Augmentation of Cytotoxicity of Chemotherapy by Human α -Interferons in Human Non-Small Cell Lung Cancer Xenografts James Carmichael, Ronald J. Fergusson, C. Roland Wolf, et al. Cancer Res 1986;46:4916-4920. Published online October 1, 1986.
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[CANCER RESEARCH 46, 4916-4920, October 1986]
Augmentation of Cytotoxicity of Chemotherapy by Human «-Interferonsin Human Non-Small Cell Lung Cancer Xenografts James Carmichael,1 Ronald J. Fergusson, C. Roland Wolf,2 Frances R. Balkwill,3 and John F. Smyth4 Imperial Cancer Research Fund, Medical Oncology Unit, Department of Clinical Oncology, Western General Hospital, Edinburgh EH4 2XV, Scotland, united Kingdom
ABSTRACT Three human non-small lung cancer xenograft lines were used to study the activity of combinations of cytotoxic drugs with human a-interferons (IFNs). Statistically significant potentiation of c»-platinum(CDDP) and cyclophosphamide (CY) given weekly in a low dose was seen when human lymphoblastoid interferon (IFN-anl) (2 x 10* /u/mouse/day) was admin
neuroblastoma (16). Balkwill and Moodie (17) showed marked synergy between human lymphoblastoid IFN and low doses of CY and Adriamycin against a human breast tumor xenograft growing in nude mice. The purpose of this study was to ascertain whether human IFN-as could potentiate the activity of cytotoxic chemotherapy in the treatment of human non-small cell lung cancers using biopsies xenografted into immune deprived mice.
istered simultaneously. The median tumor doubling times for CDDP in the three tumors (35, 22, and 29 days) increased to 52, 51, and 41 days when IFN-anl was added. A similar though less marked effect was seen with CY (median doubling time increased from 21.5, 19.5, and 27 days MATERIALS AND METHODS to 32, 27, and 35 days with the addition of IFN-anl). IFN-anl alone at this dosage was shown to have some cytotoxic activity. Similar potentia Animals. CBA mice, immune deprived by means of thymectomy, 1tion of CDDP and ifosfamide was seen in two tumors when human ,3-D-arabinofuranosylcytosine and X irradiation (18) were used in this recombinant a-2 interferon was added at a lower dose (2 x III' /¿/mouse/ study. Mice were thymectomized at 3 weeks of age and then were given day). Median doubling times for CDDP increased from 17 and 14 days 735 cGy whole body \-irradiation 3 weeks later, having received a to 27 and 18.5 days with the addition of human recombinant a-2 inter priming dose of l-/3-D-arabinofuranosylcytosine, 200 mg/kg i.p. 48 h feron, whereas for ifosfamide they increased from 11.5 and 14 days to 15 before. Implantation of the tumor was carried out the day following and 16 days. Human recombinant a-2 interferon in this dose had no effect irradiation, and the mice were treated with terramycin and neomycin as a single agent. in acidified drinking water for 14 days. The mice were housed in a separate room in the animal unit but specific pathogen-free conditions were not required. INTRODUCTION Tumors. Three non-small cell lung cancer xenograft lines were tested. The tumors were derived originally from specimens removed at surgery The outlook for the majority of patients with disseminated from previously untreated patients. Their growth characteristics and non-small cell lung cancer remains very poor. Recently, im morphology have been described elsewhere (19). The xenografts were proved response rates have been achieved with platinum-con all studied during the 5th-11th passages. The 3 xenograft lines used taining regimens (1), although the duration of these remissions were: (a) NX 002, moderately differentiated squamous carcinoma; (b) has been limited and the considerable toxicity associated with CX 117, poorly differentiated adenocarcinoma; and (c) CX 143, adenthese combinations precludes their widespread use (2). Much osquamous carcinoma. interest has been shown recently in the use of human IFNs5 All xenograft tumors had maintained the histological features of which have been shown to have modest antitumor activity in a their original tumor, and the karyotypes were confirmed to be human. number of human tumors (3-7); however, single agent activity A minimum of 6 tumors were entered in each group with the exception of one group for which there were only 4 assessable tumors (CX 117, has not been seen in either small cell lung cancer (8) or noncÃ-'s-platinumalone). Tumors were measured twice weekly using calipers
small cell lung cancer (9-10). More recently attention has been focused on the use of IFNs in combination with cytotoxic drugs. Several workers (11-13) have shown potentiation of chemotherapeutic agents by IFNs in vitro and these positive interactions have been confirmed in various animal models. In 1973 Chirigos and Pearson (14) showed that murine IFN increased the cure rate of the LSTRA murine leukemia by BCNU from 25 to 72% despite being ineffective as a single agent. Gresser et al. (15) demonstrated an additive antitumor effect between murine IFN and CY in the spontaneous lymphoma of AKR mice and a similar effect has been shown with CY and murine IFN in the C1300 mouse Received 1/4/86; revised 6/3/86; accepted 6/16/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Present address: National Cancer Institute, National Cancer Institute-Navy Medical Oncology Branch, Naval Hospital, Bethesda, MD 20892. 2 Present address: Imperial Cancer Research Fund, Laboratory of Molecular Pharmacology and Drug Metabolism, University of Edinburgh, Department of Biochemistry, Hugh Robson Building, George Square, Edinburgh, Scotland, United Kingdom. 3 Imperial Cancer Research Fund, Lincoln's Inn Fields, London, England,
and the tumor volume was estimated using the formula for an ellipsoid Volume •
x/W2
where D is the longest diameter and d is the smallest diameter. Only tumors with an estimated minimal tumor volume of 0.3 cm3 were used for drug testing. Interferons. Two human «-interférons were evaluated in these exper iments. (IFN-anl) (Namalwa) was prepared as previously described (20) and was kindly provided by Wellcome Research Laboratories (Beckenham, Kent, United Kingdom). It had a specific activity of 1-2 x 10* units/mg and was essentially pure; 2 x 10s units in 0.1 ml were injected s.c. each day for 35 days at a site distant to the tumors. This dose is equivalent to approximately 20 x IO6 units/m2 daily in humans using
the calculations of Freireich et al. (21). rIFN-a2b (Schering Corporation, Bloomfield, NJ) with specific ac tivity of 1.7 x 10* units/mg [>98% pure (2)] was injected in a dose of 2 x IO4 units/mouse/day for 35 days as above (equivalent to approxi mately 2x10* units/m2 in humans). Cytotoxic Drugs. cw-Platinum (Bristol-Myers, Ltd., Slough, United Kingdom) was dissolved in sterile normal saline and administered at a dosage of 1.4 mg/kg i.p. on days 0, 7, 14, 21, and 28. This dose was United Kingdom. 4 To whom requests for reprints should be addressed. equivalent to 20% of the maximum tolerated dose previously deter 5 The abbreviations used are: IFN, interferon; IFN-anl, human lymphoblastoid mined in these animals. Cyclophosphamide (Farmitalia Carlo Erba, interferon; r!FN-a2b, human recombinant a-2 interferon; CY, cyclophosphamide; CDDP, cis-platinum; IFOS, ifosfamide; SGD, specific growth delay; /",,, doubling Ltd., St. Albans, United Kingdom) and ifosfamide (Boehringer Ingelheim, Bracknell, United Kingdom) were dissolved in sterile water for time; MTD, maximum tolerated dose. 4916
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IFN AND CHEMOTHERAPY
COMBINATIONS IN LUNG XENOGRAFTS
injection and administered at a dosage of 40 and 60 mg/kg i.p., respectively, following an identical schedule. Study Design. Groups of tumors were randomly allocated to treat ment with drug or IFN alone, drug and IFN in combination, or a control group. CDDP and CY were assessed with the high dose IFN regime in all three tumors. CDDP and IFOS were used with the low dose IFN regimen in the NX002 and CXI 17 tumors. Response to treatment was assessed by measurement of the median tumor volume T,>after treatment and calculation of the SGD using the formula (23)
NXOO2 2.0 1.0
00
CX1
IFN
CDDP
CONTROL IFN
CDDP
CONTROL
17
CDDP CYCLO CYCLO • IFN +IFN
3.0
2.0
Tp (treated) - TD (control) SGD
1.0
To (control)
The SGD represents the number of tumor doubling times delayed by treatment and allows comparisons to be made between drugs in tumors of different growth rates. Statistical differences between the mean TDs of treated and control groups were carried out using an unpaired Student's i test. In the model, survival is not influenced by tumor growth and cannot therefore be used as an end point. Experimental results are presented up until the time of tumor doubling.
RESULTS High Dose IFN-anl and Chemotherapy. The changes in me dian TO and SGD for the high dose IFN-anl/chemotherapy combinations are illustrated in Figs. 1 and 2. NX002. Control tumors had a median TD of 16 days. IFN alone caused an increase in median TD to 21.5 days and an SGD of 0.34. The increase in mean TD was not statistically significant. Median T0 with CDDP was 35 days (SGD =1.18), (a) N>50
40 30° «
n10
n^ R Human ««!«««« j«|?*» * *%*«""£?*' human"^ «""»^ fa nude """• |nt• J-¿-a"c*r•>"•' 23.1~:135'I:;1!23°-Balkwill,F. R., Mowshowtiz,S., Seilman,S. S., Moodie,E. M., GnfTin,D. B- Fantes,K. H., andWolf,C. R. Positiveinteractionsbetweeninterférons and chemotherapydue to directtumoractivityratherthaneffectson host dragmetabolizingenzymes.CancerRes.,44:5249-5255,1984.
4920
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