Molecular and biological rationale of hyperthermia as radio- and chemosensitizer.

Mild hyperthermia, local heating of the tumour up to temperatures <43 °C, has been clinically applied for almost four decades and has been proven to substantially enhance the effectiveness of both radiotherapy and chemotherapy in treatment of primary and recurrent tumours. Clinical results and mechanisms of action are discussed in this review, including the molecular and biological rationale of hyperthermia as radio- and chemosensitizer as established in in vitro and in vivo experiments. Proven mechanisms include inhibition of different DNA repair processes, (in)direct reduction of the hypoxic tumour cell fraction, enhanced drug uptake, increased perfusion and oxygen levels. All mechanisms show different dose effect relationships and different optimal scheduling with radiotherapy and chemotherapy. Therefore, obtaining the ideal multi-modality treatment still requires elucidation of more detailed data on dose, sequence, duration, and possible synergisms between modalities. A multidisciplinary approach with different modalities including hyperthermia might further increase anti-tumour effects and diminish normal tissue damage.

Relevance of hypoxia in radiation oncology: pathophysiology, tumor biology and implications for treatment.

Tumors are characterized by an inefficient and disorganized vasculature which leads to tumor regions that are transiently or chronically undersupplied with oxygen (hypoxia) and nutrients (e.g., glucose). These adverse conditions are linked to treatment resistant and metastasizing disease with poor prognosis. Radiation sensitivity is dramatically lowered in hypoxic, yet viable and clonogenic, cells since oxygen is involved in the fixation of radiation-induced DNA damage (radiobiological hypoxia), and loco-regional tumor control is adversely affected in patients with hypoxic tumors. Hypoxia also leads to reduced sensitivity towards chemotherapeutics since drug delivery is reduced in hypoperfused hypoxic areas and hypoxic cells are quiescent, making drugs that target dividing cells ineffective. Fortunately, clinical attractive imaging and gene-expression based technologies that allows pre- and during treatment assessment of tumor hypoxia are now available. These technologies may identify patients suitable for established or emerging hypoxia-targeting treatments and, equally important; they allow us to monitor the efficacy of such intervention and may thus pave the way for effective individualized treatment. In the current review, we address 1) the causes and consequences of tumor hypoxia, 2) technologies that allow assessment of tumor hypoxia in individual patients and 3) current status of hypoxia-targeting treatments.

The new vascular disrupting agent combretastatin-A1-disodium-phosphate (OXi4503) enhances tumour response to mild hyperthermia and thermoradiosensitization.

PURPOSE: The aim of this study was to investigate the anti-cancer effect of the novel vascular disrupting agent (VDA), combretastatin-A1-disodium-phosphate (OXi4503), when combined with mild hyperthermia and/or radiation. MATERIALS AND METHODS: A C3H mammary carcinoma was grown subcutaneously in the rear right foot of female CDF1 mice, and treated when a volume of 200 mm(3) was reached. OXi4503 was administered intra-peritoneally at variable doses. Hyperthermia was administered locally to the tumour-bearing foot using a thermostat-controlled water bath. Radiation treatment was performed locally using a conventional X-ray machine. Tumour response was assessed with either a tumour growth time or a tumour control assay. RESULTS: The optimal delay between administration of 50 mg/kg of OXi4503 and hyperthermia was found to be 3 hours. The linear relationship between tumour growth time (TGT) and heating time at a specific temperature resulted in slope values between -0.003 days/min and 0.09 days/min at temperatures between 40 degrees C and 42.5 degrees C. When combined with OXi4503 this was significantly increased to 0.008 days/min and 0.03 days/min at temperatures between 39.5 degrees C and 41 degrees C, respectively. Above 41 degrees C, combined treatment did not result in significantly greater slope values. The radiation dose required to control 50% of the tumours (TCD50) was 52 Gy. Combining radiation with either heat treatment at 41.5 degrees C for 1 hour or OXi4503 reduced the TCD50 to 47 Gy and 41 Gy, respectively. Combining radiation with heat and OXi4503 further reduced the TCD50 to 37 Gy. CONCLUSIONS: OXi4503 is a highly potent VDA, which is capable of significantly enhancing the anti-cancer effect of mild hyperthermia. Mild temperature thermoradiosensitization was also enhanced.

Hyperthermia: a potent enhancer of radiotherapy.

Hyperthermia is generally regarded as an experimental treatment with no realistic future in clinical cancer therapy. This is totally wrong. Although the role of hyperthermia alone as a cancer treatment may be limited, there is extensive pre-clinical data showing that in combination with radiation it is one of the most effective radiation sensitisers known. Moreover, there are a number of large randomised clinical trials in a variety of tumour types that clearly show the potential of hyperthermia to significantly improve both local tumour control and survival after radiation therapy, without a significant increase in side-effects. Here we review the pre-clinical rationale for combining hyperthermia with radiation, and summarise the clinical data showing its efficacy.

Tumour-specific enhancement of thermoradiotherapy at mild temperatures by the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid.

The effect of combining the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) with both radiation and hyperthermia treatments was investigated in a transplanted C3H mouse mammary carcinoma and a normal mouse tissue. Tumours were grown on the right rear foot of female CDF1 mice and treated when sized 200 mm3. The foot skin of non-tumour-bearing CDF1 mice was used to assess normal tissue damage. Radiation and hyperthermia were given locally to the tumour/skin of restrained non-anaesthetized animals. DMXAA (20 mg/kg) was dissolved in saline and injected intraperitoneally 1 h after irradiating and then heating started 3 h later. The endpoints were local tumour control within 90 days or the development of moist desquamation in skin between 11 and 23 days after treatment. The radiation dose (+/- 95% confidence intervals) producing local tumour control in 50% of treated animals was 53 (51-55) Gy for radiation alone. This value was significantly (Chi-squared test; p < 0.05) decreased to 47 (42-52) Gy by DMXAA and to 47 (44-51) Gy by heating (41.5 degrees C/60 min) 4 h after irradiation. Combining both DMXAA and heating further reduced this to 30 (26-35) Gy. When the heating temperature was decreased to 40.5 degrees C, the effect of the triple combination was decreased but was still significant compared with radiation + DMXAA or radiation + hyperthermia. However, this enhancement disappeared at 39.5 degrees C. Radiation damage of normal foot skin was not enhanced by combining DMXAA and hyperthermia at 41.5 degrees C. In conclusion, adding DMXAA to thermoradiotherapy at 40.5-41.5 degrees C significantly improved local tumour control without enhancing normal tissue damage. Thus, including a vascular targeting agent in a mild thermoradiotherapy treatment regimen is a useful approach that may lead to a re-evaluation of the use of hyperthermia in cancer treatment.

Potentiation of the anti-tumour effect of hyperthermia by combining with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid.

The potential of the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) to enhance the effect of hyperthermia was investigated in a C3H mouse mammary carcinoma grown in the feet of female CDF1 mice and in normal foot skin. DMXAA, when injected intraperitoneally in restrained non-anaesthetized animals, reduced tumour perfusion, as measured using the RbCl extraction procedure, and increased necrosis in histological section, but these effects were dependent on the drug dose and time interval. At a dose of 20 mg/kg, it significantly enhanced the thermal damage of this tumour, when given 1 h or more before the start of heating, as assessed by a tumour growth assay. This enhancement became larger with increasing interval between the two treatments. No thermo-potentiation was seen at doses of 10 mg/kg or lower. These combined effects seem to be associated with the tumour vascular shut-down by DMXAA. Thermal potentiation by DMXAA was also dependent on the heating temperature, with a greater enhancement relative to hyperthermia alone obtained at the lower temperatures at 40.5 and 41.5 degreesC than at the higher temperature of 42.5 degrees C. DMXAA (20 mg/kg) also enhanced the heat damage of normal skin, and this could not be explained by any DMXAA-induced TNF-alpha production. The heat enhancement-ratio by DMXAA was larger in tumours (1.9) than in normal skin (1.3-1.5), thus giving rise to a therapeutic gain.

Combretastatin A-4 disodium phosphate: a vascular targeting agent that improves that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy.

PURPOSE: To investigate the effect of combining the vascular targeting drug combretastatin A-4 disodium phosphate (CA4DP) with hyperthermia, radiation, or mild thermoradiotherapy in a transplanted C3H mouse mammary carcinoma. METHODS AND MATERIALS: The C3H mammary carcinoma was grown on the rear foot of female CDF1 mice and treated when at 200 mm(3) in size. CA4DP was dissolved in saline and injected i.p. Hyperthermia and/or radiation were locally given to tumors in restrained nonanesthetized mice. Tumor response was assessed using either a tumor growth or a tumor control assay. Mouse foot skin was used to assess normal tissue damage. RESULTS: CA4DP significantly enhanced thermal damage in this tumor model. This effect was independent of drug doses between 25-400 mg/kg, but was strongly dependent on the time interval between drug injection and heating, with the greatest improvement seen when CA4DP preceded the heating by 1 h or less. There was also a suggestion of a temperature dependency with a 1.9-fold increase in heat damage at 42.5 degrees C and a 2.6-fold increase at 41.5 and 40.5 degrees C. Heat-induced normal tissue damage was also enhanced by combining CA4DP with heat, but the degree of enhancement was less than that seen in tumors. CA4DP (25 mg/kg) significantly increased radiation-induced local tumor control and this was further enhanced by combining CA4DP with mild temperature (41.5 degrees C, 60 min) heating. CONCLUSIONS: CA4DP improved the anti-tumor effect of hyperthermia, especially at mild temperatures. More importantly, it also increased the tumor response to mild hyperthermia and radiation, which suggests that CA4DP may ultimately have an important application in clinical thermoradiotherapy.

Improving local tumor control by combining vascular targeting drugs, mild hyperthermia and radiation.

Improvement in local control in a foot-implanted (200 mm3) C3H mouse mammary carcinoma by combining vascular targeting drugs, mild hyperthermia and radiation was investigated. The vascular targeting drug was flavone acetic acid (FAA; 150 mg/kg) intraperitoneally injected either 3 h before local tumor water-bath heating or 1 h after local tumor irradiation. For untreated tumors, the average (+/- 1 S.E.) tumor growth time (TGT; time to reach 5 x treatment volume) was 7.1 days (+/- 0.4). This was increased to 9.2 days (+/- 0.7) by using FAA. Heating also increased TGT, the effect being temperature and time dependent, and this heat response was further increased by FAA. The radiation dose (+/- 95% confidence interval) to control 50% of tumors (TCD50) 90 days after irradiation was 52 Gy (50-55) for radiation alone. This was decreased to 42 Gy (39-45) by FAA, 47 Gy (45-50) by heating (41.5 degrees C; 60 min) 4 h after irradiation, and to 28 Gy (22-35) by combining FAA and heat. Thus, vascular targeting drugs can improve the efficacy of mild hyperthermia and radiation.

The ability of hypoxia to modify the gene expression of thymidylate synthase in tumour cells in vivo.

PURPOSE: Hypoxic cells in tumours are resistant to 5-fluorouracil (5-FU). This in vivo study investigated the ability of hypoxia to regulate the gene expression of thymidylate synthase (TS), the target enzyme of 5-FU. MATERIALS AND METHODS: C3H mammary carcinomas, grown in the feet of female CDF1 mice, were used for all experiments. Mice were placed in a 10% oxygen environment for various time periods and the tumour oxygen status was determined with an Eppendorf oxygen electrode. The animals were then injected with BrdU (100 mg/kg, i.p.). Tumours were excised and immediately frozen (-80 degrees C) until isolation of total RNA. The mRNA was reversibly transcribed to complementary DNA and the resulting cDNA amplified in a multiplex PCR reaction, with beta-actin as the internal reference gene. RESULTS: One hour of low oxygen breathing made tumours significantly more hypoxic. This increase was maintained for a maximum incubation period of 48 h. In the same tumours, no change in TS gene expression was seen with up to 3 h of low oxygen breathing. At longer times it decreased, reaching significance at 12-24 h and remaining at this lower level for up to 48 h. BrdU labelling was significantly reduced after breathing low O2 for 24 h (p = 0.001). CONCLUSION: Hypoxia-induced down-regulation of TS gene expression was observed. This would be expected to make hypoxic tumour cells more sensitive to 5-FU. Other mechanisms must be responsible for the previously reported resistance to this drug.

Effects of combretastatin on murine tumours monitored by 31P MRS, 1H MRS and 1H MRI.

PURPOSE: Combretastatins have tubulin-binding activity and are being investigated for their toxicity against tumour vasculature. We report the use of 31P and 'H magnetic resonance (MR) spectroscopy and 1H MR imaging for monitoring the effects of combretastatin A-4 prodrug (100mg/kg, i.p.) on energy metabolism and necrosis, respectively, in the C3H murine mammary tumour. MATERIALS AND METHODS: The tumours (volume ca. 200mm3) were grown in the hind foot of mice. MR examinations were performed without anaesthesia within a 7.1 Tesla magnet. 31P MRS (TR = 6 s) was performed before treatment and at 1-, 2-, 3-, and 24-h after injection of drug or saline via an i.p. line. 1H MRS (PRESS; 24microl voxel; TR = 2 s; TE = 135 ms) and both T1-weighted (TR = 0.2 s; TE = 0.02 s) and T2-weighted (TR = 2 s; TE = 0.20 s) 1H MRI were performed before treatment and 2.5 and 24 h afterwards. RESULTS: The ratio beta-nucleotide triphosphate/inorganic phosphate fell by 33% within 1 h of treatment and remained constant for a further 2 h. A small but significant fall in pH (by 0.11 units) was observed at 1 h. Although an increase in the 1H MR spectroscopy signal at about 1.32 ppm (predominantly from lactate) was observed in some tumours following combretastatin treatment, this effect was not seen consistently. No changes in the intensity of T2-weighted 1H MR images or in tumour necrosis (measured histologically) were detected within 3 h of treatment. CONCLUSIONS: The reduction in tumour energetics and pH was consistent with a reduction in tumour blood flow but this occurred before any significant incidence of haemorrhagic necrosis was detected. The combretastatin dose used to achieve these effects was less than one tenth of the maximum tolerated dose in mice.

Techniques to assess the oxygenation of human tumors. State of the art.

There is now good evidence that the oxygenation status of certain types of human tumors plays an important role in determining the response of those tumors to therapy. More recent evidence suggests that oxygenation status can also influence local tumor aggressiveness and metastatic spread. As a consequence significant effort is being made to find relevant methods for assessing tumor oxygenation status and thereby allowing for the selection of patients that should be given additional/alternative therapies to improve response. In this paper we will review the importance of tumor oxygenation status and discuss the potential clinical methods for monitoring that are currently available.

Preclinical evaluation of the fluorinated 2-nitroimidazole N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) as a probe for the measurement of tumor hypoxia.

A novel probe, N-(2-hydroxy-3,3,3,-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554), has been used to detect tumor hypoxia noninvasively by 19F magnetic resonance spectroscopy (19F MRS). The compound was designed to undergo a hypoxia-dependent, one-electron reduction to metabolites that are selectively retained in tumors and has attractive pharmacokinetic, toxicological, and detection sensitivity properties. As a prelude to clinical studies, we report here for the first time on the ability to detect a MR signal following SR-4554 administration in various transplantable tumors and describe validation studies, consisting of a correlation between signal retention and radiobiological hypoxic fraction, and the effects of modulating the degree of hypoxia by hydralazine and carbogen breathing. SR-4554 was absorbed and then eliminated from EMT6 tumors with a half-life of 51 min following an injection of 180 mg/kg i.p. of SR-4554. Using a quantitative 19F MRS technique, the 19F retention index (19FRI; 19F signal level at 6 h/45 min) was determined for four commonly used murine tumors (EMT6, SCCVII, KHT, and RIF-1). The retention of high tumor concentrations of fluorinated probe at 6 h, despite the much lower (20-fold) concentration of parent SR-4554 detected by high-performance liquid chromatography, was consistent with the involvement of one or more nitroreduced metabolites and suggested that 19F MRS might give a quantitative measure of tumor hypoxia. In these murine tumors, 19FRI correlated with the reported radiobiological hypoxic fraction of the tumors (r = 0.988; P = 0.01). In addition, changes in tumor microenvironment were detected by 19F MRS. An increase in hypoxia induced by hydralazine treatment of RIF-1 tumor-bearing mice was associated with a 2.4-fold increase in 19FRI compared to untreated controls. In contrast, carbogen breathing by C3H mammary tumor-bearing mice produced a 6-fold decrease in the 19FRI compared to air-breathing mice. The data presented support the preclinical and clinical development of SR-4554 as a noninvasive probe for tumor hypoxia.

Can mild hyperthermia improve tumour oxygenation?

The oxygenation status of C3H mammary carcinomas, grown in the feet of CDF1 mice, was measured with an Eppendorf oxygen electrode. Tumours were locally heated in a water bath at temperatures of 38.5-41.5 degrees C for 1 h. Measurements made during heating showed temperature-dependent increases in tumour oxygenation. However, measurements performed after heating showed a rapid return to normal oxygenation status. Mild hyperthermia thus improves tumour oxygenation and this can explain the radiosensitization seen with low heat treatments, but only when the heat and radiation are administered concurrently, which is typically not the way that are given clinically.

The in vivo interaction between flavone acetic acid and hyperthermia.

The in vivo interaction between flavone acetic acid (FAA) and hyperthermia was studied in a C3H mammary carcinoma grown in the feet of female CDF1 mice and in normal foot skin. FAA was intraperitoneally injected prior to local tissue heating in restrained non-anaesthetized animals. Alone, FAA at doses of 100 mg/kg and above, inhibited tumour growth in a dose-dependent fashion. FAA also enhanced the tumour response to heat, the effect being dependent on both the time interval between the two modalities and the FAA dose, the greatest effect occurring when FAA doses of > or = 150 mg/kg preceeded heat by 3-48 h. These effects of FAA correlated with the drug's ability to decrease tumour blood perfusion measured using the RbCl extraction procedure. Injecting 150 mg/kg FAA 3 h before heating (42.7 degrees C) resulted in a 2.2-fold increase in tumour heat damage, but had little effect on the response of normal foot skin in non-tumour-bearing mice. However, this treatment gave a 2.0-fold increase in normal tissue damage when the skin experiments were repeated in tumour-bearing animals. These effects in skin occurred in the absence of any blood perfusion changes, but appeared to be associated with FAA-induced TNF-alpha production.

Effect of nitro-L-arginine on blood flow, oxygenation and the activity of hypoxic cell cytotoxins in murine tumours.

This study was an investigation into the ability of nitro-L-arginine to change blood flow, oxygenation status and the activity of hypoxic cell cytotoxic agents in two different transplanted murine tumours. The tumour models were the C3H mammary carcinoma grown in the feet of female CDF1 mice and the SaF grown on the backs of CBA mice. Treatments were carried out in restrained non-anaesthetised animals when tumours were about 100 to 200 mm3 in size. Blood flow was monitored using laser Doppler flowmetry; oxygen partial pressure (pO2) distributions were obtained with an Eppendorf oxygen electrode; and response to treatment with hyperthermia (43.5 degrees C; 30 min) and RB6145 (250 mg kg-1;i.p.) assessed using a tumour growth delay assay. Nitro-L-arginine (10 mg kg-1; i.v.) significantly reduced blood flow by around 40-60% within 15 min after injection in C3H tumour and by 30 min in the SaF. However, nitro-L-arginine had absolutely no effect on tumour pO2 measured at the time of maximal blood flow reduction in both tumour types. It also failed to enhance the response of the C3H tumour to heat, but did produce a small yet significant increase in the response of the SaF tumour to RB6145.

Relationship between tumour oxygenation, bioenergetic status and radiobiological hypoxia in an experimental model.

Tumour oxygenation and bioenergetic status were measured in the same tumour and these results related to radiobiological hypoxia. A C3H mouse mammary carcinoma grown in the feet of CDF1 mice was used. Bioenergetic status was assessed by 31P MRS using a SISCO 7 Tesla magnet, oxygen measurements were done by a polarographic electrode and the hypoxic fraction was determined from direct analysis of the radiation dose-response data. During all examinations restrained, non-anaesthetized mice were allowed to breathe either 100% oxygen, carbogen, normal air, carbon monoxide (CO) at 75, 220, or 660 ppm or had blood flow occluded by clamping. Results showed a significant correlation between the radiobiological hypoxic fraction and % pO2 < or = 5 mmHg under the different treatment conditions, whereas no correlation was found between beta nucleosidetriphosphate/inorganic phosphate (beta-NTP/Pi) ratio and either the hypoxic fraction or the % of pO2 values < or = 5 mmHg under the different treatment conditions. In conclusion, oxygen electrode measurements were sensitive to changes in tumour hypoxia whereas the bioenergetic status alone seemed to be a less precise measure of hypoxia in this tumour model. Furthermore, the present study demonstrated that tumour cells in vivo can actually maintain the bioenergetic status during a period of severe hypoxia.

The ability of nicotinamide to inhibit the growth of a C3H mouse mammary carcinoma.

Experimental studies have suggested that nicotinamide and its analogs may inhibit the growth of murine tumours. We have now investigated this using a C3H mouse mammary carcinoma implanted into the right rear foot of female CDF1 mice. From days 1 to 30 after implantation mice were intraperitoneally (i.p.) injected with either 100, 200, 500 or 1,000 mg/kg nicotinamide. The tumour volume (+/- 1 S.E.) after 30 days in saline-treated mice had reached 1540 mm3 (+/- 260). No change in tumour growth was seen at that time with daily doses of up to 500 mg/kg nicotinamide, but at 1,000 mg/kg tumour volume was reduced to 904 mm3 (+/- 233). However, this large dose of nicotinamide was also toxic to the mice with some 16% of animals dying during the 30-day treatment period. A similar growth inhibition was seen with daily i.p. injections of 5 mg/kg fumagillin (tumour volume at 30 days = 821 +/- 191 mm3), a known inhibitor of angiogenesis, but whether this mechanism also explains the nicotinamide effect is not clear.

Use of tetrahydraindazolone dicarboxylic acid (HIDA) to improve the therapeutic effect in vivo of combined cisplatin, heat and radiation treatment.

The effect of tetrahydraindazolone dicarboxylic acid (HIDA) on tumour response and mouse lethality after treatment with cisplatin given either alone or combined with hyperthermia (43.5 degrees C/60 min) with or without radiation, was studied in the CDF1 mouse bearing a foot transplanted C3H mouse mammary carcinoma. The tumour response to a combined heat, cisplatin and HIDA treatment was assessed by tumour growth time, while local tumour control was used when irradiation was added to that treatment scheme. Toxicity was estimated as lethality within 14 days. Cisplatin and heat exerted the highest antitumour effect when given simultaneously, but at the same time there was a substantial increase in lethality. No sensitization of the tumour response or enhanced toxicity to cisplatin was observed if heat was given sequentially (i.e. 4 h) after cisplatin. The effect of this sequential schedule being only additive. When HIDA (100 mg/kg) was given 150 min before cisplatin and tumours heated 15 min later, the lethal toxicity was significantly reduced. HIDA did not, however, influence tumour growth time results. In tumour control studies combining radiation, drug and heat, cisplatin (6 mg/kg) and heat (43.5 degrees C/60 min) were given simultaneously 4 h after local irradiating the leg of tumour-bearing mice. The lethality of this regime was more than 55%, but when HIDA was added to the protocol, the toxicity fell to 5% without affecting local tumour control. In conclusion, HIDA administered before cisplatin protects against drug-induced toxicity without reducing the drug's antitumour activity when used alone or in combination with hyperthermia and/or radiation, and thus results in a significantly improved therapeutic benefit.

Cisplatin and hyperthermia treatment of a C3H mammary carcinoma in vivo. Importance of sequence, interval, drug dose, and temperature.

The effect of combining cisplatin and hyperthermia was investigated in a C3H mammary carcinoma in vivo, using a regrowth delay assay. Cisplatin (6 mg/kg) was given i.p. at intervals ranging from 24 h before to 24 h after a 43.5 degrees C/60 min treatment. A supra-additive effect was obtained by giving cisplatin 15 min before heat, whereas an additive effect was obtained at all other intervals. The importance of cisplatin dose and heating temperature were investigated by giving variable cisplatin doses (2-8 mg/kg) 4 h or 15 min before a 60 min heating at temperatures in the range 40.5-43.5 degrees C. Linear relationships between length of regrowth delay and cisplatin dose were obtained both for cisplatin alone and for the combined treatment. The effect of the combined treatment could therefore be quantitated by a ratio (ER) between the slopes of dose-response curves. The ER values for cisplatin give 4 h before a 60 min heating at 42.5 or 43.5 degrees C were not significantly different from 1 (p greater than 0.5). In contrast, significant ER values were obtained above 40.5 degrees C (p less than 0.05) for cisplatin given 15 min before heat. The data demonstrates the possibility of achieving chemosensitization at clinically relevant temperatures.