Combined radioimmunotherapy and radiotherapy of human colon carcinoma grafted in nude mice.

The effect of combined radioimmunotherapy (RIT) and fractionated external beam radiotherapy (RT) was assessed in two human colon cancer xenografts, Co112 and LS174T in nude mice. These tumors were selected for being resistant to RIT alone, as is usually the case in the clinical situation. Tumor-bearing mice were treated with a combination of five X-ray fractions over 5 days followed by RIT with two doses of 1.5 mCi 131I-labeled anticarcinoembryonic antigen monoclonal antibody F(ab')2. In Co112 and LS174T, RIT alone achieved a regrowth delay similar to that of fractionated RT with total doses of 28 and 26 Gy, respectively. In both tumor types, an additive therapeutic effect, measured as increased regrowth delay or local control, was observed when combining RT of different dose levels with RIT. Normal tissue responses were assessed by monitoring acute peak skin reactions and blood cell count. Bone marrow depression for the combination treatment was similar to that of RIT alone; relative to skin, at equitoxic levels, no mice bearing Co112 tumors were locally controlled with a 32 Gy RT dose alone, while this RT combined with RIT gave a local control of 100%. These studies show a therapeutic benefit when external beam RT is combined with RIT.

The ribonucleoside diphosphate reductase inhibitor (E)-2'-deoxy-(fluoromethylene)cytidine as a cytotoxic radiosensitizer in vitro.

(E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC) is known as an inhibitor of ribonucleoside diphosphate reductase, a key enzyme in the de novo pathway of DNA synthesis. FMdC was tested as a modifier of radiation response in vitro on a human colon carcinoma cell line (WiDr), and the observed radiosensitization was confirmed on two human cervix cancer cell lines (C33-A and SiHa). Using the clonogenic assay, the effect ratio (ER) at a clinically relevant dose level of 2 Gy was 2.10 (50 nM FMdC), 1.70 (30 nM FMdC), and 1.71 (40 nM FMdC) for the three cell lines WiDr, C33-A, and SiHa, respectively. A more detailed analysis of the importance of timing and concentration of FMdC was done on the WiDr cell line alone, yielding an increased ER(2Gy) with increasing concentration and duration of exposure to the drug, ranging from 1.0 (6 h) to 1.8 (72 h) at 30 nM FMdC and from 1.2 (6 h) to 3.5 (24 h) at 300 nM. We investigated the effect of FMdC on the cellular deoxynucleotide triphosphate pool in WiDr cells and demonstrated a marked depletion of dATP and a significant rise of TTP levels. Cell cycle analysis showed early S-phase accumulation induced by FMdC alone, G2-M block induced by irradiation alone, and an increased accumulation of cells in G2-M if both modalities are used. Our data suggest that FMdC is a radiation response modifier in vitro on different cancer cell lines. The observed radiosensitization may in part be explained by alteration of the deoxynucleotide triphosphate pool, which is consistent with the effect of FMdC on ribonucleoside diphosphate reductase.

Accelerated radiotherapy, carbogen, and nicotinamide in glioblastoma multiforme: report of European Organization for Research and Treatment of Cancer trial 22933.

PURPOSE: A three-step phase I/II trial associating accelerated radiotherapy with carbogen (step 1, ARCO), with nicotinamide (step 2, ARN), or with both (step 3, ARCON) was conducted, the aim of which was to overcome the effects of proliferation and hypoxia as potential causes of tumor radioresistance in glioblastoma multiforme. PATIENTS AND METHODS: Radiotherapy consisted of 60 Gy delivered over 4 weeks in 1.5-Gy fractions twice daily, 5 days a week. Carbogen breathing was started 5 minutes before each fraction and continued until the end of each treatment session. Nicotinamide was given daily as a single oral dose of 85 mg/kg. RESULTS: A total of 115 patients with a median age of 55 years were registered. Of 107 eligible patients, 23 were registered in step 1, 28 in step 2, and 56 in step 3. The planned treatment was administered without any interruption in 72% of patients (86% in ARCO but 68% in ARN and ARCON). The incidence and severity of acute skin and mucous membrane toxicity were higher in patients who received nicotinamide (ie, the ARN and ARCON groups). Grade 1 to 2 gastrointestinal toxicity was observed in 44% of patients in the ARN group and 32% of patients in the ARCON group, but only in 8% of patients in the ARCO group. Eight percent of evaluated patients presented with abnormal liver test results at treatment completion. The dose of corticosteroids had to be increased in 44% of patients. Late neurologic side effects were similar in all treatment steps and were observed mostly in patients with disease progression. Median survival times for patients treated with ARCO, ARN, and ARCON were 10.1, 9.7, and 11.1 months, respectively. CONCLUSION: Feasibility of ARCO treatment was good but that of ARN and ARCON was only fair. This probably reflected the higher acute toxicity rate, particularly gastrointestinal, for patients receiving nicotinamide. The dose of corticosteroids had to be increased frequently during treatment, suggesting a higher than expected acute neurologic toxicity. Overall survival was similar in the three treatment steps and not different when compared with results of other series that used radiotherapy alone.

Vinca alkaloids: anti-vascular effects in a murine tumour.

We have investigated the blood flow modifying effects of the vinca alkaloids, vincristine and vinblastine in the murine carcinoma CaNT. Vinblastine at doses of 7.5 or 10 mg/kg induced profound and chronic reductions in tumour blood flow as measured by 86RbCl extraction. Following the maximum tolerated dose of 10 mg/kg, blood flow was reduced to 10% of pretreatment values after 2 h and remained below 20% of pretreatment values 24 h after drug administration. These findings are consistent with the early induction of necrosis by vinblastine and suggest that vascular-mediated cell death may account for a large part of the 11 day growth delay induced by this drug dose. In contrast to the large reductions in tumour blood flow, in skin, kidney, liver and muscle, blood flow reductions did not, at any time examined, exceed 40%. In all the normal tissues studied, blood flow had fully recovered by 6 h after vinblastine administration. Similar results, albeit less pronounced, have been obtained with vincristine at the maximum tolerated dose of 3 mg/kg. The results clearly show that both vinblastine and vincristine can induce, with some selectivity, a dramatic and prolonged reduction in tumour blood flow and that this may contribute to the anti-tumour effects against the CaNT tumour.

Perivascular cell protection in vivo and increased cell survival in vitro by the antihypertensive agent carvedilol following radiation.

Carvedilol, an antihypertensive drug with activity on adrenoceptors as well as on calcium channel activity, has recently been introduced. In the present study we investigated whether carvedilol interacts with the cytotoxicity induced by irradiation in vitro as well as in vivo. A daily injection of carvedilol in clinically relevant concentrations (3 mg/kg subcutaneously), 4 days before and 3 days after a single radiation dose of 20 Gy significantly decreased the inflammatory reaction in the rat lung, evaluated as number of inflammatory cells in the perivascular area. The density of mast cells was also slightly reduced. In vitro studies revealed that carvedilol caused different radio-protective effects, dependent on dose (1-7 Gy) used and cell line studied. The effects were especially pronounced in a malignant mesothelioma cell line (P-31), and somewhat less evident in a prostatic carcinoma cell line (PC-3). No significant effect was seen in a highly radiosensitive small cell lung cancer cell line (U-1690). Thus, carvedilol may under some circumstances interact with radiation-induced tissue reactions, most probably by a direct interaction at the cellular level. The specific explanation to the differences in sensitivity to carvedilol remains to be evaluated, but the known antioxidative properties and/or scavenging of free radicals of carvedilol may be a plausible mechanism of action. Secondary induced alterations in inflammatory response may also be considered. It is suggested that a potential interaction between drugs such as carvedilol and irradiation should be considered for clinical practice.

The broad spectrum of preclinical radiobiology: British contributions.

British radiobiologists have often been at the forefront in taking clinical questions into the laboratory and in taking the results back into the clinic, i.e., what is nowadays labeled as Translational Research. They have published widely and have been very active in lectures, workshops, and discussions, forming an important component of the international communication web, both within the basic science aspects and in the translation from science to medicine, and back again. Major contributions have been made at the cellular and subcellular level, and at the level of multicellular structures, both normal and malignant. The common features of the response of cells to single doses in well-defined conditions have been used to interpret the much greater complexity of tissue and tumor responses treated with repeated small doses in a fractionated course, both of photons and other radiations, with and without chemical modifiers. The many contributions to the field of cell kinetics have provided the tools with which an understanding has been gained of the latency and evolution of radiation damage in different tissues. The prolonged interest in microenvironmental gradients and compensatory responses to injury have provided a framework for designing better radiotherapy schedules, and considerable spin-off to other branches of cancer therapy.

The influence of X ray dose levels on normal tissue radioprotection by WR-2721.

A variation in the degree of radioprotection by WR-2721 with X ray dose level is detectable in several normal tissue studies. A similar effect in tumors has been attributed to differential protection of oxic and hypoxic cells. For normal tissues it was previously postulated that it resulted from greater protection of 1 hit damage at low doses, with less protection of multihit damage. However, more extensive analysis of the normal tissue data, including both single dose and fractionated results show that it is not a universal effect in all normal tissues. It now seems more likely that varying PF values result from differential protection of cells at different oxygen tensions, even though the heterogeneity of oxygenation may not be detectable in the response to X rays alone.

Superfractionation as a potential hypoxic cell radiosensitizer: prediction of an optimum dose per fraction.

PURPOSE: A dose "window of opportunity" has been identified in an earlier modeling study (1) if the inducible repair variant of the LQ model is adopted instead of the pure LQ model, and if all survival curve parameters are equally modified by the presence or absence of oxygen. In this paper we have extended the calculations to consider survival curve parameters from 15 sets of data obtained for cells tested at low doses using clonogenic assays. METHODS AND MATERIALS: A simple computer model has been used to simulate the response of each cell line to various doses per fraction in multifraction schedules, with oxic and hypoxic cells receiving the same fractional dose. We have then used pairs of simulated survival curves to estimate the effective hypoxic protection (OER') as a function of the dose per fraction. RESULTS: The resistance of hypoxic cells is reduced by using smaller doses per fraction than 2 Gy in all these fractionated clinical simulations, whether using a simple LQ model, or the more complex LQ/IR model. If there is no inducible repair, the optimum dose is infinitely low. If there is inducible repair, there is an optimum dose per fraction at which hypoxic protection is minimized. This is usually around 0.5 Gy. It depends on the dose needed to induce repair being higher in hypoxia than in oxygen. The OER' may even go below unity, i.e. hypoxic cells may be more sensitive than oxic cells. CONCLUSIONS: If oxic and hypoxic cells are repeatedly exposed to doses of the same magnitude, as occurs in clinical radiotherapy, the observed hypoxic protection varies with the fractional dose. The OER' is predicted to diminish at lower doses in all cell lines. The loss of hypoxic resistance with superfractionation is predicted to be proportional to the capacity of the cells to induce repair, i.e. their intrinsic radioresistance at a dose of 2 Gy.

Timescale of evolution of late radiation injury after postoperative radiotherapy of breast cancer patients.

PURPOSE: To evaluate the incidence and prevalence of various signs of late morbidity, their time of appearance and pattern of progression during an observation period up to 34 years in breast cancer patients treated with postoperative radiation therapy after radical mastectomy. METHODS AND MATERIALS: A group of 71 breast cancer patients received in 1963-1965 aggressive postoperative telecobalt therapy to the parasternal, axillary, and supraclavicular lymph node regions after total mastectomy and axillary clearance. None of the patients received chemotherapy either prior to, or after the irradiation as part of their primary treatment. The prescribed dose to the three lymph node regions was 44 Gy in 11 fractions. Only two of the three fields were treated per day. This total dose was given in 16-17 fractions over 3-4 weeks. Because of the overlap of the supraclavicular and axillary fields, the dose received by the brachial plexus was not the dose that was prescribed. A retrospective dose calculation showed that the total dose to the brachial plexus was 57 Gy, delivered as a complex combination of 1.8 Gy, 3.4 Gy, and 5.2 Gy fractions. This cohort of patients has now been followed to 34 years and the late side effects of the treatment evaluated and scored. RESULTS: This series is unique in the literature. There is no comparable report of a detailed long-term follow-up in a homogeneously treated group of patients with such a high survival, especially among the younger women, where it is almost 50% at 30 years. This is the reason that they were able to develop some of the very slowly evolving injuries. There was progression of many of the late effects in the period between 5 and 34 years. The more serious morbidities have increased progressively over the whole 34-year follow-up period. Ninety-two percent of the long-term survivors have paralysis of their arm. Other neurological findings included unilateral vocal cord paralysis among 5% of the patients, who developed the disease after a median time of 19 years. All of them were left-sided, indicating a mediastinal involvement of the recurrent nerve. Local recurrence or the appearance of a new primary tumor infiltrating or causing pressure on the recurrent nerve were vigorously investigated and excluded as possible causes of these symptoms. CONCLUSION: The greatest risk for all cancer patients is the inadequate treatment of their disease, because this is inevitably lethal. The aggressiveness of the therapy and the acceptable risk of complications must therefore be balanced against the risk of recurrence. The neuropathy seems to be closely linked to the development of fibrosis around the nerve trunks. The use of large daily fractions, combined with hot spots from overlapping fields contributed to the severity of the complications.

Modification of the radiation response of the mouse kidney by misonidazole and WR-2721.

The radiation response of the mouse kidney has been assayed after a range of X ray doses given with or without the nitroimidazole misonidazole or the aminothiol WR-2721. Sensitization and protection of the kidney were investigated by comparing the X ray dose needed to achieve a particular level of injury in the presence or absence of the drug. Two functional assays and kidney weight at sacrifice were used to obtain dose response curves. Urine output and 51Chromium EDTA excretion were used as functional assays at 25 and 49 weeks after irradiation. They demonstrated no radio-sensitization by misonidazole with 1, 2 or 5 fractions of X rays. Significant radioprotection was seen when 400 mg kg 1 WR-2721 was given before single X ray doses (PF = 1.34). Similar radioprotection was observed when renal weight at 1 year after irradiation was used as the third assay of damage. These results confirm that the kidney responds as a well-oxygenated normal tissue with only a small protection being afforded against radiation injury by WR-2721.

Radiation induced renal damage in mice: influence of fraction size.

Two functional assays (urine output and isotope clearance) have been used to assess the response of mouse kidneys to localized irradiation. The influence of the size of each X ray dose has been investigated by using single doses and two to 16 equal fractions. The X ray dose in each treatment ranged from 16 Gy as a single dose to 3.5 Gy (X 16 fractions). Three separate experiments were performed, one with and two without anesthetic for the irradiation. Sequential testing of the mice was used to determine the latent period before radiation damage became manifest. Latency was found to be dose dependent; functional defects appeared earlier after higher doses but there was a minimum period of 14-19 weeks before the onset of damage. The repair capacity of the kidney was assessed by comparing isoeffective doses from the dose-response curves. Within 24 hours a recovered dose of 5 Gy was obtained if 2 doses were used instead of one. The isoeffective dose increased with fractionation and a fraction number exponent of 0.42 was obtained. Analysis of the data using a linear quadratic model yielded a low alpha/beta ratio of 0-3.5 Gy. This is similar to values obtained for other late responding normal tissues and implies that the use of small dose fractions will spare the kidney relative to tumors and acutely reacting normal tissues. In conventional radiotherapy more effective sparing of the kidney should be achieved by using thin shielding with each fraction than by completely shielding the kidney for the latter part of the treatment course.

A comparison of vascular-mediated tumor cell death by the necrotizing agents GR63178 and flavone acetic acid.

A vascular component of tumor damage has been identified for the anticancer agent GR63178. The necrotizing activity of this drug and of flavone acetic acid has been compared with their ability to induce growth delay in six murine tumor models. At 24 hr, after a fixed dose of flavone acetic acid (200 mg/kg), all six tumor types appeared 80-100% necrotic histologically, although growth delays ranging from 3 to 79 days were measured. GR63178 (200 mg/kg) induced more variable degrees of necrosis (10 to 95%), but a uniformly small delay in growth (0 to 4 days). These data illustrate that the absence of a tumor-growth response should not be automatically equated with an absence of drug activity. Without assessing tumor response histologically, agents with unusual mechanisms of action may be missed, despite their potential for killing large numbers of tumor cells.

Radioprotection of normal tissues of the mouse by hypoxic breathing.

Hypoxic breathing during irradiation has been advocated as a therapeutic modality, to increase the efficacy of radiotherapy. In this form of treatment, the total and daily X-ray dose is increased by a factor of 1.25, on the assumption that all normal tissues in the beam will be protected to a similar extent by breathing gas containing a reduced oxygen concentration (usually 10%). To test this concept, we have determined the effect of varying the inspired oxygen tension on the radiosensitivity of 3 normal tissues in the mouse (kidney, jejunum and skin), and have compared these results with data from the literature for mouse lung. Reduction of the inspired oxygen tension from 21% (air) to 7-8% led to much greater radioprotection of skin (protection factor 1.37) than of lung (1.09). Protection factors for jejunum and kidney were 1.16 and 1.36 respectively. The results show that the extent of radioprotection afforded by hypoxic breathing is tissue dependent, and that great care must be taken clinically in choosing the increased radiation dose to be used in conjunction with hypoxic breathing.

Radioprotection of two mouse tumors by WR-2721 in single and fractionated treatments.

The radioprotective effect of WR-2721 has been studied in two murine tumors using single doses or five daily fractions. Single dose irradiations of the SA FA resulted in a highly variable radio-protective response. In one experiment large protection factors (1.2-2.5) were obtained, with the greatest protection at low X ray doses. In later experiments with the same tumor, there was little or no radioprotection. The Ca MT was significantly protected against single dose irradiation with both 250 mg/kg and 400 mg/kg of the drug. In a five fraction schedule the extent of radioprotection for CA MT was greater than with single X ray doses for the same drug dose per fraction. Tumor protection factors from the present work and from the literature are compared with published protection factors for normal tissues. Significant tumor radioprotection is seen in most studies. The data indicate more variability in the extent of tumor protection for a given drug dose than is seen in normal tissues. Tumor protection is often greatest at low X ray doses which may be a result of preferential protection of the better-oxygenated tumor cells.

Experimental radiotherapy with WR-2721 and misonidazole.

The data from a large series of experiments on mouse skin and tumors are summarized. Radioprotection with WR-2721 has been observed in both tumors and normal tissues. The protection factors are generally, but not always, higher for skin than for tumors. The protection observed in skin is greater for mice irradiated breathing air than for those irradiated in oxygen. It is postulated that the different protection factors observed in different normal tissues and tumors may reflect differences in tissue oxygenation levels. The combination of misonidazole and WR-2721 has been studied in terms of the modification of radiosensitivity and also as the modification of lethal toxicity. An interaction has been observed in all aspects. The toxicity of WR-2721 increases in the presence of misonidazole. The WR-2721 radioprotection of both skin and tumors decreases if the sensitizer is added. Likewise the radiosensitization with misonidazole is diminished when WR-2721 is present. These results indicate an interaction at the site of radiation injury and they also demonstrate that WR-2721 can adequately penetrate into hypoxic tumor cells.

Tumor sensitization and protection: influence of stromal injury on estimates of dose modification.

Tumor regrowth delay is an assay which reflects tumor cell kill but can be modified by growth rate changes resulting from damage to the stroma (tumor bed effects). If the stromal damage is modified by radiosensitizers and radioprotectors to a different degree from the tumor cells, the overall measurement of dose modifying factors may be influenced by the choice of a regrowth size for the growth delay analysis. We have studied the response of two mouse tumors; a fibrosarcoma which showed only a small TBE and a carcinoma which showed a very large TBE. Sensitizer enhancement ratios and protection factors have been obtained by assessing regrowth to a variety of endpoint sizes. The dose modifying effects on the stroma have then been determined by analyzing the regrowth rates of tumors after irradiation. The choice of endpoint size modified both the sensitizer enhancement ratio and the protection factor for both tumors. It appears that stromal damage may be the cause of the radioprotection observed in the carcinoma, whereas direct tumor cell radioprotection is indicated in the fibrosarcoma. Both direct tumor cell killing and cell death secondary to stromal damage will play an important role in determining the local control of irradiated tumors.

Hyperfractionation as an effective way of overcoming radioresistance.

PURPOSE: To model the influence of hypoxic radioprotection in fractionated treatments over a range of fraction sizes. To determine whether there is a "therapeutic window" of dose per fraction where hypoxic radioresistance could be reduced, and if so, where it occurs in different cell lines. MATERIALS AND METHODS: A mathematical model has been used to simulate the response of cells to low doses of radiation, in the region of clinical interest. We have used the inducible repair variant of the linear quadratic (LQ) equation, with a hypersensitive region (alphaS) at low doses that gradually transforms to the accepted "resistance" in the shoulder region (alphaR). It contains two new parameters, the ratio alphaS/alphaR, and D(C). We have accepted that the "induction dose" D(C) is modified by anoxia to the same extent as the other parameters. We have initially modeled using theoretical parameters and then checked the conclusions with 14 sets of published experimental data for cell lines investigated for inducible repair. RESULTS: We have computed the clinical hypoxic protection (OER') as a function of dose per fraction in simulations of clinical fractionated schedules. We have identified a therapeutic window in terms of dose per fraction at about 0.5 Gy, where the OER' is minimized, regardless of the precise cell survival curve parameters. The minimum OER' varies from one cell line to another, falling to about 1.0 if alphaS/alphaR = 6-10 and even far below 1.0 if alphaS/alphaR > or = 20. DISCUSSION: Hyperfractionation using 0.5 Gy fractions may therefore be more effective than oxygen mimetic chemical sensitizers, since it could even make some tumor cells more sensitive than oxic normal tissues. The tumor lines that benefit most from this type of sensitization are those with the highest intrinsic oxic radioresistance, i.e. those with high SF2 values.

The therapeutic advantage of combined X-rays and melphalan.

Early skin reactions on mouse feet and delay in growth of a mouse tumor (CA NT) were measured after combined treatments with X-rays and the cytotoxic drug Melphalan. The drug was given as a single dose (10 mg kg-1) with graded single doses of X-rays, either before or after irradiation with an interval of up to 4 days. In the mouse skin, addition of the drug increased the radiation response only slightly. The maximum Enhancement Ratio (ER) measured at a skin reaction of 1.5 (approximately 23 Gy) was 1.07 +/- 0.02 SEM for the schedule MEL 3 days before X-rays. ER's for all schedules tested were similar with a range of 1.00 to 1.07. For the tumor more enhancement was observed; the largest ER's were found when Melphalan was given before rather than after irradiation, with maximum ER's of 2.3 and 2.4 when the drug was given 3 days or 1 day before X-rays. This has been attributed to reoxygenation of hypoxic cells after drug treatment, rendering the tumor more radiosensitive. The range of ER over all schedules was 1.5-2.4. Since ER is greater for the tumor than skin for all schedules, a therapeutic advantage is indicated under these specific experimental conditions of single X-ray and drug doses.

Dose-response relationships for human tumors: implications for clinical trials of dose modifying agents.

Clinical benefit from dose modifying agents depends upon the effectiveness of the agents and the steepness of dose response curves for the local control of human tumors by radiotherapy. We have analyzed the two prospective trials and the many retrospective analyses of clinical data from the literature to determine what dose increment is needed to increase local control from 40 to 60%. This increment ranges from 3 to greater than 35%. Thus a dose modifying factor of at least 1.03 (to greater than 1.35) will be necessary for clinical detection of the benefit of a new modality, even if 135 patients are included in each arm of a trial. Two dose levels in the new treatment arm would ensure that therapeutic advantage could be assessed, and would also generate prospective dose response information.

Chemosensitization of mouse tumors by misonidazole.

The chemosensitizing action of misonidazole when used in combination with chemotherapeutic drugs has been assessed in two mouse tumors. Regrowth delay has been used as the assay, and by producing dose-response curves the effect has been classified as additive or interactive. A very small additive effect was seen with bleomycin and adriamycin. A larger additive effect was seen with cyclophosphamide, and a dose dependent interaction was seen with melphalan. The misonidazole dose needed to produce these effects has a threshold of 0.5--0.75 mg/g. Two other nitroimidazoles (Ro 05-9963 and Ro 03-8799) were no more effective than MISO when used with melphalan or cyclophosphamide, even though Ro 05-9963 was much more cytotoxic as a single agent. A distinct enhancement of normal tissue toxicity (LD50/30) was observed for MISO combined with melphalan or cyclophosphamide in two strains of mice. However, the tumor sensitization was bigger than the normal tissue effect, resulting in a therapeutic gain in 3 out of 4 comparisons.