Response to pulsed dose rate and low dose rate irradiation with and without mild hyperthermia using human breast carcinoma cell lines.

The purpose of this study was to establish whether a pulsed dose rate (PDR) treatment of 1.5 Gy given every 3 h in combination with 41 degrees C mild hyperthermia or a continuous low dose rate (LDR) treatment with mild hyperthermia could radiosensitize two isogenic human breast carcinoma cell lines in comparison to pulsed dose rate or low dose rate irradiation alone. The radiation resistant cell line was derived from the parental cell line and was transfected to over-express DNA polymerase beta. The end-points assessed were the survival of the cells using the clonogenic assay, the amount of residual DSB(s) using the comet assay and gene expression of polymerase beta using RT-PCR. Results showed that the PDR and LDR treatments combined with mild hyperthermia caused significant radiosensitization when compared to PDR and LDR irradiation alone in terms of the clonogenic and comet assays with both cell lines. RT-PCR results showed that polymerase beta levels of expression were not elevated in response to these treatments, implying that this polymerase may not be involved in sub-lethal damage repair or thermal radiosensitization. These results suggest a potential clinical advantage when combining LDR or PDR with hyperthermia, since they indicate that hyperthermia is an effective radiosensitizer.     

Response to pulsed dose rate irradiation with and without mild hyperthermia using tumour and normal cell lines

To determine whether pulsed dose rate irradiation in combination with mild hyperthermia could radiosensitize cells in comparison to pulsed dose rate irradiation alone, human ovarian carcinoma (A2780s, cisplatin- and radiation-sensitive, and A2780cp, cisplatin- and radiation-resistant) and human fibroblast (AG1522) cell lines were used. Cells were irradiated in vitro using two fraction sizes, 0.53Gy given every hour and 1.6Gy given every 3h, with an overall average dose rate of 0.53Gy/h. The data showed that 40°C hyperthermia did not radiosensitize any of the cell lines for the 0.53Gy every 1h fractionation scheme. In addition, mild hyperthermia radiosensitized both carcinoma cell lines when using the 1.6Gy fraction size for all doses tested in the A2780s and at higher doses in the A2780cp, but not the normal cell line. These results suggest a potential clinical advantage when using the 1.6Gy fraction size with 40°C mild hyperthermia, since hyperthermia radiosensitized the carcinoma cells but not the normal cells.     

Response to pulsed dose rate irradiation with and without mild hyperthermia using tumour and normal cell lines.

To determine whether pulsed dose rate irradiation in combination with mild hyperthermia could radiosensitize cells in comparison to pulsed dose rate irradiation alone, human ovarian carcinoma (A2780s, cisplatin- and radiation-sensitive, and A2780cp, cisplatin- and radiation-resistant) and human fibroblast (AG1522) cell lines were used. Cells were irradiated in vitro using two fraction sizes, 0.53 Gy given every hour and 1.6 Gy given every 3h, with an overall average dose rate of 0.53 Gy/h. The data showed that 40 degrees C hyperthermia did not radiosensitize any of the cell lines for the 0.53 Gy every 1 h fractionation scheme. In addition, mild hyperthermia radiosensitized both carcinoma cell lines when using the 1.6 Gy fraction size for all doses tested in the A2780s and at higher doses in the A2780cp, but not the normal cell line. These results suggest a potential clinical advantage when using the 1.6 Gy fraction size with 40 degrees C mild hyperthermia, since hyperthermia radiosensitized the carcinoma cells but not the normal cells.     

Comparison between fractionated high dose rate irradiation and continuous low dose rate irradiation in spheroids

Recent interest in clinical brachytherapy focuses on the possible radiobiological equivalence between fractionated high dose rate (HDR) and continuous low dose rate (LDR) irradiations. This study is designed to compare the radiobiological effects between the two in vitro using multicellular spheroids of human tumor. Both HDR and LDR irradiations were delivered by 137Cs source, the dose rates of which were as 1.18 Gy/min and 5.5 mGy/min, respectively. Fractionated HDR irradiation of various fraction sizes was applied twice a day. We found that: (1) The fractionated HDR irradiation (8 Gy/2 fr/day) was more effective radiobiologically than continuous LDR irradiation (8 Gy/day) and the ratio of radiobiological effects of these irradiations was estimated as 0.82, based on the 50% spheroid cure dose (SCD50); (2) the radiobiological effectiveness was independent of the fraction size of HDR irradiation administrated, and the repair of sublethal damage (SLD) was absent, suggesting that the sparing effect of fractionated HDR irradiations was absent in spheroids. Our findings could provide important information for the clinical usage of the fractionated HDR radiotherapy to replace continuous LDR radiotherapy.     

Cell killing,DNA polymerase inactivation and radiosensitization to low dose rate irradiation by mild hyperthermia in four human cell lines

Four human cell lines (one fibroblast, two melanoma and one glioma) were evaluated for their responses to hyperthermia and thermalradiosensitization. For mild hyperthermia (40–42°C), there was little to no chronic thermotolerance development during protracted heating for up to 72 h. In addition, there was no significant thermotolerance for polymerase inactivation during mild hyperthermia. For high temperature hyperthermia, polymerase β was more thermal sensitive than aphidicolin sensitive polymerase α + δ + ε, (termed polymerase α) but during mild hyperthermia the relative sensitivities were reversed. Polymerase β was resistant to mild hyperthermia and polymerase α was very sensitive. Within each cell line there was a correlation between polymerase α inactivation and the degree of radiosensitization (TER) and amongst the cell lines the most radiation resistant cell line had less polymerase α inactivation than the most sensitive cell line for similar values of TER's. These data indicate that, amongst the cell lines, radiosensitivity and polymerase α sensitivity may influence TER and that for a given cell line, or possibly tumour, polymerase α inactivation may have potential as an indicator to determine TER for mild hyperthermia treatments in radiosensitization to low dose rates.     

Response of cells of human origin, normal and malignant, to acute and low dose rate irradiation

Dose response curves were obtained for normal human fibroblasts and for several cell lines derived from human tumors, including melanomas and an osteosarcoma. Most of the tumor lines are similar in radiosensitivity to the normal fibroblasts, except for the melanoma lines, which are significantly more resistant. The two melanoma lines differ, one being much more radioresistant than the other. Potentially lethal damage repair (PLDR) has been studied in these cell lines as well. The extent of PLDR does not appear to correlate with radioresistance; for example, the most resistant melanoma line shows very little repair of PLD. In addition, the normal fibroblasts repair PLD at least as well as any of the tumor derived lines, which casts doubts on the wisdom of introducing into clinical practice inhibitors of PLD until a clear differential between normal tissues and tumors has been demonstrated in vivo. Low dose-rate studies with normal human fibroblasts indicate a smaller dose-rate effect than for most established cell lines of rodent origin. Indeed, in the human cells studied, the effect of sublethal damage repair is quantitatively similar to the repair of potentially lethal damage. Dose response curves for acute and protracted exposures have been obtained for cells derived from patients with cancer-prone syndromes including ataxia telangiectasia (AT) and Bloom's syndrome. Both cell lines are much more radiosensitive than normal human fibroblasts; the AT cells show a dose-rate effect, while Bloom's syndrome cells do not.     

Response of human tumor cell lines in vitro to fractionated irradiation

The surviving fraction of human tumor cell lines after 2 Gy (SF2) varies between 0.1 and 0.8. It has been postulated that differences in inherent radiosensitivity of tumor cells are a major determinant of radiation response in vivo. Assays of inherent radiosensitivity based on acute survival are being developed as predictors of tumor response which often assume that the same inherent radiosensitivity persists throughout a fractionated treatment. We have investigated the response of 2 human tumor cell lines (A549 and MCF7) with different inherent radiosensitivities to in vitro fractionated irradiation. A549 cells had an SF2 of 0.62 and a mean inactivation dose (D) of 3.07 Gy whereas MCF7 cells had an SF2 of 0.30 and a D of 1.52 Gy. Split dose repair capacity (at equal survival levels) was less for A549 than for MCF7 cells and recovery kinetics for both cell lines were substantially longer than those of rodent cell lines. Survival after 5 fractions of 2 Gy given 12 hr apart at 37 degrees C was near to that predicted from the acute survival curve, assuming complete repair and no proliferation. Acute survival of A549 cells which survived 5 fractions of 2 Gy given 12 hr apart was similar to the acute survival of unirradiated cells. When A549 cells were incubated at 22 degrees C between 5 fractions of 2 Gy given 12 hr apart, proliferation and split dose repair were substantially inhibited. These studies support the proposals to use in vitro inherent radiosensitivity assays for the prediction of in vivo response of tumors to fractionated treatment.     

Comparison of radiosensitization by 41 degrees C hyperthermia during low dose rate irradiation and during pulsed simulated low dose rate irradiation in human glioma cells

PURPOSE: Long duration mild hyperthermia has been shown to be an effective radiosensitizer when given concurrently with low dose rate irradiation. Pulsed simulated low dose rate (PSLDR) is now being used clinically, and we have set out to determine whether concurrent mild hyperthermia can be an effective radiosensitizer for the PSLDR protocol. MATERIALS AND METHODS: Human glioma cells (U-87MG) were grown to plateau phase and treated in plateau phase in order to minimize cell cycle redistribution during protracted treatments. Low dose rate (LDR) irradiation and 41 degrees C hyperthermia were delivered by having a radium irradiator inside a temperature-controlled incubator. PSLDR was given using a 150 kVp X-ray unit and maintaining the cells at 41 degrees C between irradiations. The duration of irradiation and concurrent heating depended on total dose and extended up to 48 h. RESULTS: When 41 degrees C hyperthermia was given currently with LDR or PSLDR, the thermal enhancement ratios (TER) were about the same if the average dose rate for PSLDR was the same as for LDR. At higher average dose rates for PSLDR the TERs became less. CONCLUSIONS: Our data show that concurrent mild hyperthermia can be an effective sensitizer for PSLDR. This sensitization can be as effective as for LDR if the same average dose rate is used and the TER increases with decreasing dose rate. Thus mild hyperthermia combined with PSLDR may be an effective clinical protocol.     

Thermal enhancement of low dose rate irradiation in a murine tumour system

The effects of localized hyperthermia (HT) in combination with low dose rate irradiation (brachytherapy) have been investigated in vivo using a murine mammary adenocarcinoma. Flank tumours were grown to 0.45-0.70 cm3 in volume, at which time their treatment course was initiated. Tumours were locally heated in a water bath for 15 min at either 44 or 45 degrees C. For tumour irradiations a non-invasive cap was devised to permanently house three iodine-125 sealed sources located at 120 degree intervals around the circumference of the hemispherical cap. During treatment, mice were secured in a modified syringe tube allowing mobility while restricting access to the cap which was placed over the tumour. Calculated dose rates ranged from 15 to 40 cGy/h. Brachytherapy (BT) was delivered for 48 or 72 h to obtain a dose range of 830-2378 cGy. Mice were randomized into one of 10 treatment protocols: BT alone, HT-BT, BT-HT, HT-BT-HT, 1/2BT-HT-1/2BT, four control groups of HT alone and a sham treatment group. Normalized tumour doubling volume growth delays (GDDv) were used to calculate the thermal enhancement ratios (TER). In the 44 degrees C experiments, HT before BT (TER = 1.33 +/- 0.071) was more efficacious than HT after BT (TER = 1.07 +/- 0.042). Two HT treatments, one given before and one after BT (TER = 1.38 +/- 0.152), were not different from a single HT treatment given before BT. However, a single HT treatment given in the middle of an interrupted course of BT resulted in the greatest thermal enhancement (TER = 1.64 +/- 0.072) compared to any other treatment sequence. These data suggest that potentiation of low dose rate irradiation by a single heat treatment may be maximized if the HT is given either in the middle of, or simultaneously with, the BT.     

Thermal enhancement of low dose rate irradiation in a murine tumour system

The effects of localized hyperthermia (HT) in combination with low dose rate irradiation (brachytherapy) have been investigated in vivo using a murine mammary adenocarcinoma. Flank tumours were grown to 0.45–0.70 cm³ in volume, at which time their treatment course was initiated. Tumours were locally heated in a water bath for 15 min at either 44 or 45°C. For tumour irradiations a non-invasive cap was devised to permanently house three iodine-125 sealed sources located at 120° intervals around the circumference of the hemispherical cap. During treatment, mice were secured in a modified syringe tube allowing mobility while restricting access to the cap which was placed over the tumour. Calculated dose rates ranged from 15 to 40 cGy/h. Brachytherapy (BT) was delivered for 48 or 72 h to obtain a dose range of 830–2378 cGy. Mice were randomized into one of 10 treatment protocols: BT alone, HT-BT, BT-HT, HT-BT-HT, 1/2BT-HT-1/2BT, four control groups of HT alone and a sham treatment group. Normalized tumour doubling volume growth delays (GD_Dv) were used to calculate the thermal enhancement ratios (TER). In the 44°C experiments, HT before BT (TER= 1.33 ±0.071) was more efficacious than HT after BT (TER=1.07 ±0.042). Two HT treatments, one given before and one after BT (TER = 1.38 ± 0.152), were not different from a single HT treatment given before BT. However, a single HT treatment given in the middle of an interrupted course of BT resulted in the greatest thermal enhancement (TER=1.64±0.072) compared to any other treatment sequence. These data suggest that potentiation of low dose rate irradiation by a single heat treatment may be maximized if the HT is given either in the middle of, or simultaneously with, the BT.     

Update on low dose rate irradiation for cancers of the oropharynx--May 1986

At the conclusion of our recently published article in this Journal on low dose rate irradiation in moderately extensive cancers of the oropharynx, we updated our results in May 1986. Here we report on an expanded group of 65 patients with a 2 year minimum follow-up; 32 patients were treated by low dose rate irradiation and 33 by conventional fractionation. Forty-four percent (14/32) low dose irradiation patients survived with NED vs 8/33 (24%) conventional fractionation patients. The highly significant differences in the level of local recurrences between patients treated by low dose rate irradiation, 5/32 (16%), compared with conventional fractionation, 20/33 (61%), highlights the enhanced efficacy of the low dose rate irradiation technique in the local cure of cancers of the oropharynx. This superior local control however is achieved at the cost of a number of necrosis, 5/32 (16%).     

Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u(-1), 70 keV microm(-1)) and gamma-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6-8 h(-1)), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with gamma-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, gamma-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (gamma-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.     

Interstitial pneumonitis following bone marrow transplantation after low dose rate total body irradiation

Idiopathic and infective interstitial pneumonitis (IPn) is a common complication after bone marrow transplantation (BMT) in many centers and carries a high mortality. We report here a series of 107 patients with acute leukemia grafted at the Royal Marsden Hospital in which only 11 (10.3%) developed IPn and only 5 died (5%). Only one case of idiopathic IPn was seen. Factors which may account for this low incidence are discussed. Sixty of 107 patients were transplanted in first remission of acute myeloid leukemia (AML) and were therefore in good general condition. Lung radiation doses were carefully monitored and doses of 10.5 Gy were not exceeded except in a group of 16 patients in whom a study of escalating doses of TBI (up to 13 Gy) was undertaken. The dose rate used for total body irradiation (TBI) was lower than that used in other centers and as demonstrated elsewhere by ourselves and others, reduction of dose rate to less than 0.05 Gy/min may be expected to lead to substantial reduction in lung damage. Threshold doses of approximately 8 Gy for IPn have been reported, but within the dose range of 8 to 10.5 Gy we suggest that dose rate may significantly affect the incidence. Data so far available suggest a true improvement in therapeutic ratio for low dose rate single fraction TBI compared with high dose rate.     

Modification of the effects of continuous low dose rate irradiation by concurrent chemotherapy infusion

The combined effects of continuous low dose rate irradiation (CLDRI) and concurrent infusion of bleomycin, cyclophosphamide, cis-platinum, 5-fluorouracil, actinomycin D, and mitomycin C were studied in the SCC VII/SF tumor, a squamous cell carcinoma and the jejunal crypt cells in the mouse. For the SCC VII/SF tumor, enhanced cell killing was seen with each of the six drugs when infused concurrently with CLDRI; the greatest enhancement was seen with mitomycin C and cis-platinum. For the jejunal crypt cells, enhanced cell killing was seen primarily with bleomycin. At a dose of 20 Gy, the dose effect factor (DEF) ranged from 1.13-1.64 for the SCC VII/SF tumor and 0.92-1.19 for the jejunal crypt cells. Our results suggest a therapeutic gain with concurrent CLDRI and chemotherapy infusion for five of the six chemotherapeutic drugs studied with the exception of bleomycin.     

High versus low dose rate intracavitary irradiation for adenocarcinoma of the uterine cervix.

BACKGROUND: Traditionally, low dose rate (LDR) brachytherapy has been used as a standard modality in the treatment of patients with carcinoma of the uterine cervix. The purpose of this work was to evaluate the effects of high dose rate (HDR) brachytherapy on patients with adenocarcinoma of the uterine cervix and to compare them with the effects of LDR brachytherapy. METHODS: From January 1971 to December 1992, 104 patients suffering from adenocarcinoma of the uterine cervix were treated with radiation therapy in the Department of Radiation Oncology, Yonsei University. LDR brachytherapy was carried out on 34 patients and HDR brachytherapy on 70 patients. In the LDR group, eight patients were in stage IB, six in IIA, 12 in IIB, three in IIIA and five in IIIB. External radiation therapy was delivered with 10 MV X-rays, 2 Gy fraction per day, total dose of whole pelvis 36-52 Gy (median 46 Gy). LDR radium intracavitary irradiation was performed with a Henschke applicator, 37-59 Gy targeted at point A (median 43 Gy). In the HDR group, there were 16 patients in stage IB, six in IIA, 32 in IIB and 16 in IIIB. The total whole pelvis dose of external radiation was 40-50 Gy (median 44 Gy), daily 1.8-2.0 Gy. HDR Co-60 intracavitary irradiation was performed with a remotely controlled after-loading system (RALS), 30-48 Gy (median 39 Gy) targeted at point A, three times per week, 3 Gy per fraction. RESULTS: The 5-year overall survival rate in the LDR group was 72.9, 61.9 and 35.7% in stage I, II and III, respectively and the corresponding figures for HDR were 87.1, 58.3 and 43.8% (p > 0.05). There was no statistical difference between the HDR group and the LDR group in terms of the 5-year overall survival rate from adenocarcinoma of the uterine cervix. There was a late complication rate of 12% in the LDR group and 27% in the HDR group. The incidence of late complications in stages II and III was higher in the HDR group than in the LDR group (31.6 vs 16.7% in stage II, 37.3% vs 12.5% in stage III, p > 0.05). No prognostic factors were evident in the comparison between the two groups. CONCLUSION: There was no difference in terms of 5-year survival rate in the patients with adenocarcinoma of the uterine cervix between those treated with HDR and those treated with LDR brachytherapy. Even though late complication rates were higher in the HDR group, most of them were classified as grade I. This retrospective study suggests that HDR brachytherapy may be able to replace LDR brachytherapy in the treatment of adenocarcinoma of the uterine cervix.     

Repair kinetics as a determining factor for late tolerance of central nervous system to low dose rate irradiation

The effect of continuous irradiation, delivered at four different dose rates (107.6, 14.7, 3.9 and 2 Gy.h-1) has been investigated using the rat cervical spinal cord biological system. The endpoint was the induction of foreleg paralysis at 9 months which corresponds, as has been described before, to white matter necrosis. Paralysis occurring in 50% of the animals was taken as the isoeffect, and the ED50 (radiation dose leading to paralysis in 50% of the animals) was calculated by probit analysis. There was a constant increase in the ED50 with the decrease in the dose rate, resulting from the repair of sublethal damage (SLD) occurring during irradiation. A comparison was made with the previously published results of high dose rate (100-120 Gy.h-1) fractionated irradiations (2, 4 and 10 fractions). alpha/beta (1.6 Gy for the pooled fractionation and dose rate data) and the half-time of SLD repair (82 min) were derived.     

Effect of single dose irradiation on human glioblastoma spheroids in vitro

Investigation of the predictive value of a radiosurgery-relevant treatment of glioblastoma spheroids. Organotypic multicellular spheroids were cultured and irradiated (20 Gy). Morphology, apoptosis and immunohistochemical expression of p53, p21, MIB-1, TGF-beta and VEGF were examined 4 h, 24 h, 7 days, and 14 days following treatment. Cell proliferation decreased, while apoptosis was increased. No morphological damage was observed. p53 expression was significantly increased after 4 h. TGF-beta and VEGF expression were only slightly altered. Particularly early changes in proliferation and apoptosis can be observed in spheroids. Individual response differences suggest spheroids of human gliomas to be useful for monitoring radiosurgery effects.     

Variable low dose rate irradiation (131I-anti-CEA) and integrated low dose chemotherapy in the treatment of nonresectable primary intrahepatic cholangiocarcinoma.

Previous experience using 131I anti-CEA antibody, which irradiates at a variable low dose rate in combination with a multimodality treatment program, has demonstrated acceptable toxicity and response in primary intrahepatic cholangiocarcinoma. In attempting to improve therapy, Cis-platin was added to the prior regimen. Induction therapy was unchanged. One month later, chemotherapy was given (doxorubicin, 15 mg, 5-fluorouracil, 500 mg, plus Cis-platin, 20 mg/M2) followed the next day by outpatient administration of 20 mCi 131I anti-CEA by i.v. bolus. Five days later, 10 mCi was administered. The latter regimen (chemotherapy plus 20 + 10 mCi 131I anti-CEA) was repeated every 2 months using polyclonal antibodies derived from different species (rabbit, pig, baboon, and horse). Twenty-four patients (29% with prior chemotherapy and/or metastases) were prospectively treated according to this regimen. Toxicity was limited to hematologic toxicity and was manifested by thrombocytopenia and leukopenia (17% and 4% grade 4, respectively, according to RTOG toxicity criteria). Tumor remission was evaluated by CT volumetric analysis and demonstrated a 14% response rate for the induction portion of therapy, 24% for the radioimmunoglobulin portion of treatment, and 50% remission rate when all subsequent tumor volumes were compared to the pre-treatment volume (entire program). The median survival for the entire group of patients was 10.1 months. This result is superior to previously reported trials and, in comparison to our previous study (10.1 vs 6.5 months median survival), further advancement in protocol design appears to have been made. In view of the rarity of this disorder, a randomized trial is not possible and strict statistical analyses cannot be made. The mechanism of 131I-anti-CEA variable low dose irradiation and chemotherapy interaction is discussed as well as further potential modifications for treatment improvement.