possible combination of tamoxifen and interferon. (IFN) in the treatment of breast cancer patients. The rationale in combining these drugs is that IFN induces.
C[//cer mesearch C nical 9
J Cancer Res Clin Oncol (1989) 115:465-469
9 Springer-Verlag 1989
Rationale for combining tamoxifen and interferon in the treatment of advanced breast cancer F. Porzsolt 1.2 A.M. Otto 3, B. Trausehel
1 C. Buck 1, A.W.
Wawer 1, and H. Sch~nenberger 3
1 Abteilung Innere Medizin III (H~imatologie/Onkologie) und 2 Tumorzentrum der Universit~it D-7900 Ulm 3 Lehrstuhl Pharmazeutische Chemic II der Universit~t Regensburg (SFB 234), D-8400 Regensburg, Federal Republic of Germany
Summary. Several investigators have discussed the possible combination of tamoxifen and interferon (IFN) in the treatment of breast cancer patients. The rationale in combining these drugs is that IFN induces the expression of estrogen receptors and therefore increases the sensitivity of breast cancer cells toward the growth-inhibitory activity of tamoxifen. In this paper we review the literature on the IFN-mediated expression of estrogen receptors and the postulated synergism of tamoxifen and IFN in the growth inhibition of breast cancer cell lines. Our results indicate that neither type I nor type II IFN increases the expression of estrogen receptors in MCF-7 cells. Together with tamoxifen both type I and type II IFN mediate additive but not synergistic growth inhibition of MCF-7 cells. On the basis of these results it is feasible to test tamoxifen and IFN as combined therapy in breast cancer patients. Preliminary clinical data show that the combination of 30rag tamoxifen and 2 x 106 IU IFN-e as daily doses may induce WHO grade 3 leukopenia and thrombopenia in patients who are pretreated with polychemotherapy.
Key words: Tamoxifen and interferon - Growth inhibition - Estrogen receptor - Breast Cancer Introduction
The use of tamoxifen has been well established in the treatment of breast cancer. In advanced breast cancer, non-selected patients will respond in about 30% of cases. In patients with tumors which express receptors for estrogen and/or progestin the response rate is about tenfold higher than of those which do not (Henderson 1987). Using tamoxifen as adjuvant therapy, Offprint requests to: F.Porzsolt, Tumorzentrum der Universit~it Ulm, Robert-Koeh-Strasse 8, D-7900 Ulm, FRG Abbreviations: rIFN, recombinant interferorJ; nIFN, natural interferon
extension of survival has recently been demonstrated (Early Breast Cancer Trialists' Collaborative Group 1988). The mechanism by which tamoxifen inhibits the growth of malignant breast cancer cells is unknown. However, recent results suggest that growth factors such as TGF-~ and TGF-/~ are induced by estrogen or tamoxifen, respectively (Knabbe et al. 1987). Interferons mediate antiviral, antiproliferative, and immunomodulating effects. On the basis of these properties, interferons have been introduced as the first cytokines in the treatment of malignant disorders. It has been shown that malignant diseases like hairycell leukemia (Porzsolt 1986; Quesada et al. 1984), chronic myelogenous leukemia (Talpaz et al. 1986), low grade malignancy non-Hodgkin lymphomas (Wagstaffet al. 1986) can efficiently be treaed with interferon. Although the in vitro experiments demonstrated sensitivity to interferons in solid tumors also (Porzsolt 1987; Porzsolt et al. 1988), only low response rates could be induced in clinical trials. In most malignancies, combinations of cytotoxic drugs are more efficient than single agent therapies, and combinations of interferons with cytostatic drugs also demonstrated promising results in vitro and in animal model systems (Tyring 1987). As the growth of some tumors is sensitive to hormones, it was feasible to study the efficacy of combinations of hormones and interferons in the treatment of hormone sensitive malignancies. In this article we present our own results and review the present literature on the in vitro effects of the combination of tamoxifen and interferon. The perspectives for using this drug combination in clinical trials is discussed. Materials and methods Effects of interferons and of tamoxifen on cell growth. MCF-7 cells (2 x 104/ml) were plated in Riehter's improved minimal essential me-
466
F. Porzsolt et al.: Tamoxifen and interferon
dium (IMEM) supplemented with 10% fetal calf serum. After 24 h, various sources ofinterferons, with or without 1 ~tMtamoxifen, were added. Recombinant interferon a-2a (rlFNe2a), or natural interferon fl (nlFNfl) or recombinant interferon 7 (rlFN~) were used in concentrations of 10, 100, and 1000 IU/ml. Five days later suspended nuclei were counted according to Butler (1984).
Effects of interferons on estradiolbinding. MCF-7 cellswere plated at 2 x 104/ml in Richter's IMEM (without phenol red) supplemented with 10% fetal calf serum. Interferons were added 24 h later. After a 5-day incubation, cell numbers were determined by counting suspended nuclei according to Butler (1984), and estradiol binding was determined by incubating the cells with [3H]estradiol (0.4 riM) for 30 min at 37 °C according to Olea-Serrano et al. (1985). Unspecific binding, assayed in the presence of unlabelled estradiol (0.1 gM) was less than 20%.
Calculationof synergism. To describe the degree of synergismof two agents in culture, we divided the relative effect observed in the culture treated with both agents simultaneously (O) by the calculated product of the relative effects of the single agents (E). The ratio of O/E= 1 indicates an additive effect, but not synergism(Bhuyan et al. 1985). Synergism was assumed when the ratio O/E was 2.0. An O/E ratio of < 0.5 would indicate synergismin growth inhibition while a ratio of > 2.0 would indicate synergism in growth stimulation. In viva treatment with tamoxifen and interferon. In a pilot study we treated patients suffering from metastatic breast cancer with 30 mg tamoxifen p.o. daily and 2 × 106 IU IFNa-2c s.c. daily. Safety controls included blood cell counts, coagulation tests, 12-channel biochemistry, and ECG. The protocol of the study has been approved by the ethical committee, University of Ulm. The patients signed a written consent. Results
Inhibition of cell growth by interferons and tamoxifen It is s h o w n i n T a b l e 1 t h a t t a m o x i f e n (1 IxM) r e d u c e d M C F - 7 cells to 26.5% o f u n t r e a t e d controls w i t h i n 5
days o f culture. I n t e r f e r o n s f r o m all sources tested mediated a d o s e - d e p e n d e n t r e d u c t i o n o f M C F - 7 cells. U s i n g the described culture c o n d i t i o n s , n I F N f l reduced the cell n u m b e r b y 9 0 % a n d was m o r e efficient t h a n r l F N a - 2 a or r I F N 7 , which reduced the cell n u m b e r b y only 30%. C o m b i n e d t r e a t m e n t s with t a m o x i f e n a n d different interferons resulted in additive b u t n o t synergistic i n h i b i t i o n o f cell g r o w t h as i n d i c a t e d b y O/E ratios.
Effects of interferons on estradiol binding T r e a t m e n t o f M C F - 7 cells with I F N a - 2 a , n I F N f l , or r I F N 7 in doses f r o m 1 I U / m l to 1000 I U / m l for 5 days resulted in a decrease o f cell n u m b e r s to 6 0 % , 11%, a n d 52% respectively (Table 2). However, the specific b i n d i n g o f [3H]estradiol per 10 s cells was n o t c h a n g e d w h e n unspecific b i n d i n g was a c c o u n t e d for by a control with a n excess o f u n l a b e l l e d estradiol.
In vivo treatment with tamoxifen and interferon ~-2c I n a pilot study, we treated three a d v a n c e d breast cancer p a t i e n t s with t a m o x i f e n (30 m g p.o. daily) a n d IFNc~-2c (2 × 106 I U s.c. daily). T w o p a t i e n t s h a d b e e n pretreated with c o m b i n e d cytotoxic d r u g regimens. The t r e a t m e n t with t a m o x i f e n a n d i n t e r f e r o n was well tolerated except for the occurrence o f l e u k o p e n i a (WHO grade3) and thrombocytopenia (WHO grade 1) in the two patients with cytotoxic pretreatm e n t . O w i n g to these side effects we reduced the interferon t r e a t m e n t to 2 × 106 I U twice weekly. I n two patients t r e a t m e n t h a d to be d i s c o n t i n u e d after 1.5 a n d 5 m o n t h s , respectively, because o f progressive disease. I n one patient, who h a d d e m o n s t r a t e d progressive dis-
Table 1. Combined treatment of MCF-7 cells with tamoxifen (TAM) and interferons (IFN) Interferon
Concentration
Results after in vitro treatment
Expected
Observed
rIFN~2a
(IU/ml)
10-4 x cell no.
(%)
10 × cell no.
(%)
(%)
-
47.31 45.83 40.79 33.19
100.0 96.9 86.2 70.2
12.47 12.98 12.79 11.05
26.4 27.4 27.0 23.4
25.6 22.8 18.5
1.07 1.18 1.26
47.13 41.45 27.78 5.34
100.0 87.9 59.0 11.3
12.41 11.47 9.14 2.47
26.3 24.3 19.4 5.2
23.1 15.5 3.0
1.05 1.25 1.73
44.80 44.56 39.62 30.59
100.0 99.5 88.4 68.3
12.25 12.28 10.59 8.25
27.3 27.4 23.6 18.4
27.2 24.1 18.6
1.01 0.98 0.99
10 100 1000 nIFN~
10 100 1000
rlFN~
o/E
With TAM (1 laM)
Without TAM
10 100 1000
F. Porzsolt et al.: Tamoxifen and interferon
467
Table 2. Effect of interferons on [3H] estradiol binding and cell proliferation of an estrogen-sensitive breast cancer cell line (MCF-7) Interferon
Concentration (IU/ml)
[3H] Estradiol binding (fmol/105 cells)
Cell number (% control)
Control
7.3
100
IFN~
1 10 100 1000
7.8 7.8 8.3 8.5
98 90 79 60
IFNfl
i 10 100 1000
8.1 8.1 8.7 -
98 84 44 11
IFN~
1 10 100 1000
7.6 7.9 8.0 7.8
99 89 71 52
ease after multiple polychemotherapies, a complete remission of lymph node metastases (palpation and sonography) and stable disease of bone metastases could be induced. Skin metastases were removed by surgery 18 and 31 months after beginning combined tamoxifen plus interferon therapy. Meanwhile, the patient received this combined therapy for 59 months. Except for these skin metastases, there is still no other evidence for disease progression.
Discussion It is shown in Table 1 that combined treatment with tamoxifen and interferon mediates an additive, but not synergistic, growth inhibition of MCF-7 cells. This result confirms data from previous studies (Table 3). Some investigators claim to find a synergistic growth inhibition in vitro using natural IFN~ and Toremifene (Kangas et al. 1985), recombinant IFNa and tamoxifen (Epstein et al. 1987; Van den Berg et al. 1987), natural IFNfl and tamoxifen (Sica et al. 1987), or natural IFNfl and medroxyprogesterone acetate (Sica et al. 1986). In some studies it is reported that synergism depends on a pretreatment of cells with interferon prior to the addition of the hormonal component (Epstein et al. 1987; Van den Berg et al. 1987). Although we considered this experience in our experiments, we could not confirm that tamoxifen and interferon act synergistically in the growth inhibition of MCF-7 cells. The reason for these different results may be only a different interpretation of data. According to Bhuyan et al. (1985), we distinguished additive from synergistic effects as described in Materials and methods. These calculations were not done in such studies by other investigators. Our results were interpreted as synergism if the observed effect was more than twofold the expected effect. Lower than twofold effects were considered as additive. An interesting model to explain the effects of a combined treatment with tamoxifen and interferon
Table 3, Reported effects of interferons on cellular estradiol binding and proliferation of breast cancer cell IFN
Cells
rIFNc~2
BT-20 ZR-75.1 CG-5 MCF-7
(ER-) (ER +) (ER +) (ER §
ZR-75.1 (ER +) 5 x 104 cells/weIl 20 x 104 cells/well
Dose of IFN IU/ml
Incubation time (days)
Binding of [3H] estradiol (% of contr01)
Cell number (% of control)
500 500 500 111-6000
14 ? 6 5
n.d. n.d. 91 100
75 48 85 ?c
= IFNe n.d. = TAM >TAM
2; 6" 2
Increasedb 100
90 n.d.
>TAM n.d.
van den Berg et al. (1987)
6 5
63 150 100
55 80 Up to 35
> TAM
Jacobelli et al. (1986)
100 100 n.d. 65
60 55 75 61
=IFN7 =IFN? > TAM > TAM
10-1000 10
nlFNfl
CG-5 CG-5
(ER +) (ER +)
500 10 100-1000
rIFNy
BT-20 ZR-75.1 MCF-7 CG-5
(ER-) (ER +) (ER +) (ER +)
500 500 500 500
4; 14 a 4; 14 a 14 a 6
a Second time refers to determination of cell number b Data with very high unspecific binding c Not given in reference (abstract) a Comparison with the compound which gave the higher inhibitory effect
Combination of IFN with tamoxifen(TAM): rel. growth inhibition d
n.d.
Ref.
Marth et al. (1985) Jacobelli et al, (1986) Epstein et al. (1987)
Sica et al. (1987) Marth et al. (1985)
Jacobelli et al. (1986)
468
would be the postulate that interferon stimulates the expression of hormonal receptors, which increase the susceptibility of cells to effects of tamoxifen. The in vitro data that had been reported in the literature are summarized in Table 3. There it is shown that various sources ofinterferons and various doses do not consistently induce an increased binding of [3H]estradiol. Together with our own data, shown in Table 2, we suggest that interferons do not increase the expression of estrogen receptors in hormone sensitive cells. The hypothesis that interferon might stimulate the expression of hormone receptors was derived from observations by Pouillart et al. (1982), who treated patients suffering from advanced breast cancer with IFN/3. The binding of estradiol and progestin in biopsies of skin metastases was determined before and after the IFNfl treatment. The authors found increased estradiol binding in two out of two patients and markedly increased progestin binding in five out of six patients tested. It was not described whether the same or different metastases were selected for receptor analyses before and after interferon therapy. This might be critical, as the expression of hormone receptors may vary in different metastases in the same patient (Hull et al. 1983). As the expression of hormonal receptors is controlled by sex steroids (Saez et al. 1978), it should also be considered in premenopausal patients that different phases of the menstrual cycle could influence the receptor expression in tumor biopsies. Unspecific binding of estradiol in the presence of interferon has been considered by others (Dimitrov et al. 1984; Filipic et al. 1977). Clinical studies on the combined treatment with tamoxifen and interferon in advanced breast cancer have not yet been done. Available data suggest that combinations of tamoxifen and interferon produce additive inhibition of tumor growth. Although the mechanism of the additive growth inhibition is not understood it is feasible to test the combination of tamoxifen and interferon in clinical trials. Our preliminary data suggest that even low doses of these drugs may cause peripheral blood cytopenia in patients pretreated with cytotoxic drugs. Acknowledgements. The excellent technical assistance of Ms S.S. Scholz and B. Neuhguser is appreciated. Interferons were kindly provided by Hoffmann-La Roche, D-7889 Grenzach (rIFN~-2a), Dr. Rentschler GmbH, D-7957 Laupheim (nIFN/3), and Ernst Boehringer Institute, A-1020 Wien/Dr. Karl Thomae GmbH, D7950 Biberach (IFNct-2c and rIFN~,).
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Received 15 March 1989/Accepted 5 June 1989
