Clinical Oncology(1992) 4:388-393 © 1992The Royal Collegeof Radiologists

Clinical Oncology

Review Article C o m b i n a t i o n T h e r a p y with Cisplatin: M o d u l a t i o n of Activity and T u m o u r Sensitivity H. J. Guchelaar 1, D. R. A. Uges 1, E. G. E. de Vries 2, J. W. Oosterhuis 3 and N. H. Mulder 2 Departments of 1pharmacy and Toxicology and 2Medical Oncology, University Hospital, Oostersingel 59, 9713 EZ Groningen and 3Dr Daniel Den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands

Abstract. Although cisplatin is applied with success in clinical oncology, this success is limited because some cancers are initially unresponsive to cisplatin or become so during treatment. In this review, some strategies to overcome this problem are discussed. Among these are combination with the differentiation inducing agent, retinoic acid, combination with radiotherapy, and the use of hyperthermia. Keywords: Chemoradiotherapy; Cisplatin; Differentiation induction; Hyperthermia; Retinoids

INTRODUCTION Cisplatin is one of the most effective cytotoxic drugs and has earned a place in many multidrug chemotherapy regimens, especially those for the treatment of disseminated germ cell tumours [1]. The success of cisplatin is limited because some cancers are initially unresponsive to it or become so during treatment [2]. In order to overcome these limitations one might attempt to modify the malignant potential of the tumour. One way of doing this is by induction of differentiation, preferably in a non-invasive and non-metastatic growth [3]. In human cancers, differentiation induction alone has shown disappointing clinical results so far [4]. Combination with chemotherapeutic drugs might help to eliminate the bulk of the most rapidly growing and malignant cell population, offering a better chance for induction of differentiation in the remaining tumour cells. The tumour can also be modulated towards a higher degree of cisplatin sensitivity, such as by combining cisplatin treatment with hyperthermia. Another approach is the combination of cisplatin with radiotherapy, as the drug has been shown to potentiate radiation induced cell killing. Correspondence and offprint requests to: Dr N. H. Mulder, Division of Medical Oncology,Department of Internal Medicine, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Netherlands.

DIFFERENTIATION THERAPY OF CANCER The clinical behaviour of a tumour is correlated with the degree of differentiation. A patient with a poorly differentiated ovarian carcinoma has a shorter life expectancy compared with a patient with a more differentiated tumour [5]. Furthermore, there are well documented reports on the spontaneous regression of neuroblastoma with terminal differentiation [6]. Induction of cellular differentiation by chemicals might therefore have a place in the treatment of cancer [7]. The above mentioned clinical observations also indicate that, at least in some cancers, tumour cells have not lost the genes that control normal growth and differentiation. Substances from several chemical and pharmacological classes are known to induce differentiation in vitro. These include polar-planar compounds (dimethyl formamide, dimethyl sulphoxide), hormones, regulatory peptides and chemotherapeutic drugs [7]. The most prominent are the retinoids.

Retinoids The induction of differentiation of the embryonal carcinoma cell line F9 by all-trans-retinoic acid (RA) was first demonstrated by Strickland et al. [8]. Thereafter, many authors have used retinoids to study the differentiation of (murine) embryonal carcinoma cells [9-11]. Studies with leukaemic cells also have shown differentiation in response to RA. The human promyelocytic leukaemia cell line HL-60 differentiates into granulocytes on incubation with R A in vitro [1215] and proliferation is inhibited in concentrations in the nanomolar range [16]. Furthermore, exposition of Friend erythroleukaemia cells to R A results in the appearance of a population of quiescent, non-proliferating cells [17]. Inhibition of cellular proliferation and increased expression of the differentiated phenotype of human melanoma cell lines is observed upon exposure to

Combination Therapy with Cisplatin retinoids [18]. However, different susceptibilities of cell lines, and even RA resistant subclones, are documented [19,20]. The mechanism of action of retinoids is not yet fully understood. The most widely accepted hypothesis is that they exert their action via a steroid hormone-like mechanism [21,22]. Cytosolic retinoid binding proteins have been found which are synthesized on exposure to retinoids [23]. The efficacy of retinoids to bind to these proteins correlates well with their biological activity. However, there are cells which respond to retinoids without having detectable binding proteins [24]. It is thought that the binding proteins are involved in the transportation of retinoids to the cell nucleus. In the nucleus the complex interacts with the nuclear retinoic acid receptors, resulting in alteration of gene expression. Three types of retinoic acid receptors (RAR-cr, RAR-fi and RAR-T) have been described [25-29]. The fact that retinoids show receptor subtype specificity may be useful in the design of more potent and less toxic derivatives [30]. In this way, retinoids control the expression of many proteins which are either direct constituents of the cytoskeleton and extracellular matrix or participate in the formation of these organelles [31].

Clinical Efficacy of Retinoids Alone in Cancer Therapy Clinical efficacy of retinoids alone [4,32,33] is disappointing with the exception of dermatological malignancies and acute promyelocytic leukaemia (APL). In the (topical and systemic) treatment of (metastasized) basal cell carcinoma with either 13-cisretinoic acid (13cRA), R A or etretinate, 20%-65% partial responses (PR), and 16%-31% complete remissions (CR) were observed [34,35]. In squamous cell carcinoma of the skin [36,37] retinoids may be effective. An overall response rate cannot be given for this type of tumour, because the number of treated patients is too limited. Topical treatment [33,38] but also systemic treatment [39] of malignant melanoma with R A or 13cRA showed responses in individual cases. Systemic treatment shows a remission rate of 15%, albeit without CR. Cutaneous T-cell lymphoma (mycosis fungoides) responds relatively well to retinoid therapy [40]. Combined data of published reports reveal a 15% CR rate and 40% PR rate for 13cRA, and a 38% CR and 34% PR for etretinate [41]. In solid turnouts, retinoids have only been tested on a limited scale. No responses were observed in 18 advanced breast cancer patients treated with 13cRA [41] and in 15 patients with advanced germ cell tumours [42]. In the latter study, mature teratoma was found at autopsy in a patient who experienced stabilization of disease, whereas pure embryonal cell carcinoma was diagnosed at original pathology. Taking these studies together, the remission rate never exceeded 15% [43]. Clinical trials with retinoids in patients with leukaemia are limited, but some (especially APL) show promising results. Case reports of 13cRA in patients

389 with APL showed transient effects and sometimes CR; increased peripheral granulocyte counts were found [44,45]. After relapse, cells were found to be resistant to 13cRA [44]. Recently, Warrel et al. showed a complete remission with oral RA (45 mg/ m 2) in nine of 11 patients with APL [46]. Clinical response was correlated with the existence of an aberrant RAR-o: receptor and transcript, both of which are characteristic of APL [47]. Also, in acute nonlymphocytic leukaemia, responses were observed using retinoids in combination with chemotherapy [4].

Combination of Retinoids with Chemotherapeutic Drugs Most solid tumours contain a fraction that is responsive to chemotherapy. It is likewise conceivable that many tumours contain a population inducible by differentiation. There is at present no indication that cross resistance between the two treatment modalities exists [48], therefore this combination could be interesting. Additive antitumour effects might be expected and, furthermore, differentiation of drug resistant cells could alter the sensitivity of the tumour to chemotherapy [49]. When drugs of each modality are chosen with non-overlapping toxicities, combination may allow the lowering of each dose without decreasing response rate. Few studies on the combination of retinoids and chemotherapeutic agents have been done. Effects of combinations of retinol palmitate and six different cytotoxic drugs on the life span of mice bearing ascites sarcoma 180 or P388 leukaemia were examined. With the sarcoma, enhanced antitumour effects were observed with 5-fluorouracil, methotrexate and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl3-(2-chloroethyl)-3-nitrosourea (ACNU), but not with doxorubicin and 6-mercaptopurine. With the leukaemia cell line, enhanced antitumour effects were seen with 6-mercaptopurine, methotrexate, doxorubicin, ACNU and cisplatin, but not with 5fluorouracil [50]. Wouda et al. studied transplanted murine teratocarcinoma cell lines with different grades of somatic differentiation in nude mice, and found an improved antitumour effect of concurrent RA and cisplatin, which was dependent on the initial level of differentiation [51]. Some other studies have also shown an enhancement of cisplatin cytotoxicity, although with other differentiation inducers. Dexter et al. showed an increase of cisplatin and Mitomycin-C sensitivity of cultured human colon cancer cells after incubation with the differentiation inducers N-methyl formamide (NMF) and its metabolite dimethyl formamide (DMF) [52]. Enhancement of the sensitivity for cisplatin, 1,3-bis(2-chloroethyl)-l-nitrosourea and melphalan was also observed in a murine hepatocarcinoma cell line after pre-treatment with NMF [53]. The antitumour effect of cisplatin in combination with NMF was shown to be additive against the M5076 sarcoma implanted in mice [54]. Contradictory results were found with the combination of NMF and cisplatin in vitro and in vivo. Against a murine carcinoma cell line, enhancement of cisplatin cyto-

390 toxicity was observed after pre-treatment with NMF. Administration of NMF to MCA-K murine mammary carcinoma-bearing mice did not enhance cisplatin induced tumour growth delay, unless NMF was administered after cisplatin [55]. This study demonstrated the significance of the timing of differentiation induction therapy combined with chemotherapy. Nogae et al. showed potentiation of the effect of vincristine by retinyl acetate against a vincristine sensitive and resistant leukaemia [56]. NMF also showed chemosensitization for doxorubicin of a human melanoma cell line in vitro and in vivo (transplanted in mice) [57]. The results of combining another differentiation inducer, dimethyl sulphoxide (DMSO), with doxorubicin, vinblastine, 5-fluorouracil and cisplatin, respectively, were found to vary between additive and synergistic effects [58]. Combination of the differentiation inducer sodium butyrate and doxorubicin resulted in a combined inhibitory effect against a cell line derived from the ascitic fluid of a patient with ovarian carcinoma. The cells were relatively insensitive to cisplatin but regained sensitivity when treated with sodium butyrate [59]. In a transplanted murine neuroblastoma cell line, enhancement of cisplatin antitumour activity was observed when combined with the differentiation inducing agent vitamin E [60].

HYPERTHERMIA In some early reports, patients with 'spontaneous' regression of advanced tumours were described. These patients had deliberate infections of erysipelas, with concomitant high fevers. Many years later it was recognized that application of supranormal temperatures has a selective lethal effect on malignant cells and may explain these 'spontaneous' cures [611. Hyperthermia regained clinical interest after Hahn showed that many currently used anticancer drugs, as well as radiotherapy, demonstrate increased cell killing at elevated temperatures [62]. Cisplatin is a promising agent to combine with hyperthermia [63,64]. With this drug there appears to be no threshold temperature at which sensitization appears; a gradual increase of cytotoxicity at temperatures of 37-43 °C is observed [62,65-67]. Above 42-43 °C no further enhancement of cisplatin cytotoxicity is observed [67]. Herman showed not only an increased cisplatin cytotoxicity at supranormal temperatures, but also decreased toxicity with hypothermia [68]. This finding offers the opportunity to target the cytotoxic effect of cisplatin by selectively heating the turnout region and cooling normal tissues. A maximal effect of hyperthermia on cisplatin cytotoxicity in vitro was observed when both modalities were applied simultaneously [65-67,69]. In the in vivo situation, scheduling of hyperthermia and chemotherapy is a complex issue. Hyperthermia can have a profound effect on factors such as drug distribution, clearance, vascular function, pH and

H.J. Guchelaar et al. oxygen tension, which can have an impact on drug effect [70,71] and the combined drug-heat effect [72]. In the in vivo situation, toxicity to normal cells may depend on the schedule applied. For example, although simultaneous hyperthermia and cisplatin treatment appears to have an optimal effect in vitro, this schedule was found to be extremely nephrotoxic in mice [73] as well as in patients [74]. The mechanism of cisplatin thermosensitization is not yet fully understood. Increased cellular (Pt) accumulation resulting from increased membrane permeability has been found [65,66,75]. Herman et al. described an increased reaction rate of cisplatin with D N A during hyperthermia. This may be due to higher cellular Pt accumulation, but can also be explained by altered thermodynamics at elevated temperatures [76]. Furthermore, hyperthermia is known to change the conformation of chromatin, which may lead to more acceptable D N A [77] and hence the formation of more D N A - P t cross links. Indeed, more D N A - P t cross links were found during supranormal temperatures [76,78]. It is known that hyperthermia can inhibit DNA repair [79]; although, this could explain thermosensitization, such a phenomenon was not found in vitro [78]. A general finding with hyperthermia is that blood circulation to the tumour is increased initially, but is subsequently diminished to below normal levels when heating is continued [69]. In SCK tumours in mice, heating to 41.5-45.0 °C has led to severe vascular occlusion in the tumour and to a down shift of pH [71]. Vascular effects of hyperthermia may influence drug distribution, while a lower pH enhances cisplatin cytotoxicity [80]. Hyperthermia also influences the pharmacokinetics of cisplatin. Drug clearance, volume of distribution and elimination half-life are increased during hyperthermic conditions [70]. Clinically, hyperthermia can easily be applied during isolated regional perfusion [81]. With this treatment massive drug doses can be administered to the perfused region, while systemic concentrations are low [82].

CISPLATIN AND RADIOTHERAPY Wodinsky et al. [83] were the first to report potentiation of radiation by cisplatin. They found an increase in life span in mice bearing P388 lymphoCytic leukaemia when treated with a combination of radiation and cisplatin. Douple et al. [84] subsequently found therapeutic synergism of this combination in a murine mammary adenocarcinoma and an intracerebral rat brain tumour. Following these observations, many investigators have tested this combination in vitro as well as in vivo. In these studies, which differed in dose, timing, tumour type and species, both additive [85,86] and supra-additive [87-90] interactions were found. Timing is an unresolved problem in chemotherapy. Kovacs et al. [85] showed in vitro that, in combined therapy, where cisplatin precedes radiotherapy for 12 hours, the response is additive, whereas at 24 hours it is supra-additive. In a study with a transplanted

Combination Therapy with Cisplatin tumour in mice, exposure to cisplatin immediately following irradiation and for 2 hours previously, resulted in the most effective tumour cell kill [88]. In a recent review, it was concluded that cisplatin concurrent with radiation is most effective [91]. Von der Maase showed that cisplatin administered 24 hours prior to radiation resulted in a maximal tumour response [92]. However, normal tissues generally show a more pronounced enhancement of radiation response. Furthermore, toxicity of normal tissue was highly dependent on the time of cisplatin administration [92]. Radiation sensitization is only one rationale for the combination of radiotherapy and chemotherapy. The addition of chemotherapy may also result in better control of micrometastatic disease, while radiotherapy is given as treatment for local control. The mechanism of enhancement of radiation induced cell kill by cisplatin has not yet been fully explained. From studies with bacterial cells under hypoxic conditions it was concluded that the formation of a radiolytic product ('radiation chemical based potentiation') from cisplatin (and analogues) may be involved [91]. The formation of such a product cannot be excluded in mammalian cells. However, Coughlin and Richmond present arguments that, in the context of clinical radiation therapy, a 'radiation biochemical-based mechanism', such as the inhibition of repair of radiation damage by cisplatin, is primarily responsible [91]. The first argument is that the concentration required for biochemical-based potentiation seems to be less than that for chemical-based potentiation, the latter requiring a minimum of about 10 #M. After clinical doses of cisplatin, the free cisplatin concentration is assumed to be lower. A second argument is that potentiation is not only observed when cisplatin is administered before, but also when given soon after, irradiation. A third argument is that in several (but not all) studies it was shown that the shoulder in the survival curve was affected when irradiation was combined with cisplatin treatment. As the shoulder originates from the recovery of sublethal damage it seems reasonable that cisplatin treatment interacts with these recovery processes. Several experiments have indeed shown that cisplatin inhibits repair of sublethal radiation damage [93,94]. According to this hypothesis it is expected that the extent of radiation induced chemical-based potentiation can be increased by administering higher cisplatin doses, resulting in higher tissue concentrations. This may also be achieved by locoregional drug administration or the use of less toxic derivatives in high doses. Studies with cisplatin analogues have shown that the property of radiation sensitization is not unique for cisplatin, but also exists for other analogues [91,95]. Coughlin and Richmond have summarized clinical trials which included both radiation and cisplatin. Cisplatin was used either alone or in combination with other chemotherapeutic drugs [91]. This review reveals that survival benefits (in comparison to radiation alone) are shown, especially in trials with high dosage cisplatin regimens. These studies do not clarify whether this benefit is due to potentiation of radiation or to additional tumour kill by cisplatin. An extensive

391 review of results in the combined treatment of radiation and cisplatin in vitro and in vivo, as well as in clinical studies, is reported by Dewit [96]. It has been shown that the inhibition of repair of radiation induced damage by cisplatin is greater in cisplatin sensitive tumour types than in drug resistant tumours [97]. For this reason it is suggested that future clinical trials should be carried out with cisplatin and radition in cisplatin sensitive tumours, such as head and neck, small cell lung, non-small cell lung, prostatic, cervical, ovarian, bladder and oesophageal cancers, and malignant melanoma [91]. In a recent study in patients with non-metastatic inoperable nonsmall cell lung cancer improved survival was observed in those treated with combined radiotherapy and low-dose cisplatin (30 mg/m 2) compared with radiotherapy alone [98]. Furthermore, future clinical trials of a trimodality therapy with local hyperthermia, radiotherapy and selected anticancer drugs have been suggested [69].

Acknowledgements. This study was supported by grants G U K C 90-18 and 91-09 of the Dutch Cancer Society.

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Received for publication April 1992 Accepted following revision August 1992