Influence of time interval between surgery and radiotherapy on tumor regrowth.

UNLABELLED: In order to evaluate the influence of time intervals between tumor cell injection and radiotherapy on tumor control and regrowth after surgery, we performed two kinds of experiments on C3D2F1 mice bearing a mammary carcinoma inoculated in the foot or leg. 1st experiment: tumor in foot. END POINT: Tumor Control Probability (TCP). Single dose radiation treatments (RT) were administered at different period times from injection time of tumor cells (day 1). 1st group: unirradiated control, 2nd group: RT on day 2 (TCP50 29 +/- 2.1 Gy), 3rd group: RT on day 7 (TCP 52.5 +/- 2.9 Gy), 4th group: RT on day 12 (TCP50 61.9 + 2.4 Gy). 2nd experiment: tumor in leg. END POINT: percentage of tumor regrowth. Mice were randomly assigned to three groups: 1st control group (tumor growth in all mice), 2nd surgical excision of macroscopically evident tumor on day 7-9 from injection (tumor regrowth in 85% of mice), 3rd as the previous group plus 30 Gy radiation treatment within 24 hours from excision (tumor regrowth in 33% of cases). The radiation dose was selected on the basis of TCP50 observed in the 1st experiment for mice with sub-clinical disease. These data indicate that the radiation dose able to control 50% of tumors increases with the time interval between tumor cells injection and RT. A short time interval between surgery and RT should increase the probability of local control, supporting the rationale of intraoperative radiation therapy (IORT) as adjuvant therapy after surgical resection, when subclinical residual cells are suspected.

Combined use of gemcitabine and radiation in mice.

The aim of this study was to explore, in a murine tumor, if the effectiveness of radiation, in doses and schedules commonly used in clinical practice is potentiated by the combined use of the recently developed drug gemcitabine. Gemcitabine (30-360 mg/kg b.w.) was administered i.p. in female C3D2F1 mice bearing a mammary adenocarcinoma alone or combined with X-rays. Firstly, gemcitabine (single administration) was administered alone or at 20 min, 4 h, and 24 h before X-ray treatments. The significant effect observed only at 24 h time interval, depended on the X-ray dose and not on the gemcitabine dose. Secondly, 4 gemcitabine administrations every 3 days were used in fractionated combined schedules (overall treatment time of 10 days). We studied the relationship among different doses of gemcitabine, alone or combined with 10 daily X-ray treatments (2 Gy/fraction). We observed an interactive effect of gemcitabine up to its threshold dose of 60 mg/kg/fraction. Furthermore, 10 X-ray daily treatments and 4 X-ray treatments every 3 days (total doses 20-40 Gy) were performed with gemcitabine 60 mg/kg/fraction to study the effect of different doses and schedules of X-rays. Tumor growth delays increase with higher X-ray doses, and this occurs more with 4 X-ray treatments than with 10 X-ray treatments. Our results re-affirm the uselessness of high gemcitabine doses, and indicate the effectiveness of combined gemcitabine-radiation fractionated protocols.

Schedule dependent toxicity and efficacy of combined gemcitabine/paclitaxel treatment in mouse adenocarcinoma.

Increased interest in combining drugs with different targets has emerged over recent years. Our study aims at evaluating the effectiveness of combined gemcitabine/paclitaxel treatment taking into consideration doses, schedules, and toxicity. A spontaneous mammary carcinoma was transplanted into the right-hind foot of C3D2F1 mice. Paclitaxel (in doses from 20 to 80 mg/kg b.w.) and gemcitabine (in doses from 30 to 480 mg/kg b.w.) were administered i.p. in single or fractionated treatments. Toxicity and tumor growth delay (TGD) were the endpoints. TGDs for different gemcitabine doses in single administration (120, 240, and 360 mg/kg) overlapped (TGD approximately = 2.5 days). Toxicity was very high in daily administration. Results with gemcitabine alone showed the efficacy of treatments every 3 days. TGDs in fractionated treatments of 60 and 120 mg/kg x 4 were of approximately equals 16 days. Also in this case, tumor growth curves overlapped pointing out the uselessness of the high drug doses. For combined treatments, we used only fractionated protocols, administering gemcitabine every 3 days. Paclitaxel was administered alone in one or two fractions and with different sequences in respect to gemcitabine administration. With 120 mg/kg of gemcitabine all the protocols showed an increased unacceptable toxicity. The best result was obtained administering paclitaxel 40 mg/kg on days 1 and 15 and gemcitabine 60 mg/kg on days 3, 6, 9, and 12 (TGD = 38.2 days). The light toxicity and the high efficacy obtained with this protocol indicate the possible use of gemcitabine/paclitaxel treatment in clinical practice.

Hyperthermia and paclitaxel--epirubicin chemotherapy: enhanced cytotoxic effect in a murine mammary adenocarcinoma.

Multimodality therapy is considered of great interest in the treatment of locally advanced solid tumours. In previous experiments, paclitaxel (TX) and epirubicin (EP) were combined with different schedules, obtaining a superadditive effect on the growth of a murine mammary carcinoma. In the present study, the authors have analysed the possible use of hyperthermia (HT) to increase the efficacy of TX and EP combinations. Tumours were transplanted into the right hind foot of female hybrid (C3D2F1) mice. Both TX and EP were administered i.p in two different doses. Hyperthermia was applied using a water bath at 43.2 degrees C for 1 h. Results were analysed in terms of Tumour Growth Delay (TGD). The maximum tolerated doses in combined protocols were TX 45 mg/kg and EP 9 mg/kg, with an interval time of 24h between the two administrations. TGDs of some of the schedules performed are reported: EP + HT = 11 days, TX + HT = 16 days, TX + EP (with an interval time of 24 h) = 14 days, and TX + EP + HT = 22 days. In the experimental model, HT significantly increases the effects of both TX and EP. TX + EP + HT treatment is the most effective (significantly different from TX + EP), but not in a significant way when compared to TX + HT treatment. These results suggest the possible use of a TX + HT protocol for local tumour response, whereas EP could be added in order to achieve a better systemic control.

Hyperthermia enhances the response of paclitaxel and radiation in a mouse adenocarcinoma.

PURPOSE: The aim of our study was to investigate if the efficacy of paclitaxel and paclitaxel-radiation treatments in vivo could be enhanced by hyperthermia. MATERIALS AND METHODS: Paclitaxel was administered i.p. in doses from 30 to 60 mg/kg b.w. to (C3D2F1) mice bearing spontaneous mammary carcinoma. Local hyperthermia (41 degrees, 42 degrees, 43 degrees C) was carried out by immersing tumor-bearing legs in a water bath for 1 h. Single X-ray treatments from 10 to 90 Gy were performed. Tumor growth delay (TGD) or tumor control dose (TCD(50), radiation dose needed to induce local tumor control in 50% of irradiated animals) were the endpoints. RESULTS: A significant increase of dose-dependent growth delay was observed in paclitaxel and 43 degrees C hyperthermia combined treatments, and a superadditive effect was seen with paclitaxel 45 mg/kg. Combined treatments with hyperthermia at 41 degrees and 42 degrees C were less effective. Administration of paclitaxel 24 h, 4 h, and 15 min before or 15 min and 4 h after hyperthermic treatments produced similar results (TGDs varying from 22.1 to 17 days), and administering paclitaxel 48 h before or 24 h after hyperthermic treatments decreased TGDs (about 10 days). Trimodality treatment (paclitaxel 45 mg/kg, hyperthermia, and X-ray), with a TCD(50) of 14. 1 Gy, in respect to the TCD(50) of 53.1 obtained with X-ray alone, was the most effective. CONCLUSIONS: Hyperthermia enhanced the effectiveness of paclitaxel in all the tested protocols. Our results show a superadditive effect of paclitaxel 45 mg/kg combined with a hyperthermic treatment of 1 h at 43 degrees C. Trimodality treatment, evaluated in terms of percentage of cures, shows a very high enhancement ratio.

Greater antitumor efficacy of paclitaxel administered before epirubicin in a mouse mammary carcinoma.

Combined treatment with paclitaxel and anthracyclines is increasingly being tested in clinical practice. Epirubicin is in general administered before paclitaxel. We have investigated, using a murine mammary adenocarcinoma, whether the efficacy and toxicity of this combination is influenced by treatment sequence, different time intervals and dose intensity. The tumor was transplanted into the right hind foot of C3D2F1 female mice. Paclitaxel was administered i.p. in doses ranging from 15 mg/kg to 75 mg/kg and epirubicin (i.p. or i.v.) in doses from 9 mg/kg to 30 mg/kg. The hepatic and peritoneal toxicity observed with epirubicin administration increased in combined treatments (stronger with i.p. than i.v. epirubicin administrations) and was dose-dependent. When paclitaxel and epirubicin were administered simultaneously or paclitaxel was given 24 h before epirubicin, the same tumor growth delays were obtained in all groups. A smaller effect was observed when paclitaxel was administered 24 h after epirubicin. Increasing the epirubicin or paclitaxel dose led to higher tumor growth delays but also an increased toxicity. In conclusion, in this experimental model, the administration of 45 mg/kg paclitaxel before 15 mg/kg epirubicin was very effective and the increased toxicity can be limited by introducing an interval of 24 h between drug administrations. These results should be considered when designing clinical trials.

Enhancement of radiation response by paclitaxel in mice according to different treatment schedules.

PURPOSE: The aim of our study was to determine if paclitaxel could be used as a radiosensitizer in vivo. MATERIALS AND METHODS: Paclitaxel was tested as a single agent and combined with an X-ray treatment. Paclitaxel was administered i.p. in doses from 30 to 120 mg/kg b.w. to (C3D2F1) mice bearing spontaneous mammary carcinoma. Tumor growth delay (TGD) or tumor control dose (TCD50, radiation dose needed to induce local tumor control in 50% of irradiated animals) and moist desquamation dose (MDD50, radiation dose needed to induce serious moist desquamation in 50% of the non-tumor-bearing feet) were the endpoints. DNA flow cytometric analysis was performed. RESULTS: DNA analysis demonstrated a G2/M block of tumor cells and a depletion of cells in S phase, with a maximum at 24 h from paclitaxel administration. Administering paclitaxel, in graded doses, 15 min before a 10-Gy X-ray treatment resulted in a linear regression line, almost parallel to that with paclitaxel alone, with a growth delay of about 6 days. In contrast, varying the X-ray dose with a constant paclitaxel injection (45 mg/kg b.w.) treatment showed some degree of synergism as the linear regression curves diverged. Interval time and sequence between paclitaxel administration and a 10 Gy X-ray treatment did not influence TGD. Protocols with paclitaxel at 30, 45, or 60 mg/kg were combined with radiation treatments at various doses (from 10 to 65 Gy). Values of TCD50 varied from 50.8 Gy for X-ray alone to 31.8 Gy for paclitaxel 60 mg/kg + X-ray. No differences were observed among MDD of different protocols. CONCLUSIONS: These results suggest that, under some conditions, paclitaxel combined with radiation can show superadditive effects and this result combined with the lack of severe normal tissue damage indicate that a favorable therapeutic gain can be obtained.

4'-Epi-doxorubicin and hyperthermia in a murine mammary carcinoma: influence of the dose and fractionation.

Our aim was to analyse the dose-response rate of 4'-epi-doxorubicin (EP) alone and of EP combined with hyperthermia (HT) treatments in tumor-bearing mice. A spontaneous mammary carcinoma, transplanted into the right foot of female hybrid (C3H/RIxDBA/2J) mice, was used. EP (from 5 to 30 mg/kg) was administered i.p. and local HT (45-60 minutes at 42 or 43 degrees C) was carried out. Mice were treated with EP and/or HT in 1, 2 or 3 doses at 8 day intervals; in the case of 3 HT treatments EP was administered before the first or before each HT session (same EP total dose). When EP was given alone, in 1 or 2 fractions, results showed a clear dose-response relationship: tumor growth delay depended on the total dose only. Combining different EP single doses and 1 HT treatment (43 degrees C), an additive effect and perhaps a synergistic effect at the highest doses was observed. Among all tested combinations, the best results were observed combining 3 HT with only 1 EP treatment.

Invivo control of tumor-growth with the association of ionidamine with radiation and or hyperthermia.

The effect of lonidamine (LND) in association with fractionated doses of radiation and/or hyperthermia on tumor growth has been evaluated. The results may be summarized as follows: (i) the fractionation of the radiation dose, in spite of the higher dose delivered (20 Gy) does not increase tumor growth delay when compared to that obtained with a single irradiation treatment. A similar behaviour was also found when the fractionated irradiation was associated with LND. (ii) Hyperthermia is less effective than radiation in controlling tumor growth, but its effect is potentiated by LND although to a lesser extent that with radiation. (iii) LND delivered together with hyperthermia and radiation arrests tumor growth, and leads to the disappearance of the tumor in 75% of mice. The possible mechanisms underlying these effects are discussed.

Combined radiation and hyperthermia: effects of the number of heat fractions and their interval on normal and tumour tissues.

The effect of one or more heat treatments at 43 degrees C combined with conventional multifraction irradiation was studied in murine normal and tumoral tissues. The endpoint of this study was the TCD50 of the C3H mammary carcinoma, inoculated into the foot. For normal tissue the reaction of the skin of the foot was assessed according to a graded scoring system. Twenty X-ray fractions delivered in 20 or 26 days were combined with one, four or eight fractions of quasi-simultaneous or sequential hyperthermia (i.e. heat applied 4 h after irradiation). Tumour dose-response curves show a decrease in TCD50 and in increase in TER linked to the increasing number of heat sessions. A qualitatively similar effect of the number of sessions was observed for the different protocols (quasi-simultaneous and sequential) and for the two overall times. No significant difference was measured between one and four HT sessions plus 20 fx of RT in the normal tissue damage, while a slight difference was observed between one, four and eight HT sessions delivered with 20 fractions of RT in 26 days.

Effect of in vivo hyperthermia on thymocyte maturation and selection.

Two-month-old male mice were exposed to whole-body hyperthermic treatment in a circulating water bath. After 1 h exposure to 41 degrees C, mice were kept at room temperature for 24, 48, 72, and 96 h before thymus examination. The total thymocyte number progressively decreased after 24 and 48 h, reached a minimum value at 72 h, and returned to almost normal value after 96 h. Similar changes occurred in the CD4+CD8+ cell subset. Conversely, the percentage of CD4-CD8- cells rose to a maximum at 48 h and then declined to normal value at 96 h. The percentage of CD4-CD8+ and CD4+CD8- cell subsets increased to a maximum at 48 h and then declined to normal value at 96 h. These variations in single positive cell subsets correlated well with similar changes in the thymocyte mitotic response to concanavalin A. The observed effects on thymocytes were heat dose dependent, and suggest that hyperthermia induces the development of the most primitive thymocytes and positively selects mature T cells with high mitotic responsiveness. It is proposed that heat shock proteins might be involved in the hyperthermia-induced alterations of thymocyte maturation and selection.

Invivo control of tumor-growth by lonidamine and radiations - influence of the timing and sequence of drug administration and irradiation treatment.

It has been well established that Lonidamine (LND), [1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid], affects tumor growth and enhances the effect of X-ray both in vitro and in vivo. Nevertheless, difficulties arise if the available experimental data should be utilized to design clinical trials since schedules, routes of administration as well as dosages greatly differ from those currently employed in the clinic. With the aim to overcome these difficulties, experiments with modalities similar to those employed in the current clinical practice have been undertaken to evaluate: (i) the influence of the LND dosage on the antitumor effect; (ii) the time lenght of its administration for the optimal effect; (iii) the best schedule of treatment when LND is associated with radiations. The results may be summarized as follows: (i) antitumor effectiveness of LND, in terms of growth delay, increases with LND dosage. Moreover, the drug administered from the day of transplant significantly decreases the tumor takes. (ii) to exert the antineoplastic effect LND must be administered continuosly because if the treatment is interrupted the tumor regrows like an untreated one. (iii) the maximal response of the association X-ray-LND is elicited when the drug is given after irradiation treatment.

Radiotherapy and hyperthermia. Analysis of clinical results and identification of prognostic variables.

Site- and tumor-specific data obtained from two groups of patients with head and neck and melanoma lesions, respectively, showed that both immediate response and response duration were enhanced by the addition of heat. Two important variables, however, such as tumor volume and "isoeffect thermal dose" appeared to influence local tumor control. The volume effect was less pronounced in the lesions treated with radiotherapy plus heat than in those treated with radiotherapy alone, suggesting that the addition of heat was more damaging to the large than to the small lesions. Furthermore, a striking isoeffect thermal dose-response relationship was shown in head and neck lesions. Those data were collected and used to design a mathematical model relating the probability of local control to clinical and treatment variables. The analysis shows that, by using the same radiation parameters, the probability of local tumor control is a function of both "isoeffect thermal dose" and tumor volume.

Tumour response to heat and radiation: prognostic variables in the treatment of neck node metastases from head and neck cancer.

A total of 38 patients with 81 multiple neck node metastases from squamous cell carcinoma of head and neck were treated with radiotherapy alone or with radiotherapy plus hyperthermia. Irradiation was delivered following a three fractions per day schedule of 2 + 1.5 + 1.5 Gy/day, with 4 h intervals between fractions, up to a total dose of 60 Gy. Heat was applied by means of a 500 MHz apparatus. Temperature data were converted to equivalent minutes at 42.5 degrees (Eq 42.5). Initial complete response rates and local control distribution were compared for subgroups of tumour volume and thermal dose. The data indicated that the volume effect was less pronounced in the combined modality than in the radiation alone arm, suggesting that the addition of heat was more damaging to the large than to the small lesions. A striking thermal dose-response relationship was shown, although complete response rates increased only after a certain thermal dose was accumulated, clearly indicating the presence of a threshold dose.

Orgotein as a radioprotector in normal tissues. Experiments on mouse skin and a murine adenocarcinoma.

The effects of Orgotein (a superoxide dismutase) on the radiation response of normal and malignant murine tissue in vivo were evaluated. The observations were made on the mouse hind leg bearing, in some cases, an adenocarcinoma. The following irradiation protocols were tested: 1) single dose (e.g., 35 and 53 Gy), 2) conventional fractionation (3 Gy/day, 5 days a week) and 3) multiple fractions per day (2 X 3 Gy/day, 3 h fractionation interval, 5 days a week). Radiation was either delivered alone or preceded by a subcutaneous injection of 20, 100 or 400 mg/kg Orgotein administered 1 or 2 h before the beginning of irradiation. No effects of Orgotein on tumor radiation response were detected. A protective effect on normal tissue was observed when radiation was delivered according to aggressive protocols and a relatively high dosage of Orgotein was administered. Furthermore, an accelerated trend of recovery of normal tissue was observed.

Problem of sequence and fractionation in the clinical application of combined heat and radiation.

Tumor control, thermal enhancement, and therapeutic effect have been evaluated in a series of studies on 77 patients with a total of 163 multiple superficial lesions by using different protocols of combined radiotherapy and local external hyperthermia. Local tumor control and recurrence rate were constantly better in lesions treated with combined treatment in comparison with those treated with radiotherapy alone, regardless of treatment schedule. The enhancement of tumor control appeared to be related to both the magnitude of the applied heat and the size of radiation fractions, in that an increase in either produced an increase in tumor control. When tumor and critical normal tissue were heated to the same temperature, the immediate combination of heat and large radiation fractions resulted in a pronounced enhancement of tumor control, but the concomitantly heated normal tissue showed an increased percentage of radiation reaction, resulting in a low therapeutic advantage. By introducing an interval of 4 hr between the two modalities or by delivering few heat fractions during the course of a conventional fractionation radiotherapy, the enhancement of tumor control was lower, but the increase in skin reaction was minimal, resulting in a clearly improved therapeutic effect. When the tumor could be preferentially heated with respect to normal tissue, the immediate combination of the highest hyperthermic treatment and the largest radiation fractions resulted in the best therapeutic advantage, since no increase of skin reaction was observed.

Tumor control and therapeutic gain with different schedules of combined radiotherapy and local external hyperthermia in human cancer.

Tumor control and therapeutic gain have been evaluated in a series of studies on patients with multiple lesions employing different protocols of combined radiotherapy (RT) and local external hyperthermia (HT). Tumor response has been evaluated during a follow-up ranging 6 to 18 months. Therapeutic enhancement factor (TEF) was defined as the ratio of thermal enhancement (TE) of tumors to TE of skin, where TE was clinically evaluated as the ratio of percent response (i.e., complete tumor clearance and moist desquamation, respectively) after combined modality to percent response after RT alone. Local tumor control was constantly better in lesions treated with any combined modalities in comparison with RT alone. The use of high RT dose per fraction appeared to increase tumor control only in the combined modalities groups, the immediate (so called "simultaneous") schedule (HT at 42.5 degrees C/45 min, applied immediately after each RT fraction, twice a week) being more effective than the delayed (so called "sequential") treatment (HT at 42.5 degrees C/45 min, delivered 4 h after each RT fraction, twice a week). The combination of high RT dose per fraction with high temperature HT (45 degrees C for 30 min) achieved the best tumor control. No increased radiation skin reaction was observed when a conventional fraction size of RT was used (3 daily fractions of 1.5-2 Gy, 4 h interval between fractions) in association with HT (42.5 degrees C/45 min, every other day, immediately after the second daily RT fraction). A remarkable enhancement of skin reaction was observed, however, when using high RT doses per fraction in association with 42.5 degrees C HT, especially with the immediate treatment schedule. No enhancement of skin reaction was obtained after high RT doses per fractions and 45 degrees C HT because an active skin cooling by means of circulating cold water was used in these cases. Consequently, a good TEF (1.58) was obtained when conventional RT doses per fraction were used in association with 42.5 degrees C HT. TEF values of 1.40 and 1.15 were observed when high RT doses per fraction were employed in association with the delayed and immediate 42.5 degrees C HT, respectively. HT at 45 degrees C can be safely employed only when tumors can be heated selectively or at least preferentially in comparison with normal tissue; in the lesions treated with such a schedule a TEF of 2.10 was obtained.

Clinical results after different protocols of combined local heat and radiation.

Since 1977, 69 patients with 138 multiple lesions have been treated with combined radiotherapy and hyperthermia, according to 3 protocols. Firstly, radiotherapy was given following a thrice-a-day fractionation scheme of 1.5 to 2 Gy/fraction, up to 60 Gy. Hyperthermia (42.5 degrees C/45 min) was applied each other day, immediately after the 2nd radiation fraction. Immediate response resulted significantly higher in the combined group (76% clearances in comparison with 46% after radiotherapy alone). Secondly, tumors received 40 Gy/8 fractions, twice a week, and hyperthermia (42.5 degrees C/45 min) was applied with each radiotherapy fraction, either immediately after irradiation (simultaneously) or 4 h later (sequentially). A remarkable improvement of radiation response was obtained, especially with the simultaneous treatment. Thirdly, tumors received 30 Gy/6 fractions, twice a week. Hyperthermia (45 degrees C/30 min) was applied simultaneously with each radiotherapy fraction and the surrounding skin was cooled. Complete tumor clearance was achieved in 88% lesions in comparison with 31% after radiotherapy alone. As expected, the incidence of thermal damage on uncooled skin was also increased. In conclusion, the best therapeutic ratio was obtained with low fractional radiotherapy doses and low temperature hyperthermia.