Molecular Biology to Radiation Oncology: A Model for Translational Research? Opportunities in basic and translational research. From a workshop sponsored by the National Cancer Institute, Radiation Research Program, January 26-28, 1997, Bethesda, Maryland.

Many exciting discoveries are being made that are providing new insights into how molecules, cells and tissues respond to ionizing radiation. There remains a need, however, to translate these findings into more effective treatments for cancer patients, including those treated with radiation therapy. This complex task will require the collaboration of scientists studying molecular, cellular and tissue responses, and those performing clinical trials of emerging therapies. The Radiation Research Program of the National Cancer Institute sponsored a workshop entitled "Molecular Biology to Radiation Oncology: A Model for Translational Research?" to bring together basic scientists and clinicians to exchange ideas and fundamental concepts and to identify opportunities for future research and collaboration. Four broad topics were addressed: signal transduction and apoptosis, the cell cycle, repair of radiation damage, and the microenvironment. The development, selection and use of appropriate experimental models is crucial to finding and developing new therapies, and opportunities exist in this area as well. This paper and the accompanying paper by Coleman and Harris that provides the viewpoint of radiation oncologists (Radiat. Res. 150, 134-147, 1998) summarize the background concepts and opportunities for translational research identified by the workshop participants.

Oxygen in human tumors: correlations between methods of measurement and response to therapy. Summary of a workshop held November 19-20, 1992, at the National Cancer Institute, Bethesda, Maryland.

Knowledge about the oxygenation of human tumors and its importance in the response to radiotherapy is crucial to the effort to develop improved treatment methods for radiotherapy. The measurement of oxygenation of human tumors and correlations with response to radiotherapy were the subjects of a recent workshop sponsored by the National Cancer Institute. The following methods for measuring oxygen or hypoxia, or a parameter related to either, were presented: polarographic oxygen electrodes, the comet and alkaline elution assays for radiation-induced DNA damage, nitroimidazole binding assays, hemoglobin saturation assays, magnetic resonance spectroscopy, electron spin resonance spectroscopy, phosphorescence imaging, and an assay for tumor interstitial pressure. The electron spin resonance, alkaline elution, and phosphorescence imaging methods have not been used in human tumors. The comet assay, nitroimidazole binding assays, magnetic resonance spectroscopy, cryospectroscopy, and near-infrared spectroscopy have been employed in human tumors, but correlations to treatment response have not been made. Polarographic measurements have indicated that the presence of hypoxia correlates with a poor response to radiotherapy in cervical cancers, but additional data are needed on early-stage disease, and with long-term follow-up on local control and survival. If these confirm the correlation between hypoxia and poor response to radiotherapy, additional tumor sites should be studied. Future clinical trials of treatments that sensitize, exploit, or kill hypoxic cells should identify and include the individual patients with hypoxic tumors. Fundamental unanswered questions regarding the assessment of tumor oxygenation concern the need for invasive procedures, the spatial resolution needed for prediction of response to radiotherapy, the importance of reoxygenation, differences between tumors in rates and degrees of reoxygenation, whether measurements made during a course of therapy are of value, and correlations among methods and with other predictive assays such as intrinsic radiosensitivity and potential doubling time.

Pharmacological modification of tumor blood flow: lack of correlation between alteration of mean arterial blood pressure and changes in tumor perfusion.

The correlation between mean arterial blood pressure (MABP) and vascular perfusion in SCC-VII/St tumors in mice was compared following administration of three vasoactive drugs: flavone acetic acid (200 mg/kg), hydralazine (5 mg/kg), or nicotinamide (500, 750, and 1000 mg/kg). MABP was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Vascular perfusion of the tumor was measured using the 86RbCl extraction method. Body temperature was maintained at 36 degrees to 37 degrees C after drug administration when necessary. All three drugs reduced MABP from a control value of 125 +/- 2 (s.e.) mm Hg in mice without tumors. Flavone acetic acid at this dose had the least effect on blood pressure, with a minimum of 86% of control values at 10 to 20 min, and a return to control values by 1 hr. However, it produced a profound reduction in tumor perfusion that lasted more than 48 hr. Hydralazine and nicotinamide reduced blood pressure to minima between 55% and 69% of control values within 30 min, followed by a gradual return toward control values by about 8 hr. The reduction in tumor perfusion by hydralazine paralleled its effect on blood pressure. However, nicotinamide produced a transitory, although not statistically significant, increase in tumor perfusion at the highest dose given. These data demonstrate that tumor blood flow modification by drugs is not necessarily the result of changes in MABP, and blood pressure changes alone do not inevitably lead to changes in tumor perfusion.

A comparison of radiosensitization by etanidazole and pimonidazole in mouse tumors.

Radiosensitization by pimonidazole (Ro 03-8799) was tested in three murine tumors, EMT6/SF using the excision assay, SCC-VII/SF using the excision and growth delay assays, and MDAH-MCa-4 using TCD50 assays with both single doses and 6 fractions of radiation with a 24-hr interfraction interval. Results were compared with those using etanidazole (SR-2508), both at equitoxic doses and at doses giving tumor concentrations similar to those achievable in the clinic. In excision assays with EMT6/SF and SCC-VII/SF tumors, pimonidazole and etanidazole gave similar radiosensitization at similar concentrations in the tumors. Pimonidazole, however, did not demonstrate radiosensitization in SCC-VII/SF tumors in the growth delay assay, despite tumor concentrations that gave maximum sensitization in the excision assay. Furthermore, pimonidazole gave less than expected sensitization in single dose and 6-fraction TCD50 assays with MDAH-MCa-4 tumors, and less sensitization than comparable levels of etanidazole in this tumor line. When the concentration of pimonidazole in the tumors was approximately 0.36 mumoles/g the dose modification factor (DMF = dose without sensitizer/dose with sensitizer to give an isoeffect) was 1.56 (1.40-1.74, 95% c.l.) in single dose TCD50 assays. Etanidazole, however, gave a DMF of 1.92 (1.59-2.32) with a tumor concentration of approximately 0.32 mumoles/g and 1.69 (1.46-1.96) with a tumor concentration of approximately 0.21 mumoles/g. Thus, etanidazole gave more consistent sensitization for different tumors and different endpoints than did pimonidazole. The results appear to confirm the disappointing performance of pimonidazole in the clinic.

Thermal enhancement of radiation-induced leg contracture.

Early and late damage in the normal tissues of the legs of mice was compared following treatment with radiation alone or radiation followed immediately by hyperthermia. Hyperthermia was given by immersing the hind leg in a water bath at 43.0 degrees, 43.3 degrees, or 43.5 degrees C for 1 hr. Damage was assayed by measuring leg contracture at various intervals from 5 to 365 days after treatment. At 5 days after treatment, only hyperthermia-induced contracture was observed. At 10 and 20 days, contracture increased with radiation dose in heated legs, but little contracture had developed in mice treated with radiation alone. By 45 through 365 days, however, contracture correlated with radiation dose both in mice treated with radiation alone as well as in those treated with radiation and hyperthermia. The greatest differential in the slopes of the dose response curves, suggesting hyperthermic radiosensitization, was seen 20 days after treatment. Nevertheless, at 365 days, contracture was still significantly greater in the mice treated with radiation and hyperthermia (43.5 degrees bath) than in the irradiated controls. Thermal enhancement ratios (TERs) were calculated from LCD50 values (LCD50 = radiation dose that would give a stated level of leg contracture in 50% of the mice). For greater than or equal to 3 mm contracture, TERs were 4.1 to 7.9 at 30 days, depending on bath temperature, but only 1.1 to 1.5 at 365 days. For an isoeffect of greater than or equal to 7 mm contracture, TERs were 1.9 to 5.3 at 30 days, and 0.8 to 1.8 at 365 days. Thus, contracture was enhanced more at 20 to 30 days after treatment with radiation and hyperthermia than at 120 through 365 days. Radiation damage not only appeared earlier in mice treated with hyperthermia than in those treated with radiation alone, but after the highest temperature tested (43.5 degrees bath), contracture was greater from 5 through 365 days after treatment than in controls treated with radiation alone.

Misonidazole in fractionated radiotherapy of a murine mammary carcinoma: comparison of tumor and normal tissue response.

The potential therapeutic benefit of misonidazole was tested in radiotherapy with 1, 2, 5, and 10 equal fractions, using as endpoints local tumor control (TCD50) of murine mammary carcinoma MDAH-MCa-4 and leg contracture at the TCD50, measured 120 days after irradiation. In controls and misonidazole-treated mice, the TCD50 increased with the number of fractions, from 66.7 to 114.6 Gy in controls, and from 43.3 to 75.7 Gy with misonidazole. At doses of greater than or equal to 0.1 mg/g body weight, misonidazole reduced the TCD50 in all fractionation schedules; however, because of toxicity, 1.0 and 0.6 mg/g could be given with only 1 or 2 fractions. Leg contracture at the TCD50 was greatest (14.5 mm) in control mice treated with a single dose of radiation, and was least (7.2 to 7.4 mm) in those treated with a single dose of radiation preceded by 1.0 or 0.6 mg misonidazole/g body weight. With 0.1 mg misonidazole/g, the leg contracture at the TCD50 was less (9.8 to 12.2 mm with the various schedules) than in controls (12.0 to 14.5 mm) for 1, 5, or 10 fractions. Therefore, a therapeutic gain could be obtained by using misonidazole with 1, 2, 5, or 10 fractions, but the greatest gain occurred with 1 fraction, with high doses of misonidazole, that is, 0.6 to 1.0 mg/g.

Enhancement of radiation-induced normal tissue damage by a fibrosarcoma.

Normal tissue damage in the legs of mice was enhanced by the presence of a fibrosarcoma (FSa I) in the leg at the time of treatment with single doses of 30 to 70 Gy. Damage was assessed by measuring leg contracture 23 to 365 days after irradiation of 8 mm diameter tumors. Animals with and without tumors were treated with 1.0 mg misonidazole/g body weight 30 min before irradiation to ensure that the tumors did not recur and interfere with the assessment of contracture. The data suggest that in some cases, late damage may be enhanced by destructive effects of the tumor on the tumor bed.

Leg contracture in mice after single and multifractionated 137Cs exposure.

This is a report of studies of time-dose relationships for post-irradiation leg contractures in mice. The isoeffect doses for various degrees of contracture, measured 250 days after irradiation, increased with the number of fractions, but not with the overall treatment times, throughout 30 days. The isoeffect curves relating the total doses for given levels of responses to the doses per fraction were steeper for leg contractures than for acute skin reactions. The alpha/beta ratios ranged from 1.4 to 5.0 Gy, depending on the degrees of contracture. They were less than the 7.5 to 50 Gy for acute skin reactions as determined in previous experiments using the same animals and irradiation systems. Thus, the data resembled those from other slowly-responding normal tissues such as the spinal cord, kidney and lung. The leg contracture consisted of dermatogenic, myogenic, and arthrogenic components; after the mice were sacrificed there was residual contracture following removal of the skin and muscle. Inhibition of bone growth accounted for only a small proportion of the contracture. The overall response reflected responses of several tissue types.

Sensitization by SR-2508 plus Ro 03-8799.

The primary toxicity of Ro 03-8799 is a central nervous system toxicity, whereas that of SR-2508 is a peripheral neuropathy. The feasibility of reducing overall toxicity while maintaining maximal radiosensitization by using the two sensitizers together was tested. The LD50/2 of Ro 03-8799 was 0.68 mg/g body wt (mg/gbw) after intravenous (i.v.) administration, and that of SR-2508 was 4.4 mg/gbw after i.v. administration. When both drugs were given together in equitoxic proportions, the LD50/2 was 0.45 mg of Ro 03-8799 plus 2.9 mg of SR-2508/gbw. These doses are 66% of the respective LD50/2 values of the drugs when given separately. Radiosensitization was evaluated using in vivo-in vitro assays with EMT6/SF tumors in BALB/c mice. At drug doses between 10 and 60% of the LD50/2, sensitization was generally maximal and similar to that from misonidazole, but there was less sensitization below this dose, both with the drugs given separately and together. If chronic toxicities of these drugs overlap as do the acute toxicities there will be little or no additional benefit from using these drugs in combination, compared to using them separately.

Reversible injury after mild hyperthermia.

Skin contraction and leg contracture resulted from immersion of mice legs in a water bath at temperatures of 42.5 degrees to 43.7 degrees C for 45 to 90 minutes. The maximum contracture was observed between 5 and 15 days after treatment, but little damage remained after about 30 days. After healing of the early tissue damage, there was no progression of residual damage in skin up to 490 days after treatment. In contrast, radiation-induced contracture develops rapidly after 14 days, and may continue to progress for 100 days or more. In the present studies, leg contracture could be attributed primarily to injury in the skin, because skinning the legs before measuring eliminated most of the contracture. Temperature differences between subcutaneous tissue and deep muscle were not consistently observed or statistically significant, and probably made little or no contribution to the difference in thermal response of these tissues.

Leg contracture in mice: an assay of normal tissue response.

Leg contracture, defined as the difference in extensibility of the control and irradiated hind legs of mice, was found to correlate with single doses of radiation from about 20 to 80 Gy. The time of development of the early phase of the response coincided with that reported for the appearance of the acute skin response, and in some cases, partially reversed as this reaction healed. The contracture then progressed again at a moderate rate through 90 days, and then more slowly through one year. Skin contraction, measured by decrease in intertattoo distance, was assayed in the same mice. It followed the same time course as leg contracture, but had a different dose-response relationship. Maximal contraction occurred following doses of 30 Gy or more, reaching this level sooner following higher doses. The early reactions in individual mice were not reliable in predicting late response for either assay. To determine the contribution of skin contraction to the overall leg contracture response, mice were sacrificed and the leg contracture measured before and after the removal of the skin of the leg. After doses of up to 30 Gy, little contracture remained from skinning the leg, indicating that skin contraction was largely responsible for leg contracture in this dose range. After doses of about 45 Gy and above, some contracture remained in the skinned legs, although less than in intact legs. This indicated that injury to the deeper tissues of the leg as well as to the skin was responsible for contracture at these higher doses. There was little or no enhancement of either skin contraction or leg contracture by the hypoxic cell sensitizers metronidazole or misonidazole.

Enhancement of local tumour control by misonidazole and hyperthermia.

Combination treatment of a C3H mammary carcinoma with misonidazole, hyperthermia and radiation resulted in greater local tumour control than with any of these agents singly or in pairs. Dose modification factors for the 3-fold combinations were 3.28 when tumours were immersed in a 42.5 degree C waterbath for 1h after irradiation, and 5.03 at 43.0 degrees C. Cytotoxicity of misonidazole alone was slight, as reflected histologically and in tumour growth. Hyperthermia had a marked effect on these two parameters. Foot damage by hyperthermia was greatest when feet were taped.

Modification of radiation responses of murine tumors by misonidazole (Ro 07-0582), host immune capability, and Corynebacterium parvum.

The hypoxic cell sensitizer misonidazole (Ro 07-0582),1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol, significantly enhanced the local control of the weakly immunogenic C3H mouse mammary carcinoma MDAH-MCa-4 (8-mm diameter) by single doses of radiation. The dose modification factor (DMF) was 2.33 when the drug was given ip to inbred C3Hf/Bu mice in a dose of 1 mg/g body weight 30 minutes before irradiation of the tumor. The DMF in a highly immunogenic 3-methylcholanthrene-induced C3H fibrosarcoma (FSa) was 1.65 in normal mice and 1.86 in mice immunosuppressed by 600 rads whole-body irradiation 1 day before tumor transplantation. In mice treated iv with 0.25 mg Corynebacterium parvum when tumors were 6 mm in diameter and irradiated at 8 mm, local control of FSa was enhanced at low doses of radiation but was similar to that in normal mice at higher doses. In mice treated with both misonidazole and C. parvum, local control at lower doses of radiation was similar to that in mice treated with C. parvum alone but was enhanced at higher doses in mice that failed to respond to C. parvum. Cytotoxicity of misonidazole, as reflected in tumor growth, was not detected.

Metronidazole: effect on radiosensitivity of tumor and normal tissues in mice.

Local control of a mammary carcinoma in air-breathing C3H mice was enhanced by giving metronidazole before radiotherapy; When 0.5-1.0 mg metronidazole/g body weight was given ip 10 minutes before irradiation, the dose required for local control of 50% of the tumors (TCD50) was reduced by a factor of 1.37-1.38. The TCD50 was reduced by a factor of 1.48-1.50 when tumors were irradiated 30-60 minutes after 1.0 mg/g. Metronidazole alone, at a dose of 1.0 mg/g, only slightly inhibited tumor growth. The acute skin response of the leg, as determined by the radiation dose required to produce complete moist desquamation in 50% of the animals (DD50), was enhanced by a factor of 1.26 when mice were irradiated 10 minutes after a dose of 1.0 mg metronidazole/g. Clonogenic cells in the jejunal epithelium were not sensitized after metronidazole administration in either air-breathing mice or those irradiated in O2 30 pounds per square inch, but the drug appeared to exert a slight protective effect at higher doses of radiation.