Wound healing morbidity in STS patients treated with preoperative radiotherapy in relation to in vitro skin fibroblast radiosensitivity, proliferative capacity and TGF-beta activity.

BACKGROUND AND PURPOSE: In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-beta) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-beta activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms. PATIENTS AND METHODS: Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: approximately 0.02 Gy/min) and TGF-beta assays (high dose-rate: approximately 1.06 Gy/min) following gamma-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF(2.4)) and binucleation index (BNI), respectively. Active and total TGF-beta levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined. RESULTS: Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after irradiation compared with those from individuals in the treatment group with normal wound healing, consistent with the results of our previous study. No link was observed between fibroblast radiosensitivity and WHC. Neither active nor total TGF-beta levels in cultures were significantly affected by irradiation. Fibroblast proliferation in unirradiated and irradiated cultures, as well as radiosensitivity, was not influenced by TGF-beta content. TGF-beta expression in fibroblast cultures did not reflect wound healing morbidity. CONCLUSIONS: These data are consistent with our previous study and combined the results suggest that in vitro fibroblast proliferation after irradiation may be a useful predictor of wound healing morbidity in STS patients treated with preoperative radiotherapy. TGF-beta levels in culture do not predict WHC, suggesting that the role of TGF-beta in wound healing is likely controlled by other in vivo factors.     

Clonogenic survival and cytokinesis-blocked binucleation of skin fibroblasts and normal tissue complications in soft tissue sarcoma patients treated with preoperative radiotherapy.

BACKGROUND AND PURPOSE:To evaluate the clonogenic and cytokinesis-blocked assays in skin fibroblast cultures for their utility as tools for predicting normal tissue responses in soft tissue sarcoma (STS) patients treated with preoperative radiotherapy. PATIENTS AND METHODS:Dermal fibroblast strains were established from skin biopsies of 26 STS patients who received preoperative radiotherapy. Cultures were subjected to the colony forming and cytokinesis-blocked assays after low (approximately 0.02 Gy/min) dose-rate 60Co -irradiation. Fibroblast radiosensitivity was expressed as the dose for 1% clonogenic survival, D0.01, based on colonies/clusters with >or=10 cells. Fibroblast proliferative capability was represented by binucleation index (BNI) and genomic damage was expressed in terms of micronucleus frequency. Wound healing complications (WHC) and subcutaneous fibrosis were the clinical endpoints examined. The ability of each in vitro parameter to detect patients at high risk of a given normal tissue complication was assessed using receiver operating characteristic (ROC) analysis. RESULTS:While fibroblasts from patients without WHC were marginally more radiosensitive than fibroblasts from patients with WHC (P=0.08), the reduction in BNI following a dose of 2.4 Gy was significantly higher in strains from patients without WHC compared to those from patients with WHC (P=0.01). The area under the ROC curve (c-index) is indicative of the power of discrimination of D0.01 and BNI for WHC, and was found to be 0.68 and 0.79, respectively. Subcutaneous fibrosis was not associated with D0.01 (rs=0.09, P=0.66) and the percent reduction in BNI after 2.4 Gy (rs=-0.19, P=0.36). Micronucleus frequency did not reflect differences in normal tissue responses. CONCLUSION:These data suggest that it is the ability of fibroblasts to undergo one-three divisions in vitro following radiation treatment that may reflect the development of wound healing morbidity or subcutaneous fibrosis in this population of patients.     

Analysis of telomere damage by fluorescence in situ hybridisation on micronuclei in lymphocytes of breast carcinoma patients after radiotherapy

Radiotherapy has become an indispensable tool in the effective management of most of the cancers. There have been efforts earlier to study the differential radio-sensitivity patterns in patients undergoing radiation treatment to correlate with treatment induced complications such as tissue injury, cell death, and chromosomal aberration frequencies etc. The present study is an attempt to correlate the radiation-induced damage in the peripheral blood lymphocytes (PBLs) of breast cancer patients with the frequency of telomere mediated chromosomal damage. Blood samples from 55 patients with (Gr-II and Gr-III) CA-breast were obtained pre- and post-radiotherapy. The patients were treated with external beam radiotherapy of 50.4 Gy over a period of 6 weeks. Chromosome damage was measured by analysing micronucleus (MN) frequency in PBLs. The MN-frequency of the irradiated patients increased significantly compared to the patients being self-controls. The micronuclei were hybridized with telomere probes to study the extent of telomere damage. The fluorescence signals of the telomere regions in the first generation of the binucleated cells were significantly higher in the post-radiotherapy patients. There was also significant correlation observed in the patients with higher-grade tumours. Inter-individual variability was observed in the radiation-induced MN frequency in lymphocytes of patients after six weeks of radiotherapy. There was a significant correlation between functionally intact telomeres and the cellular response to ionising radiation. Our findings suggest that fluorescence in situ hybridisation on micronuclei could be effectively used as routine clinical application to determine the individual sensitivity to ionising radiation with respect to telomere damage.     

Improvement of the breaking strength of wound by combined treatment with recombinant human G-CSF, recombinant human M-CSF, and a TGF-beta1 receptor kinase inhibitor in rat skin

Effective doses of ionizing radiation during preoperative radiotherapy occasionally cause wound complications after subsequent surgery. The authors attempted to accelerate radiation-impaired wound healing in animal models. Recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human macrophage colony-stimulating factor (rhM-CSF), and an inhibitor of transforming growth factor (TGF)-β1 receptor kinase, SB431542, were injected s.c. into a full-thickness incisional wound site in the dorsal skin of rats after local irradiation of X-ray (30 Gy). Wound healing of irradiated skin was assessed using the breaking strength of the wound and histological analyses. The impaired wound healing in irradiated skin was found to be associated with impaired mobilization of bone marrow-derived cells and enhanced expression of TGF-β1 mRNA. The breaking strength of the wound in the irradiated skin was approximately one-eighth of that in the non-irradiated skin; however, following combined treatment with the above three compounds the breaking strength increased to approximately one-half of that in the non-irradiated skin. Histological analysis of the wounded skin revealed an increase in formation of collagen fibers and the panniculus carnosus following the combined treatment. Moreover, the increased breaking strength was associated with an increase in a subpopulation of fibrocytes (collagen I/ED1 double positive cells). These findings suggested that a combined treatment with rhG-CSF, rhM-CSF, and SB431542 is promising as a means of improving radiation-impaired wound healing. ( Cancer Sci 2008; 99: 1021–1028)     

Severe normal tissue complication correlates with increased in vitro fibroblast radiosensitivity in radical prostate radiotherapy: a case report

The radiation responses of fibroblasts taken from four patients undergoing radiotherapy for prostate cancer were evaluated in vitro. One patient exhibited a severe normal tissue late reaction after radiotherapy, and the fibroblasts from this patient also showed increased radiosensitivity. The other three patients exhibited a normal clinical response, and their fibroblast response in vitro was also considered normal when compared with previously published data from our laboratory. When cells were allowed to repair potentially lethal damage (PLDR), the differences between cell lines were reduced; therefore, a deficiency in PLDR did not cause the differences in radiosensitivity. Fractionated radiation resulted in elevated survival due to repair of sublethal damage (SLDR), and the differences in radiosensitivity were increased. In addition, the survival curves displayed an upward bending nature, indicative of an adaptive response. Thus, adaptive response and ability to modify it may play an important role in fractionated radiotherapy. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 336-342 (2000).     

In vitro radiosensitivity of skin fibroblasts can identify a group of patients with complications in various health tissues after radiotherapy]

PURPOSE: A retrospective study of the in vitro radiosensitivity of skin fibroblasts derived from two groups of patients treated by definitive radiotherapy for a variety of tumors who either displayed or did not display severe complications. PATIENTS AND METHODS: Seven radiotherapy patients were selected: three were treated for head and neck, prostate and non-Hodgkin lymphoma tumors, and did not develop any significant complications (control group); four patients were treated for bladder, thyroid, head and neck and anal canal tumors and developed serious acute and especially late reactions (hypersensitive group). Primary cell cultures of skin fibroblasts were established and their radiosensitivity studied by the clonogenic assay after exposing to single radiation doses ranging between 1 and 8 Gy. RESULTS: The survival fraction at 2 Gy (SF2) ranged from 0.27 to 0.38, with a mean of 0.33 for the control group, and from 0.10 to 0.20 with a mean of 0.17 for the hypersensitive group. The Mann-Whitney non-parametric test showed that the difference between the two means was statistically significant (p = 0.03). CONCLUSION: The data are in favor of a correlation between the radiosensitivity of patients' fibroblasts and the reactions of different normal tissues to radiotherapy. This association supports the use of the clonogenic survival, or a surrogate test, as a predictive assay. The multiplicity of normal tissues and organs implicated in this association suggests the existence of genetic factors that determine, at least in part, the radiosensitivity of target cells involved in the expression of normal tissues complications following radiotherapy.     

Wound complications following pre-operative radiotherapy for soft tissue sarcoma.

AIMS: We analysed wound complications in 43 patients with soft tissue sarcoma who were treated with combined pre-operative radiotherapy and surgery. METHODS: All patients received the same protocol of pre-operative radiotherapy at our institution. RESULTS: Thirty-six (84%) patients developed acute skin toxicity following radiotherapy. After wide local excision, 15 patients required primary soft tissue reconstruction with vascularized muscle transfer and four patients underwent free skin flap to enable wound closure as part of their primary surgery. Nineteen patients (44%) developed post-operative wound complications including 10 (23%) patients who required an additional surgical procedure. Four (27%) patients developed flap necrosis in a group of 15 who underwent primary vascularized soft tissue transfer. All required a second vascularized muscular flap. One elderly patient, who had grade 3 acute radiation skin toxicity, had an arterial graft and total hip arthroplasty for a femoral artery aneurysm and an avascular necrosis of the hip, respectively. In our series, age (> or = 40 years) was the only impact factor influencing wound complication after surgery following radiotherapy (P=0.06). CONCLUSIONS: Site of tumour, radiation field size, surgical resection volume, grade of acute radiation toxicity, co-morbidity, and smoking were not demonstrated to have predictive value in wound complication following pre-operative radiotherapy. Although previous papers suggested that vascularized soft tissue transfer could be useful reducing wound morbidity, our results could not confirm this.     

Partial volume rat lung irradiation; assessment of early DNA damage in different lung regions and effect of radical scavengers.

PURPOSE: These studies were designed to examine radiation-induced in-field and out-of-field DNA damage in rat lung as a function of dose and various volumes of irradiation. They also determined whether superoxide dismutase (SOD) and nitro-L-arginine methyl ester (L-NAME) protected against this damage. METHODS AND MATERIALS: The whole lung, or various volumes of the lower or upper lungs of Sprague-Dawley rats were exposed to doses up to 20 Gy of 60Co gamma rays. Radiation-induced DNA damage was quantified in fibroblasts obtained at 18 h after irradiation from both irradiated and shielded lung regions using a micronucleus assay. The radioprotective role of SOD (CuZnSOD: 10 mg/kg body weight; MnSOD: 50-100mg/kg body weight) and L-NAME (0.2 mg/kg body weight.) in vivo was determined by injecting them into rats 30 min before or immediately after a dose of 10 Gy. RESULTS: Micronucleus formation was approximately linear with dose up to 15 Gy. When 70% of the lung volume was irradiated with 10 Gy, irradiated lower lung gave similar numbers of micronuclei (MN)/binucleate cell (BN) to that observed following whole lung irradiation (0.91 MN/BN), whereas the irradiated upper lung gave only 0.66 MN/BN. Following lower lung irradiation, the shielded upper lung (30% of lung volume) showed substantial (out-of-field) damage (0.43 MN/BN). When 30% of the lung was given 10 Gy, irradiated upper or lower lung showed similar amounts of in-field damage (0.43 MN/BN) but this was smaller than that seen following irradiation of 70% of the lung volume. For 30% lower lung irradiation, the shielded upper lung showed only a small out-of-field effect (0.1 MN/BN). For both volumes of irradiation there was a similar or smaller effect in the shielded lower lung after upper lung irradiation. Injection of SOD before or L-NAME after 10 Gy to the lower 70% lung volume resulted in a reduction in DNA damage both in-field and out-of-field but the percentage was much greater for out-of-field damage (50-60%) than for in-field damage (10-30%). Following whole lung irradiation (10 Gy) significantly greater DNA damage was observed in fibroblasts from the left lung than from the right lung (0.93 MN/BN vs. 0.82 MN/BN). Following whole lung irradiation there was no significant difference in DNA damage observed in fibroblasts from the lower lung and the upper lung. CONCLUSIONS: With partial lung irradiation the lower lung sustains more in-field DNA damage following irradiation than the upper lung, whereas out-of-field effects are observed primarily in the upper lung (i.e. following lower lung irradiation). Following whole lung irradiation the left lung sustains more damage than the right lung but there is no difference between the upper and lower lung. The protective effects of SOD and L-NAME suggest that inflammatory cytokines induced by the irradiation may be involved in the initiation of a reaction resulting in the production of reactive oxyradicals and nitric oxide that cause indirect DNA damage both in and out of the radiation field.     

Fractionated irradiation of five human lung cancer cell lines and prediction of survival according to a radiobiology model

BACKGROUND: This study evaluates a predictive radiobiology model by measurements of surviving fraction (SF) by the clonogenic assay or the extrapolation method and the proliferation rate in vitro. It is hypothesized that incorporating proliferation to intrinsic radiosensitivity, measured by SF, to predict radiation responsiveness after fractionated irradiation adds to the model's accuracy. Materials and Methods. Five lung cancer cell lines with known SF after 1 Gy (SF1), and also SF2 and SF5, were irradiated with three different fractionation regimes; 10 × 1 Gy, 5 × 2 Gy or 2 × 5 Gy during the same total time to achieve empirical SF. In addition, the SF1, SF2 and SF5 after fractionated irradiation was calculated for each cell line based on the already known single fraction SF and with or without a proliferation factor. The results were compared to the empirical data. Results and Discussion: By using the clonogenic assay to measure radiosensitivity, prediction of radiosensitivity was improved after fractionated radiotherapy when proliferation was used in the radiobiology model. However, this was not the case in the cell lines where the extrapolation method was used to calculate SF. Thus, a radiobiology model including intrinsic radiosensitivity, measured by the clonogenic assay, as well as proliferation, is better at predicting survival after fractionated radiotherapy, compared to the use of intrinsic radiosensitivity alone.     

The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability.

A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B-IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 x 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08-0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60-610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer.     

Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients

About 5% of oncology patients treated by radiation therapy develop acute or late radiotoxic effects whose molecular mechanisms remain poorly understood. In this study, we evaluated the potential role of DNA repair proteins in the hypersensitivity of cancer patients to radiation therapy. The expression levels and focal nuclear distribution of DNA repair proteins, hMre11, Rad50 and Rad51 were investigated in skin fibroblasts strains derived from cancer patients with adverse early skin reaction to radiotherapy using Western blot and foci immunofluorescence techniques, respectively. Cells from cancer patients with normal reaction to radiotherapy as well as cells from apparently healthy subjects served as controls. Cellular radiosensitivity after in vitro irradiation was assessed by the clonogenic survival assay. The clonogenic survival assay and Western blot analysis of the DNA repair proteins did not reveal any abnormalities in cellular radiosensitivity in vitro and in protein expression levels or their migration patterns in the fibroblasts derived from cancer patients with hypersensitive reaction to radiotherapy. In contrast, in vitro irradiated cells from radiosensitive patients exhibited a significantly higher number of nuclei with focally concentrated Rad50 protein than in both control groups. The observed alteration of the distribution of radiation-induced Rad50 foci in cells derived from cancer patients with acute side reactions to radiotherapy might contribute to their radiation therapy outcome. These data suggest the usefulness of the Rad50 foci analysis for predicting clinical response of cancer patients to radiotherapy.     

Lack of correlation between residual radiation-induced DNA damage, in keratinocytes assayed directly from skin, and late radiotherapy reactions in breast cancer patients

PURPOSE: To study the relationship between the severity of late reactions to radiotherapy in breast cancer patients, and the extent of residual radiation-induced DNA damage, using a rapid assay of keratinocytes obtained directly from skin biopsies. METHODS AND MATERIALS: A review was made of 32 patients with breast cancer, treated uniformly by radiotherapy between 1983 and 1988, following breast-conserving surgery. Their late radiotherapy reactions were scored (9-14 years post-radiotherapy) using a modified LENT SOMA scale, and a 5-mm buttock skin punch biopsy was obtained. Intact skin was irradiated at room temperature, and after allowing 24 h for repair, the tissue was disaggregated and the cells processed for pulsed field gel electrophoresis (PFGE). Residual DNA damage was expressed as the fraction of DNA released (FDR) following 150 Gy. RESULTS: Studies using flow cytometry on disaggregated breast skin showed that over 90% of the cells were keratinocytes. The PFGE assay was robust with low background FDRs in unirradiated skin samples (mean 3.2%) and a wide range of FDRs following irradiation from 11.5% to 26.6%. No correlation was found between the FDR at 150 Gy (FDR 150) and any of the late reaction scores or retrospective acute reaction scores. There was, however, a borderline significant correlation for family history and FDR 150 (p = 0.059). CONCLUSION: Rapid measurement of residual DNA damage in irradiated differentiated keratinocytes, the predominant cell population in skin biopsies, showed no correlation with the severity of symptomatic early or documented late reactions in a retrospectively studied group of 32 breast cancer patients.     

DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation

Predictive markers of intrinsic radiosensitivity in healthy individuals are needed in monitoring their occupational or environmental radiation exposure and may predict a patient's response to radiotherapy. Ionizing radiation can induce a large spectrum of DNA lesions, but under optimal DNA repair conditions, the principal residual lesions of importance are misrepaired double-strand breaks. The micronucleus (MN) assay represents a useful test in measuring radiosensitivity since it reflects non-repaired DNA breaks at the time of cell division. Spontaneous and radiation-induced MN vary greatly between individuals, and little is known about the molecular mechanisms of this variability. DNA repair and apoptosis processes are involved in the cellular response to radiation-induced DNA damage, and variation in gene expression related to these cellular pathways could be linked to individual radiosensitivity. In this study we analysed by real-time quantitative RT-PCR the basal expression of 12 genes involved both in DNA repair and apoptosis in a series of blood samples obtained from 32 healthy male donors. Relationships between basal RNA expressions and MN frequency and distribution per bi-nucleated cell were studied after ex vivo irradiation of total blood samples. Our results indicate that the variability of mRNA gene expression among the 32 subjects appears to be of the same magnitude or higher than that found for spontaneous or radiation-induced MN frequency and that RAD51 gene expression is negatively correlated with radiation-induced MN frequency.     

Intrinsic radiosensitivity of normal human fibroblasts and lymphocytes after high- and low-dose-rate irradiation.

The existence of heritable radiosensitivity syndromes and clinical observations in radiotherapy patients suggests that human cellular radiosensitivity differs among individuals. We report here an in vitro study of radiosensitivity in 30 fibroblast and 29 lymphocyte cultures obtained from cancer patients and controls. In 25 cases, both fibroblasts and lymphocytes were obtained from the same donors. Fibroblasts were cultured from skin biopsy samples, and peripheral T-cell lymphocytes were cultured from blood. Clonogenic survival assays were performed by using high- and low-dose-rate irradiation; lymphocytes were in G0 phase and fibroblasts in confluent plateau phase. Various end points were calculated and compared (i.e., surviving fraction at 2 Gy, initial slope of the survival curve, and doses resulting in 10 and 1% survival, respectively). Depending on the end point, the coefficient of variation of the survival parameters ranged from 31 to 68% for lymphocytes and 21 to 41% for fibroblasts following high-dose-rate irradiation. Similar ranges were obtained after low-dose-rate irradiation. Variance analysis performed on replicate assays in cultures derived from the same patient showed that variation due to technical or sampling errors was significantly lower than variation between individuals (P = 0.00034 and 0.014 for fibroblasts and lymphocytes, respectively). No correlation was observed between the radiosensitivity of lymphocyte and fibroblast cultures derived from the same donors. We conclude that there is significant variation in normal cell radiosensitivity among individuals. On the other hand, comparisons of lymphocyte and fibroblast radiosensitivities suggest that tissue-specific characteristics, such as differentiation status, may variably modulate radiosensitivity.     

Correlation between normal tissue complications and in vitro radiosensitivity of skin fibroblasts derived from radiotherapy patients treated for variety of tumors

PURPOSE: To assess the relationship between fibroblast intrinsic radiosensitivity in vitro and late reactions of normal tissues in patients treated by definitive radiotherapy for variety of tumors. PATIENTS AND METHODS: Ten patients were selected for this study. They were treated by radical radiotherapy for variety of tumors, including non-Hodgkin's lymphoma, prostate, glottic larynx, anal canal, cervix, bladder, thyroid gland, and tonsil pillar. Five patients did not develop any significant late reactions (normally sensitive group, NS). The other five developed late complications in different normal tissues and organs that proved to be fatal in one patient (clinically hyper-sensitive group, HS). Fibroblast cultures were established from punch skin biopsy and radiosensitivity in vitro was measured. The survival fraction at 2 Gy (SF2) was calculated and compared between the two groups. RESULTS: SF2 ranged between 0.10 and 0.38 with a mean of 0.24. The mean SF2 for each of the NS and the HS groups were 0.31 and 0.17, respectively. The non-parametric rank test of Mann-Whitney shows that the difference between the two groups is statistically significant (p = 0.01). CONCLUSION: This study indicates that the in vitro radiosensitivity of skin fibroblasts is correlated with late complications in different organs and normal tissues following radiotherapy for variety of tumors. It also lends support to the existence of a common genetic component determining the radiosensitivity of cells targeted by the late effects of ionizing radiation.     

Radiosensitivity of mouse skin epithelial cell line established in serum-free culture: an alternative to animal use.

A cultured line of murine skin epithelial cells was established to investigate the potential use of cultured cells as an alternative to animal use in radiation research. C3Hf/Sed newborn mouse skin cells have been successfully cultured in serum- and Ca(2+)-free medium with no terminal differentiation to keratinized cells. Presently, more than 25 passages have passed with no loss of stem cell capability. The radiosensitivity and repair of sublethal and potentially lethal radiation damages were investigated in this epithelial cell line. The population cell doubling time was 25 +/- 2.9 hr at 37 degrees C. The clonal growth of epithelial cells after irradiation was performed in the serum-free medium in the presence of lethally irradiated skin fibroblasts. Single dose survival curves of exponentially growing epithelial cells were investigated from the seventh to the twenty-third passages, and no significant changes in radiosensitivity and doubling time were found. The confluent epithelial cells also showed an identical sensitivity to radiation. The alpha/beta ratios of survival curves fitted by the linear quadratic model were 6.1 +/- 1.0 and 5.9 +/- 1.3 Gy for cells in exponential and confluent phases, respectively. The survival curve of epithelial cells left in confluence for 8 hr after irradiation showed a smaller beta value than that of cells plated immediately after irradiation with a resultant alpha/beta ratio of 9.5 +/- 3.8 Gy. This alpha/beta ratio was identical to those found in many animal experiments, suggesting a potential use of this cell line as an alternative to animal use. The magnitude of repair of sublethal damage following 6 Gy was greater than that following 3.9 Gy. Survival curves were also obtained following twice-a-day irradiations with no sign of rapid repopulation. These results are discussed by comparing with published in vivo and in vitro data.     

Contribution of Lethal Mutations to Excision Assays for Tumour Cell Survival

Conventional assays of cell survival determine only the proportion of colony-forming cells, assuming that all such cells are equivalent. However, cells surviving irradiation are reported to have lower plating efficiencies than unirradiated controls, suggesting an additional component of cellular damage that is ignored in conventional survival assays, but which could contribute to therapeutic outcome. Therefore we have examined the contribution of this additional form of damage to excision assays for cell survival in experimental tumours following both single dose and fractionated irradiation (10F/5 days) in vivo. Plating efficiencies were considerably lower for the long-term descendents of irradiated compared with non-irradiated cells. Expression of delayed reproductive death was reduced after fractionated radiation doses, only appearing after a substantial number of 3.4 Gy fractions had accumulated. Thus estimates of survival derived from single clonogenicity assays may underestimate the reduction in cell viability from a particular treatment. This could compromise assays for intrinsic radiosensitivity and mathematical modelling of the efficacy of treatment regimens.     

Intraoperative radiotherapy given as a boost after breast-conserving surgery in breast cancer patients

Conventional radiotherapy after breast-conserving therapy is confined to 50-55 Gy external beam radiation therapy (EBRT) to the whole breast and 10-16 Gy external boost radiation to the tumour bed or brachytherapy to the tumour bed. Local recurrence rate after breast-conserving surgery varies between 5 and 18%. External boost radiation can partially miss the tumour bed and therefore can result in local failure. Intra-operative radiotherapy (IORT) as a high precision boost can prevent a 'geographical miss'. From October 1998 to December 2000, 156 patients with stage I and stage II breast cancer were operated upon in a dedicated IORT facility. After local excision of the tumour, the tumour bed was temporarily approximated by sutures to bring the tissue in the radiation planning target volume. A single dose of 9 Gy was applied to the 90% reference isodose with energies ranging from 4 to 15 MeV, using round applicator tubes 4-8 cm in diameter. After wound healing, the patients received additional 51-56 Gy EBRT to the whole breast. No acute complications associated with IORT were observed. In 5 patients, a secondary mastectomy had to be performed because of tumour multicentricity in the final pathological report or excessive intraductal component. 2 patients developed rib necroses. In 7 patients, wound healing problems occurred. After a mean follow-up of 18 months, no local recurrences were observed. Cosmesis of the breast was very good and comparable to patients without IORT. Preliminary data suggest that IORT given as a boost after breast-conserving surgery could be a reliable alternative to conventional postoperative fractionated boost radiation by accurate dose delivery and avoiding geographical misses, by enabling smaller treatment volumes and complete skin-sparing and by reducing postoperative radiation time by 7-14 days.     

Radiation-induced DNA damage and repair in lymphocytes from breast cancer patients and their correlation with acute skin reactions to radiotherapy

PURPOSE: Repair of radiation-induced DNA damage plays a critical role for both the susceptibility of patients to side effects after radiotherapy and their subsequent cancer risk. The study objective was to evaluate whether DNA repair data determined in vitro are correlated with the occurrence of acute side effects during radiotherapy. METHODS AND MATERIALS: Breast cancer patients receiving radiation therapy after a breast-conserving surgery were recruited in a prospective epidemiologic study. As an indicator for clinical radiosensitivity, adverse reactions of the skin were recorded. Cryo-preserved lymphocytes from 113 study participants were gamma-irradiated with 5 Gy in vitro and analyzed using the alkaline comet assay. Reproducibility of the assay was determined by repeated analysis (n = 26) of cells from a healthy donor. A coefficient of variation of 0.3 was calculated. RESULTS: The various parameters determined to characterize the individual DNA repair capacity showed large differences between patients. Eleven patients were identified with considerably enhanced DNA damage induction, and 7 patients exhibited severely reduced DNA repair capacity after 15 and 30 min. Six patients were considered as clinically radiosensitive, indicated by moist desquamation of the skin after a total radiation dose of about 50 Gy. CONCLUSIONS: Using the alkaline comet assay as described here, breast cancer patients were identified showing abnormal cellular radiation effects, but this repair deficiency corresponded only at a very limited extent to the acute radiation sensitivity of the skin. Because impaired DNA repair could be involved in the development of late irradiation effects, individuals exhibiting severely reduced DNA repair capacity should be followed for the development of late clinical symptoms.     

Radiosensitivity of jejunal mucosa after whole abdomen irradiation and CDDP pretreatment

Pretreatment of mice with a single radiation dose of 11.5 Gy or with fractionated irradiation (2 x 6.5 or 7.5 Gy, with an interval time of 12 hr) led to a relative decrease in the radiosensitivity of jejunal crypt cells when a second single dose of radiation was delivered 2 months later. When the same irradiation pretreatment was combined with CDDP (6 mg/kg, i.p.) injected 12 hr after single radiation or between two equal doses of radiation, similar crypt cells resistance was obtained. The combination of CDDP with irradiation did not modify the radiosensitivity of jejunal crypts in comparison with irradiation alone, even when the total radiation dose was delivered in 2 split doses. This induced radioresistance was demonstrated to be a reflection of late injury on intestinal tissue, presumably due to hypoxia resulting from vascular damage. It seems that the administration of CDDP did not change the possible hypoxia suspected in crypt cells of mice pretreated with single radiation dose. However, mouse lethality increased significantly when CDDP was combined with irradiation.