[en] Background and purpose: Using pulsed field gel electrophoresis (PFGE) a significant correlation was demonstrated between residual DNA double-strand breaks (dsbs) and the development of late radiation fibrosis in a group of 39 breast cancer patients studied retrospectively. This group formed a training cohort generating a hypothesis that there is a relationship between residual radiation-induced DNA dsbs in cultured fibroblasts and late radiotherapy reactions in breast cancer patients. The aim of this study was to retest and validate the hypothesis. Materials and methods: The study was retrospective. Skin biopsies were taken from a validation cohort of 50 breast cancer patients and PFGE was used to examine residual radiation-induced dsbs in cultured fibroblasts. Late morbidity was measured clinically as fibrosis and using the late effects on normal tissues scales that incorporate subjective, objective management and analytic data (LENT SOMA). Results: PFGE data were obtained for 49 biopsies. In the 49 patients there was no correlation between residual DNA damage and either fibrosis (r=-0.027, P=0.85) or LENT SOMA (r=-0.10, P=0.48) scores. There was no significant relationship between residual damage and fibrosis for the combined training and validation cohorts of 88 patients (r=0.20, P=0.063). Conclusions: This study did not validate the hypothesis that there is a relationship between fibroblast residual DNA damage and late morbidity in breast cancer patients. The PFGE assay on fibroblasts is not a suitable test of the degree of late radiation-induced fibrosis in the breast

[en] Purpose: The aim of the work was to establish to what extent a variety of human severe-combined-immunodeficiency (SCID) disorders are associated with in vitro cellular hypersensitivity to ionizing radiation. Materials and methods: A study was made of fibroblast strains established from individuals with adenosine deaminase deficiency, T(-)B(-) SCID, Omenn's syndrome and a SCID heterozygote. For comparison, an assessment was also made of the radiosensitivity of a series of fibroblast strains derived from: normal donors, a patient with ataxia-telangiectasia (A-T) and an A-T heterozygote. Radiosensitivity was determined using a clonogenic assay following both high (HDR) and low (LDR) dose-rate irradiation. Results: Following HDR irradiation, the fibroblast strains derived from the different human SCID disorders displayed a wide range of radiosensitivity: the adenosine deaminase deficiency cells were similar in radiosensitivity to normal fibroblasts, T(-)B(-) cells were as hypersensitive to radiation as A-T cells and the Omenn's syndrome cells showed intermediate radiosensitivity. However, whereas all four normal cell strains studied showed significant LDR sparing, none of the SCID fibroblasts did. Conclusions: These data indicate that human SCID is variable in terms of radiosensitivity depending on the particular defect. In addition, the lack of LDR sparing of radiation-induced damage suggests the involvement of some form(s) of DNA repair defect in all the human SCID syndromes

[en] Purpose: To investigate the relationship between radiation-induced apoptosis and clonogenic cell kill in 9 cervical cancer cell lines. Methods and Materials: Cells were irradiated with 0, 2, 8, and 30 Gy. The level of apoptosis was evaluated using flow cytometry (Annexin-V binding), light microscopy (morphology), gel electrophoresis (DNA ladder formation), and TUNEL assay. Cell survival was measured using a clonogenic assay. Results: Of the 9 cervical carcinoma cell lines analyzed, 3 underwent radiation-induced apoptosis: CaSki, HT3, and 778. The levels of apoptosis, obtained 72 h after a dose of 30 Gy, were 49%, 28%, and 26%, respectively. All cell lines exhibited some level of background apoptosis measured by Annexin-V binding (mean = 2.6%±0.8; range, 0.2-6.9%) that correlated with the level of radiation-induced apoptosis (r=0.92, p=0.001). In 6 of the 9 lines, necrosis was the dominant form of cell death. A significant inverse relationship was found between the level of radiation-induced apoptosis and necrosis after 30 Gy (r=-0.87, p=0.002). No relationship was found between radiation-induced apoptosis and intrinsic radiosensitivity measured, using a clonogenic assay, as surviving fraction at 2 Gy (SF2). Conclusion: Cervical carcinoma cells do not readily undergo radiation-induced apoptosis in vitro. There is no relationship between ability to undergo apoptosis and intrinsic radiosensitivity measured using a clonogenic assay

[en] Purpose: The aims of the work were to study the intrinsic radiosensitivity of tumor biopsies from patients with cervical carcinoma and to correlate the data with information on patient age, disease stage, differentiation status, tumor volume, and tumor ploidy. Methods and materials: Radiosensitivity was assessed for 145 tumors in vitro as surviving fraction at 2 Gy (SF₂) using a clonogenic assay. Results: Although the clonogens in tumors classified as Stage I or II tended to be more radiosensitive than in Stage III or IV disease, the difference was not statistically significant (p > 0.15). There was also no significant difference in the intrinsic radiosensitivity of well, moderately, or poorly differentiated tumors or between squamous cell carcinoma and adenocarcinoma (p > 0.53). There was no correlation between patient age and tumor radiosensitivity (p = 0.49). Large volume (≥ 4 cm) disease was more radioresistant than small volume (< 4 cm) disease, but the difference was not significant (p = 0.08). Finally, diploid tumors tended to be more radioresistant than aneuploid tumors (p = 0.07). Conclusion: The intrinsic radiosensitivity of cervix tumors is independent of disease stage, tumor grade, and patient age. Weak trends, however, were observed of increased tumor radioresistance for large volume disease and diploid tumors, suggesting that tumor SF₂ may not be a completely independent parameter

[en] Purpose: A retrospective study was made of the role of apoptosis in determining radiotherapy outcome in 39 adenocarcinoma of the cervix. A comparison was also made of the detection of apoptosis by morphology and the TdT dUtp nick end-labeling (TUNEL) assay. Methods and Materials: The level of apoptosis was assessed in paraffin-embedded sections by cell morphology, the TUNEL assay, and a combination of the two. A total of 2,000 cells were counted per section, to obtain apoptotic (AI) and mitotic (MI) indices. Results: Patients with a high AI had a higher survival rate than those with a low AI, however, the difference was not significant. Using a ratio of apoptosis to proliferation indices, patients with an AI:MI > median had significantly better survival than those with AI:MI < median. This was true where the AI was quantified by morphology alone (p = 0.030) or in combination with the TUNEL assay (p = 0.008). Where the AI was quantified by a combination of morphology and TUNEL, the 5-year survival rates for women with AI:MI greater or less than the median were 81% and 25%, respectively. Conclusion: A high ratio of AI:MI in adenocarcinoma of the cervix indicates a good prognosis. A combination of the TUNEL assay and morphology provided the best discrimination between outcome groups

[en] Purpose: To determine whether pretreatment plasma-transforming growth factor beta 1 (TGFβ1) levels are prognostic for tumor control and late morbidity following radiation therapy in carcinoma of the cervix. Methods and Materials: The study was comprised of 79 patients undergoing radiotherapy with curative intent for Stage I-III carcinoma of the cervix. TGFβ1 levels were analyzed using ELISA. Late morbidity was measured using the Franco-Italian glossary. Data were available for the pretreatment levels of circulating tumor markers that represent disease burden, and for peripheral blood lymphocyte radiosensitivity measured as SF2. Results: Pretreatment TGFβ1 levels were a significant prognostic factor for survival and local control. There were weak significant correlations of TGFβ1 levels with disease stage and the levels of circulating tumor markers (CA125, TPA). There was a weak significant correlation between TGFβ1 levels and normal cell radiosensitivity (lymphocyte SF2). There was no relationship between TGFβ1 levels and grade of morbidity and pretreatment TGFβ1 levels were not a significant prognostic factor for the probability of developing late morbidity. Conclusion: In carcinoma of the cervix, pretreatment TGFβ1 levels reflect tumor burden and are a significant prognostic factor for survival. Despite an underlying weak relationship of TGFβ1 levels with intrinsic normal cell radiosensitivity, pretreatment levels are not prognostic for the probability of developing late complications. This finding does not rule out the possible usefulness of measurements toward the end of treatment once tumor burden has been reduced

[en] Background and purpose: To assess the correlation between the LENT (late effects on normal tissue) SOMA (subjective objective management analytic) system and the Franco-Italian glossary scores of late morbidity in cervical cancer patients treated with radiation, and to compare the ability of the scoring systems to detect differences between radiation treatment groups. Materials and methods: The study was retrospective. Patients, invited to take part in the study, had radiotherapy for cervical cancer and had a minimum of 3 years follow-up with no evidence of recurrence. One hundred patients agreed to take part. LENT subjective data were obtained using a patient questionnaire approach in order to complete the scales as published. The LENT objective, management and Franco-Italian glossary scores were obtained by a physician. Correlations between scores and differences between treatment groups were examined using non-parametric tests. Results: The average LENT SOMA scores had a greater resolution than the maximum scores, and using the maximum score alone underestimated treatment morbidity. The Franco-Italian glossary scores correlated strongly with the LENT objective scores (ρ=0.61, P<0.0005), and less strongly with the LENT subjective scores (ρ=0.45, P<0.0005). Significant differences in morbidity between the radiation treatment groups were measured using both the LENT SOMA system and the Franco-Italian glossary. Conclusions: The maximum and average LENT scores should be reported for each subsite. The LENT objective scores correlated well with the scores obtained using the established Franco-Italian glossary, but the LENT system provided additional information on subjective treatment effects. Both systems were able to measure significant differences in morbidity between radiation treatment groups. In conclusion, the LENT SOMA system is a valid and comprehensive approach for scoring the late normal tissue effects of radiotherapy

[en] Purpose: To assess whether incorporation of measurements of surviving fraction at 2 Gy (SF₂) and colony-forming efficiency (CFE) into a tumor control probability (tcp) model increases their prognostic significance. Methods and Materials: Measurements of SF₂ and CFE were available from a study on carcinoma of the cervix treated with radiation alone. These measurements, as well as tumor volume, dose, and treatment time, were incorporated into a Poisson tcp model (tcp_α,ρ). Regression analysis was performed to assess the prognostic power of tcp_α,ρ vs. the use of either tcp models with biologic parameters fixed to best-fit estimates (but incorporating individual dose, volume, and treatment time) or the use of SF₂ and CFE measurements alone. Results: In a univariate regression analysis of 44 patients, tcp_α,ρ was a better prognostic factor for both local control and survival (p<0.001 and p=0.049, respectively) than SF₂ alone (p=0.009 for local control, p=0.29 for survival) or CFE alone (p=0.015 for local control, p=0.38 for survival). In multivariate analysis, tcp_α,ρ emerged as the most important prognostic factor for local control (p<0.001, relative risk of 2.81). After allowing for tcp_α,ρ, CFE was still a significant independent prognostic factor for local control, whereas SF₂ was not. The sensitivities of tcp_α,ρ and SF₂ as predictive tests for local control were 87% and 65%, respectively. Specificities were 70% and 77%, respectively. Conclusions: A Poisson tcp model incorporating individual SF₂, CFE, dose, tumor volume, and treatment time was found to be the best independent prognostic factor for local control and survival in cervical carcinoma patients

[en] 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

[en] Purpose: To study the relationship between residual DNA damage and clonogenic measurements of radiosensitivity in fibroblasts from pretreatment cervix cancer patients. Methods and Materials: Early passage vaginal fibroblasts from nine preradiotherapy cervix cancer patients and two radiosensitive skin fibroblast cell strains were studied. Cell survival was measured by clonogenic assay following both high and low dose rate irradiation. Residual DNA damage was measured using pulsed-field gel electrophoresis (PFGE) after irradiating radiolabeled, plateau-phase cells at 37 deg. C and allowing 24 h for repair. DNA damage was expressed both in terms of the residual damage slope (fitted to data from 60 to 150 Gy) and the fraction of activity released (FAR) following 150 Gy. Results: The surviving fraction at 2 Gy (SF₂) values after high dose rate irradiation for the vaginal fibroblasts ranged from 0.15 to 0.32 (a 2.2-fold difference). When the two radiosensitive cell strains were included, residual damage, expressed as the residual damage slope, correlated with α (r = 0.82, p = 0.002), D bar (r = -0.91, p < 0.001) and SF₂ (p = -0.79, p = 0.004), and when the vaginal fibroblasts alone were studied, the residual damage slope again correlated with clonogenic survival, although less strongly [α (r 0.66, p = 0.053), D bar (r = -0.83, p = 0.006), and SF₂ (r = -0.63, p 0.07)]. Within the group of vaginal fibroblasts there was a 4.0-fold difference in residual DNA damage slope. When residual damage was expressed as FAR at 150Gy, then for all cell strains the correlations were α: r 0.78, p 0.004, D bar: r = -0.86, p = 0.001, and SF₂: r = -0.78, p = 0.004, and for the vaginal fibroblast strains alone the correlations were α: r = 0.60, p = 0.088, D bar: r = -0.75, p = 0.02, and SF₂: r = 0.62, p = 0.077. Conclusion: This study confirms previous findings that residual DNA damage correlates with clonogenic survival in fibroblasts. In addition, it demonstrates a correlation for fibroblasts from pretreatment cervix cancer patients demonstrating a relatively small range of SF₂ values