with human HL-60 leukemic cells [15]. Cell morphology and production of lysozyme The number of cells with different morphologies before and after culture was determined on cytocentrifuge smears stained with May-GrOnwald°Giemsa by counting 300 cells. The number of myeloblasts and promyelocytes was combined and referred to as blasts. Lysozyme activity within the cells before and after culture (intracellular) and in the culture medium of the cultured cells was determined by a turbidometric method with Micrococcus lysodeikticus [8] using hen egg-white lysozyme as standard, as previously described [I ll. The total amount of lysozyme is expressed as I,tg equivalent per 5 x 10' cells. The variations in the assays were up to _+ 15% of the values given.

RESULTS T r e a t m e n t o f s o m e p a t i e n t s w i t h a c u t e m y e i o b l a s t i c l e u k e m i a w i t h a low d o s e o f c y t o s i n e a r a b i n o s i d e has p r e v i o u s l y s h o w n t h a t it was p o s s i b l e to o b t a i n r e m i s s i o n that lasted f o r s e v e r a l m o n t h s . T h i s t r e a t m e n t c o n s i s t e d o f 2 d a i l y s u b c u t a n e o u s i n j e c t i o n s o f 10 m g / m 2 c y t o s i n e a r a b i n o s i d e ( e v e r y 12 h o u r s ) f o r 17-25 d a y s [3, 101. W e h a v e n o w f o u n d t h a t r e m i s s i o n was a c h i e v e d in a 7 5 - y e a r - o l d p a t i e n t w i t h a c u t e m o n o b l a s t i c

Cell differentiation and low dose therapy in human myeloid leukemia 100

o

z

~

785

A.

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O

~ 3 01 e I , 7"~...AIO 20 40 60 20 40 60 No. of cloys ofter b e g i n n i n g of t r e a t m e n t

Fie,. I. Blood composition in patient MS (AMoL) treated with low dose cytosine arabinoside. (A) Number of white blood cells (WBC), platelets, and amount of hemoglobin (Hb) were determined at the beginning of treatment and then 14 and 56 days later. Cytosine arabinoside (40 rag/day) was given for 4 successive days at 3 week intervals, starting on days I, 22 and 43. (B) Blood differential counts v,ere carried out on the same days as in (A). PMN, polymorphonuclear cells; Ly, lymphocytes; Mo, monocytes; BI, monoblasts.

leukemia (patient MS) who was injected subcutaneously with 3 courses of low dose cytosine arabinoside (40 mg cytosine arabinoside per day for 4 successive days). The time between each course of treatment was 3 weeks. This patient's clinical status improved already l0 days after the first 4-day course (Fig. l) and remission was achieved after the third course. The patient continued to receive one 4-day course of low dose cytosine arabinoside every month and the remission lasted for l I months, at which time the patient died from urinary sepsis without any signs of leukemic involvement o f bone marrow. Since we had not cultured this patient's cells before treatment, we decided to test the ability of the bone m a r r o w cells to be induced to differentiate in culture by a low dose of cytosine arabinoside in 2 other newly diagnosed leukemic patients. At the same time these patients were treated with low dose cytosine arabinoside by the same schedule as the patient described above. The results have shown that in a 65-year-old patient with acute myeloblastic leukemia (patient KE), the total number of cells was 15o70 less in cells cultured with cytosine arabinoside compared to cells cultured without this c o m p o u n d (Table 1). This small decrease in the total number of cells was associated with a 2-fold increase in the total number of metamyeiocytes (Table 1, Fig. 2B). Unlike monocytes and macrophages, these cells did not attach to the Petri dish. This differentiation was accompanied by a 6-fold decrease in the number of myeioblasts and promyelocytes (these 2 types of cells will be referred to as blasts) and there was an I l-fold increase in the ratio of differentiated myeloid cells to blasts (Table l). The results, therefore, indicate that the decrease in the number of blast cells included their differentiation to metamyelocytes. The culture of cells without cytosine arabinoside also showed some increase in metamyelocytes c o m p a r e d to cells that had not been cultured, but this increase was lower than with cytosine arabinoside, and there was no decrease in the number of blasts (Table 1). Four courses of a low dose of cytosine arabinoside injected subcutaneously (50 mg per day for 4 days). 3 weeks apart, resulted in a partial remission in this patient (KE) with 5-7°7o bone marroxv blasts and an increase in the number of platelets from 20 x l0 ~ to 50 x 109/l. This patient was still in remission 14 months after the beginning of treatment.

73F

L.R

AMoL

AML

Type o f leukemia

Before: culture Af¢¢r culture None AraC (100) A c t . D (5) Dauno.(30) Adria.(50)

Before culture After culture None AraC (100)

Compound added to culture (ng/ml)

5.5 2.2 0.2 I. I 3.4

I 1.0 9.4 8.6 7.6 9.8

2.6 3.0 1.3 1.4 0.9

2.1

i .4 2.2

1.6

Myelocytes

0.5 0.6 0.7 0.6 0.1

0.8

0.8 1.6

0.3

Metamyelocytes

0.5 0.7 I. I 0.6 0.6

1.3

0.5 0.7

0.7

PMN

0.7 2.0 4.0 2.2 2.8

0.2

0 0

0

Monocytes and macrophages

0.4 0.3 0.4 0.9 0.9

0.8

3.3 2..5

3.0

Lymphocytes

0.8 0.6 0.9 0.8 I.I

1.3

0.4 0.2

2.3

Erythroid precursors

18.6 36.4 44.9 43.8 38.3

14.3

NT NT

NT

0.8 2.9 35.5 4.4 1.3

0.9

! .0 I 1.0

1.2

LysoRatio of zyme differentiated myeloid cells to blasts

5 x 10 ~ cells/ml in 2 ml m e d i u m / 3 5 m m Petri dish and cultured for 4 days in RPMI 1640 medium from h u m a n P H A treated leukocytes. Induction o f differentiation by cytosine or d a u n o m y c i n (Dauno.) was measured as described in Patients and Methods. NT, not I0' cells. Differentiated myeloid cells includes myelocytes, metamyelocytes, polymorpho-

5. I

2.6 0.4

9.0 7.6 I 1.6

2. I

Blasts

10.0

Total no. o f cells

Bone marrow cells were separated on FicoiI-Hypaque, seeded at culture medium with 20~/0 fetal calf serum and 10~0 conditioned arabinoside (AraC), actinomycin D (Act.D), adriamycin (Adria.) tested. The a m o u n t of lysozyme is given as pg equivalent per 5 x nuclear cells (PMN), monocytes and macrophagcs.

65M

Age/sex

KE

Patient

No. o f cells per Petri dish ( x 10 ')

TABLE i . INDUCTION OF DIFFERENTIATION IN CUI.TURES OF BONE MARROW CELLS FROM MYEI.OID LEUKEMIC PATIENTS

z

ea

to

.r

"r'

--4 3."

u

Fro. 2. Induction of differentiation in culture of cells from patients KE (AML) and LR (AMol). Cells from patient KE (A) before and (B) 4 days after the addition of 100 ng/ml cytosine arabinoside. Cells from patient LR (C) before and (D) 4 days after the addition of 5 ng/ml actinomycin D. (A-D) x 670.

787

Cell differentiation and low dose therapy in human myeloid leukemia

789

The third patient's cells (a 75-year-old patient, LR, with acute monocytic leukemia) showed differentiation in culture to monocytes and macrophages. However, in contrast to the patient KE where culture with cytosine arabinoside induced an 11-fold increase in the ratio of differentiated myeloid cells to blasts, in this patient there was only a 3-fold increase in the ratio (Table 1). The testing of other compounds in culture has shown that there was a much better effect in patient LR with a low dose of actinomycin D (5 ng/ml) than with cytosine arabinoside. Daunomycin gave similar results to those with cytosine arabinoside and adriamycin produced the lowest effect (Table 1). All compounds showed a similar total number of cells in the cultures (Table 1). All the compounds tested also induced similar amounts of lysozyme (Table 1) and we have already shown that synthesis of lysozyme can be induced without apparent morphological differentiation [15]. In patient LR, like in patient KE, an increase in the ratio of differentiated myeloid cells to blasts was due to an increase in the total number of differentiated cells and a decrease in the total number of blasts (Table 1). In patient LR, except for an increase in the number of platelets from 7 x 109 to 30 x 10~/1, there was no improvement in the patient's condition even after 4 courses of treatment with a low dose of cytosine arabinoside (40 mg per day).

DISCUSSION The present results show that a low dose of cytosine arabinoside can induce differentiation in culture and can affect to different degrees, depending on the patient, the number of blasts in cultures of bone marrow cells from patients with acute myeloid leukemia (myeloblastic and monocytic). The successful induction in vivo of remission by this compound used at a low dose may thus also involve the induction of differentiation. This strengthens our previous suggestion to use low dose chemotherapy based on the differentiation-inducing potential rather than only on the cytotoxic effect of currently used chemotherapeutic agents [ 15, 20, 21 ], although a combination of both effects may be necessary. The present and other encouraging reports on the use of low dose cytosine arabinoside in treatment of patients with acute myeloid leukemia [3, 10] or refractory anemia with excess of myeloblasts [1, 3], make this type of treatment a promising alternative to the high dose treatment at least in some patients with these disorders. However, the present results as well as others [5, 9] also indicate that the use of a low dose of cytosine arabinoside does not induce remission in every treated patient. This treatment should therefore be considered after in vitro data have established that the cells respond to the compound to be used. The use of low doses of other compounds such as actinomycin D, which is not currently used in myeloid leukemia therapy but has been used with other tumors [7] and is a potent inducer of differentiation in vitro in mouse myeloid leukemia [12, 20] and erythroleukemia cells [17, 24] the human myeloid leukemic cell line HL-60 [4, 14, 15] and in cells of a patient such as the one in our present study (patient LR), should be considered especially when low dose cytosine arabinoside treatment has failed. Anthracyclines different from adriamycin or daunomycin, that induce differentiation in the human HL-60 cells [23], may be of help in some of these patients. With the currently used most successful high dose protocols remission is not always achieved [25, 26] and such patients may also be candidates for the use of the appropriate low dose treatment. A low dose of cytosine arabinoside produced remission in patients over 60 years of age with acute myeloid and acute monocytic leukemia. In view of the higher incidence of severe complications and death during current high dose protocols for leukemic patients above 60 years [26], the use of the appropriate differentiation-inducing compounds, either single or in combination, could be especially beneficial for these patients. Such therapy may also be considered even in younger patients whose cells can be effectively differentiated. We trust that these results will stimulate larger scale trials with cytosine

790

RITA MICHALEWlCZ, JOSEPH LOTEM and LEO SACHS

arabinoside, actinomycin D and other compounds. Induction of differentiation in malignant cells after pre-screening in culture of the compounds to be used may be a useful approach to the therapy of other types of tumors. A c k n o w l e d g e m e n t - - T h i s research was supported by a grant from the Jerome A. and Estelle R. Newman Assistance Fund.

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