Comparison of the radiosensitivity of normal-tissue cells with normal-tissue reactions after radiotherapy

Purpose : To investigate whether the in vitro radiosensitivity of normal lymphocytes and fibroblasts evaluated by the micronucleus (MN) assay predicts acute and late reactions after radiotherapy in cancer patients. Materials and methods : Studies were performed on blood samples from 31 cervical and head and neck cancer patients and on skin fibroblasts from eight of the cancer patients. The radiosensitivity of lymphocytes and of fibroblasts was also assessed in 24 and five healthy donors, respectively. Radiosensitivity was measured after in vitro irradiation with doses ranging from 2 to 5 Gy using micronucleus frequency (the number of micronuclei per single binucleated (BN) cell) and the percentage of BN cells with micronuclei. The in vitro results were compared with the maximum grade of acute and late reactions. Results : There was no significant difference in cellular radiosensitivity between cancer patients and healthy donors. Although cancer patients differed considerably in normal-cell radiosensitivity, no correlation was found between radiosensitivity, either of lymphocytes or fibroblasts, and acute and late clinically observed side effects. In addition, no relationship was observed between the radiosensitivity of lymphocytes and fibroblasts derived from the same donors. Conclusion : The data do not support the usefulness of the MN assay in predicting normal-tissue response to radiotherapy in cancer patients.     

Comparison of chromosomal radiosensitivity of normal cells with and without HRS-like response and normal tissue reactions in patients with cervix cancer

PURPOSE: In our previous study, using the micronucleus (MN) assay, the low- and high-dose radiation response of fibroblasts and keratinocytes from cancer patients was assessed. We reported that a hyper-radiosensitivity (HRS)-like phenomenon was observed for fibroblasts of two and keratinocytes of four of the 40 patients studied. In this paper, we report the comparison of these in vitro results and normal tissue reactions in patients with cervix cancer and answer the question of the predictive value of the MN assay. MATERIALS AND METHODS: Of the 40 patients with cervix cancer whose cells were previously studied in vitro, 32 received radiotherapy. The treated group included two patients with HRS-like positive fibroblasts and four patients with HRS-like positive keratinocytes. In 26 patients both types of cells were HRS-like negative. The in vitro results (MN induction measured in patients' fibroblasts and keratinocytes after in vitrogamma-irradiation with doses ranging from 0.05-4 Gy) were compared with the maximum grade of acute and late reactions. RESULTS: Five of the six patients whose cells demonstrated low-dose chromosomal hypersensitivity in vitro, did not suffer from any mild or severe side effects after radiotherapy. Although individual variations in the grade scores of normal-tissue reactions were observed in cancer patients, no significant relationship was found between MN induction, either in fibroblasts or keratinocytes, and acute and late effects. CONCLUSION: Since the MN assay showed no predictive value, it is rather impossible that the severe late complication observed in one of the six HRS-like positive patients reflects her low-dose chromosomal hypersensitivity in vitro.     

In vitro cellular radiosensitivity in relationship to late normal tissue reactions in breast cancer patients: a multi-endpoint case-control study

Purpose: A minority of patients exhibits severe late normal tissue toxicity after radiotherapy (RT), possibly related to their inherent individual radiation sensitivity. This study aimed to evaluate four different candidate in vitro cellular radiosensitivity assays for prediction of late normal tissue reactions, in a retrospective matched case-control set-up of breast cancer patients.

Methods: The study population consists of breast cancer patients expressing severe radiation toxicity (12 cases) and no or minimal reactions (12 controls), with a follow-up for at least 3 years. Late adverse reactions were evaluated by comparing standardized photographs pre- and post-RT resulting in an overall cosmetic score and by clinical examination using the LENT-SOMA scale. Four cellular assays on peripheral blood lymphocytes reported to be associated with normal tissue reactions were performed after in vitro irradiation of patient blood samples to compare case and control radiation responses: radiation-induced CD8+ late apoptosis, residual DNA double-strand breaks, G0 and G2 micronucleus assay.

Results: A significant difference was observed for all cellular endpoints when matched cases and controls were compared both pairwise and grouped. However, it is important to point out that most case-control pairs showed a substantial overlap in standard deviations, which questions the predictive value of the individual assays. The apoptosis assay performed best, with less apoptosis seen in CD8+ lymphocytes of the cases (average: 14.45%) than in their matched controls (average: 30.64%) for 11 out of 12 patient pairs (p?p?p?Conclusion: This matched case-control study in breast cancer patients, using different endpoints for in vitro cellular radiosensitivity related to DNA repair and apoptosis, suggests that patients’ intrinsic radiosensitivity is involved in the development of late normal tissue reactions after RT. Larger prospective studies are warranted to validate the retrospective findings and to use in vitro cellular assays in the future to predict late normal tissue radiosensitivity and discriminate individuals with marked RT responses.     

In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients

Purpose : To compare colony-forming and comet assays on fibroblasts and lymphocytes of 32 breast cancer patients irradiated after breast-conserving operations and to correlate the results with acute clinical radiation reactions in the skin. Material and methods : Skin fibroblasts were isolated and cultivated before radiotherapy and lymphocytes were drawn prior to the first and directly after the final external irradiation. The colony-forming assay was performed with fibroblasts and the comet assay with lymphocytes and fibroblasts of breast cancer patients according to standard protocols. The clinical radiation reactions of the patients were graded according to the RTOG system. Results : No significant correlation (p =0.09) was detected between clinical acute skin reactions and the in vitro clonogenic data in fibroblasts. Results of the comet assay in lymphocytes, however, showed a significant correlation (p <0.05) with the clinical data when patients were divided into two groups with average and elevated acute reactions. Apart from initial damage, fibroblasts did not show significant differences between the two patient groups. Repeated comet assays in lymphocytes of the same patient drawn before treatment and before and after external radiotherapy demonstrated good reproducibility of the test and no significant impact of preceding radiation treatment. There was a good correlation (r =0.65) between the comet assay results in fibroblasts and lymphocytes of the same individual. Conclusions : In this cohort of patients, a significant correlation between the in vitro results of the comet assay in lymphocytes and clinical acute reactions was detected. The results of the comet assay and of fibroblast colony formation did not correlate with in vitro radiosensitivity.     

Comparison of chromosomal radiosensitivity of normal cells with and without HRS-like response and normal tissue reactions in patients with cervix cancer

Purpose: In our previous study, using the micronucleus (MN) assay, the low- and high-dose radiation response of fibroblasts and keratinocytes from cancer patients was assessed. We reported that a hyper-radiosensitivity (HRS)-like phenomenon was observed for fibroblasts of two and keratinocytes of four of the 40 patients studied. In this paper, we report the comparison of these in vitro results and normal tissue reactions in patients with cervix cancer and answer the question of the predictive value of the MN assay.

Materials and methods: Of the 40 patients with cervix cancer whose cells were previously studied in vitro, 32 received radiotherapy. The treated group included two patients with HRS-like positive fibroblasts and four patients with HRS-like positive keratinocytes. In 26 patients both types of cells were HRS-like negative. The in vitro results (MN induction measured in patients' fibroblasts and keratinocytes after in vitroγ-irradiation with doses ranging from 0.05–4 Gy) were compared with the maximum grade of acute and late reactions.

Results: Five of the six patients whose cells demonstrated low-dose chromosomal hypersensitivity in vitro, did not suffer from any mild or severe side effects after radiotherapy. Although individual variations in the grade scores of normal-tissue reactions were observed in cancer patients, no significant relationship was found between MN induction, either in fibroblasts or keratinocytes, and acute and late effects.

Conclusion: Since the MN assay showed no predictive value, it is rather impossible that the severe late complication observed in one of the six HRS-like positive patients reflects her low-dose chromosomal hypersensitivity in vitro.     

In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients

PURPOSE: To compare colony-forming and comet assays on fibroblasts and lymphocytes of 32 breast cancer patients irradiated after breast-conserving operations and to correlate the results with acute clinical radiation reactions in the skin. MATERIAL AND METHODS: Skin fibroblasts were isolated and cultivated before radiotherapy and lymphocytes were drawn prior to the first and directly after the final external irradiation. The colony-forming assay was performed with fibroblasts and the comet assay with lymphocytes and fibroblasts of breast cancer patients according to standard protocols. The clinical radiation reactions of the patients were graded according to the RTOG system. RESULTS: No significant correlation (p =0.09) was detected between clinical acute skin reactions and the in vitro clonogenic data in fibroblasts. Results of the comet assay in lymphocytes, however, showed a significant correlation (p <0.05) with the clinical data when patients were divided into two groups with average and elevated acute reactions. Apart from initial damage, fibroblasts did not show significant differences between the two patient groups. Repeated comet assays in lymphocytes of the same patient drawn before treatment and before and after external radiotherapy demonstrated good reproducibility of the test and no significant impact of preceding radiation treatment. There was a good correlation (r =0.65) between the comet assay results in fibroblasts and lymphocytes of the same individual. CONCLUSIONS: In this cohort of patients, a significant correlation between the in vitro results of the comet assay in lymphocytes and clinical acute reactions was detected. The results of the comet assay and of fibroblast colony formation did not correlate with in vitro radiosensitivity.     

Relationships between acute reactions to radiotherapy in head and neck cancer patients and parameters of radiation-induced DNA damage and repair in their lymphocytes

PURPOSE: To study the relationship between lymphocyte radiosensitivity measured in vitro and acute reactions to radiotherapy in patients with head and neck cancer. MATERIALS AND METHODS: Acute reactions were measured in 34 patients using the Dische scale. Lymphocyte radiosensitivity was measured using the alkaline comet assay, the micronucleus assay, the nuclear division index and morphological assessment of apoptosis. RESULTS: There was a weak, statistically significant correlation between in vitro radiosensitivity measured as the rate of DNA damage repair and the cumulative radiation dose exerting the maximum acute reaction scored (r = -0.366, p = 0.039, n = 34). Subgroup analyses showed that for patients with a low level of radiation-induced DNA damage there was a statistically significant relationship between lymphocyte radiosensitivity measured as inhibition of proliferation and acute toxicity (r = -0.621, p = 0.007, n = 18). For patients with a high level of residual DNA damage, there was a relationship between lymphocyte radiosensitivity measured using the micronucleus assay and acute toxicity (r = -0.597, p = 0.023, n = 14). CONCLUSIONS: Combining two measures of radiosensitivity improves the ability to correlate in vitro lymphocyte radiosensitivity and acute radiotherapy toxicity data.     

Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers

Purpose: The chromosomal radiosensitivity of a selected group of familial breast cancer patients carrying a mutation in BRCA1 (n=11) or BRCA2 (n=9) and a group of healthy mutation carriers (n=12) was investigated and compared to a reference group of breast cancer patients without a BRCA1/2 mutation (n=78) and a group of healthy women carrying no mutation (n=58).

Materials and methods: The chromosomal radiosensitivity was assessed with the G2 and the G0‐micronucleus (MN)‐assay on fresh blood samples and on Epstein‐Barr virus (EBV)‐transformed lymphoblastoid cell lines. For the MN‐assay, lymphocytes were exposed in vitro to 3.5?Gy and 2?Gy ⁶⁰Co γ‐rays at a high dose rate (HDR) or low dose rate (LDR). 70‐h post‐irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. For the G2‐assay lymphocytes were irradiated in vitro with a dose of 0.4?Gy ⁶⁰Co γ‐rays after 71h incubation. Cultures were arrested 90?min after irradiation and chromatid breaks were scored in 50 metaphases.

Results: The group of breast cancer patients with a BRCA1 or 2 mutation was on average more radiosensitive than the control group, but not different from breast cancer patients without a BRCA mutation. The radiation response of healthy BRCA1/2 carriers was not significantly different from the control group and also not different from relatives without a BRCA mutation. Comparing the radiation response in EBV cell lines derived from breast cancer patients with or without a BRCA1 mutation revealed no significant difference. Conclusions: Our results reveal that chromosomal radiosensitivity observed in breast cancer patients heterozygous for BRCA1 or 2 mutations, could not be demonstrated in healthy BRCA1/2 mutation carriers. This suggests that mutations in BRCA1 or 2 genes are not playing a main role in chromosomal radiosensitivity, this although BRCA1 and 2 are both involved in DNA repair/signalling processes.     

Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers

PURPOSE: The chromosomal radiosensitivity of a selected group of familial breast cancer patients carrying a mutation in BRCA1 (n=11) or BRCA2 (n=9) and a group of healthy mutation carriers (n=12) was investigated and compared to a reference group of breast cancer patients without a BRCA1/2 mutation (n=78) and a group of healthy women carrying no mutation (n=58). MATERIALS AND METHODS: The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus (MN)-assay on fresh blood samples and on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. For the MN-assay, lymphocytes were exposed in vitro to 3.5 Gy and 2 Gy 60Co gamma-rays at a high dose rate (HDR) or low dose rate (LDR). 70-h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. For the G2-assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co gamma-rays after 71h incubation. Cultures were arrested 90 min after irradiation and chromatid breaks were scored in 50 metaphases. RESULTS: The group of breast cancer patients with a BRCA1 or 2 mutation was on average more radiosensitive than the control group, but not different from breast cancer patients without a BRCA mutation. The radiation response of healthy BRCA1/2 carriers was not significantly different from the control group and also not different from relatives without a BRCA mutation. Comparing the radiation response in EBV cell lines derived from breast cancer patients with or without a BRCA1 mutation revealed no significant difference. CONCLUSIONS: Our results reveal that chromosomal radiosensitivity observed in breast cancer patients heterozygous for BRCA1 or 2 mutations, could not be demonstrated in healthy BRCA1/2 mutation carriers. This suggests that mutations in BRCA1 or 2 genes are not playing a main role in chromosomal radiosensitivity, this although BRCA1 and 2 are both involved in DNA repair/signalling processes.     

DNA Double-Strand Break Induction and Repair in Irradiated Lymphoblastoid, Fibroblast Cell Lines and White Blood Cells from ATM, NBS and Radiosensitive Patients

BACKGROUND AND PURPOSE: DNA double-strand breaks (dsbs) in lymphoblastoid cell lines (LCLs), fibroblasts and white blood cells from healthy donors, cancer patients with and without late effects of grade 3-4 (RTOG) as well as donors with known radiosensitivity syndromes were examined with the aim to detect dsb repair ability as a marker for radiosensitivity. MATERIAL AND METHODS: LCLs from six healthy donors, seven patients with a heterozygous or homozygous genotype for ataxia-telangiectasia (ATM) and Nijmegen breakage syndrome (NBS), two patients with a late toxicity of grade 3-4 (RTOG), and one cell line with a ligase IV-/- status and its parental cell line were examined. Furthermore, fibroblasts from patients with ATM, NBS, two healthy control individuals, and leukocytes from 16 healthy and 22 cancer patients including seven patients with clinical hypersensitivity grade 3 (RTOG) were examined. Cells were irradiated in vitro with 0-150 Gy. Initial damage as well as remaining damage after 8 and 24 h were measured using constant field gel electrophoresis. RESULTS: In contrast to cells derived from patients homozygous for NBS, impaired dsb repair ability could be detected both in fibroblast and lymphoblastoid cells from ATM and ligase IV-/- patients. The dsb repair ability of all 38 leukocyte cell lines (patients with grade 3-4 late effects and controls) was similar, whereas the initial damage among healthy donors was less. CONCLUSION: Despite showing a clinically elevated radiosensitivity after irradiation, the DNA repair of the patients with clinical hypersensitivity grade 3 (RTOG) appeared to be normal. Other mechanisms such as mutations, altered cell cycle or defective apoptosis could play a critical role toward determining radiosensitivity.     

Chromosomal Radiosensitivity, Cancer Predisposition and Response to Radiotherapy

AIM: This paper briefly summarizes the research on this topic, undertaken in the Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, England, over the previous 6 years. PATIENTS AND METHOD: Patients with the recessively-inherited disease, ataxia-telangiectasia (A-T), who are cancer-prone and suffer severe reactions after radiotherapy, also have highly radiosensitive cells, particularly when chromosome damage is used as the measure of radiosensitivity. Enhanced chromosomal radiosensitivity is also a feature of many other cancer-prone disorders. We have investigated the possible role of such radiosensitivity as a marker of cancer predisposition and response to radiotherapy in the general population. RESULTS: We found that 42% (57/135) of breast cancer patients exhibit chromosomal radiosensitivity when lymphocytes are irradiated in the G2 phase of the cell cycle, compared with 6% (6/105) of healthy controls (Figure 1). These figures are much higher than the estimated frequencies of carriers of the ataxia-telangiectasia gene (heterozygotes) amongst breast cancer patients (< 5%) and controls (0.5%). We have also obtained evidence of heritability of G2 sensitivity by studying relatives of breast cancer cases (Figures 2 and 3). The pattern of inheritance is relatively simple and attributable to 1 or 2 genes segregating in each family (Figure 4). In a prospective study of 123 breast cancer patients, 9 (7%) had severe acute reactions to radiotherapy and their mean G2 sensitivity was significantly greater (p = 0.001) than that of the remaining patients (Figure 5). In 16 patients with adverse acute reactions we found no mutations of the ataxia-telangiectasia gene (ATM). Using another chromosomal assay (micronucleus induction in G0 lymphocytes) we found that the mean radiosensitivity of patients with severe late reactions was higher than that of normal reactors. For example, 8 patients with severe fibrosis were more sensitive (p = 0.055) than 39 patients with a normal response (Figure 6). However, the discriminatory power of these chromosomal assays is too low for them to be used alone in a clinical setting. CONCLUSION: Our results provide good evidence that genes other than ATM, that confer chromosomal radiosensitivity, are involved in low penetrance predisposition to breast cancer in a high proportion of cases and contribute to adverse reactions after radiotherapy.     

Chromosomal radiosensitivity study of temporary nuclear workers and the support of the adaptive response induced by occupational exposure

Purpose : To study chromosomal radiosensitivity in a population of radiation workers and investigate the possibility of an adaptive response in lymphocytes of workers after short-term occupational exposure to ionizing radiation. Materials and methods : The studied group comprised 41 workers temporarily employed at the Nuclear Power Plant Doel (Belgium) for reactor maintenance. A blood sample was taken before and directly after the exposure period of about 1 month. Chromosomal radiosensitivity was assessed in vitro by the G2 assay and the G0 micronucleus (MN) assay. For the MN assay, a low dose-rate (LDR) in vitro irradiation protocol was applied in addition to high dose-rate (HDR) irradiation of the blood samples in order to determine the dose-rate sparing (DRS) effect. Results : No statistically significant effect of the occupational exposures (up to 10 mSv) on the baseline MN frequencies without in vitro irradiation was observed. A comparison of the number of chromatid aberrations pre- and post-exposure shows no effect of the occupational exposure. On the other hand, the G0-MN assay with the LDR irradiation protocol reveals a systematic reduction in chromosomal radiosensitivity by the exposure, increasing with dose. For workers who received the highest dose (4-10 mSv) a statistically significant (p < 0.05) decrease of the in vitro induced MN yields and increase of the dose-rate sparing was observed. Conclusions : Short-term low-dose occupational exposure may act as an in vivo adaptive dose and stimulate repair in G0 lymphocytes.     

Chromosomal radiosensitivity study of temporary nuclear workers and the support of the adaptive response induced by occupational exposure

PURPOSE: To study chromosomal radiosensitivity in a population of radiation workers and investigate the possibility of an adaptive response in lymphocytes of workers after short-term occupational exposure to ionizing radiation. MATERIALS AND METHODS: The studied group comprised 41 workers temporarily employed at the Nuclear Power Plant Doel (Belgium) for reactor maintenance. A blood sample was taken before and directly after the exposure period of about 1 month. Chromosomal radiosensitivity was assessed in vitro by the G2 assay and the G0 micronucleus (MN) assay. For the MN assay, a low dose-rate (LDR) in vitro irradiation protocol was applied in addition to high dose-rate (HDR) irradiation of the blood samples in order to determine the dose-rate sparing (DRS) effect. RESULTS: No statistically significant effect of the occupational exposures (up to 10 mSv) on the baseline MN frequencies without in vitro irradiation was observed. A comparison of the number of chromatid aberrations pre- and post-exposure shows no effect of the occupational exposure. On the other hand, the G0-MN assay with the LDR irradiation protocol reveals a systematic reduction in chromosomal radiosensitivity by the exposure, increasing with dose. For workers who received the highest dose (4-10 mSv) a statistically significant (p <0.05) decrease of the in vitro induced MN yields and increase of the dose-rate sparing was observed. CONCLUSIONS: Short-term low-dose occupational exposure may act as an in vivo adaptive dose and stimulate repair in G0 lymphocytes.     

Chromosomal radiosensitivity of HIV positive individuals

Purpose:?Radiosensitivity in relation to the human immunodeficiency virus (HIV) status is important in South Africa as the prevalence of HIV infections is high. In this study the in?vitro chromosomal radiosensitivity of HIV positive individuals was investigated and compared with that of HIV negative individuals.

Materials and methods:?Blood samples from 59 HIV positive and 39 HIV negative individuals were exposed in?vitro to doses of 6MV X-rays ranging from 1–4?Gy. Chromosomal radiosensitivity was assessed with the micronucleus assay. Micronuclei are a measure of chromosomal damage and were quantified in at least 500 binucleated lymphoblasts (BN) per sample. Un-irradiated control samples from each donor were also analysed.

Results:?In 47% of HIV positive individuals difficulties with cell stimulation by adding phytohaemagglutinin (PHA) to blood cultures were noticed which resulted in insufficient yield of BN for microscopic analysis. Micronuclei frequencies were consistently higher in irradiated lymphocytes obtained from HIV positive individuals compared to that observed in cells from HIV negative donors. Data for both groups were fitted to the linear-quadratic equation Y?=?αD?+?βD² where Y is the number of micronuclei in 500 binucleated cells and D is the dose in Gy. The fitted parameters for respectively HIV positive and HIV negative lymphocytes are α?=?80.17?Gy^?1, β?=?14?Gy^?2 and α?=?54.5?Gy^?1, β?=?16.2?Gy^?2. The confidence ellipses of these parameters are separated indicating that the increase in radiosensitivity is statistically significant.

Conclusion:?T-lymphocytes of HIV infected individuals were considerably more sensitive to X-rays compared to that of HIV negative donors. This may have implications for normal tissue tolerance during radiotherapy as well as for the radiological health of radiation workers.     

The use of cryopreserved lymphocytes in assessing inter-individual radiosensitivity with the micronucleus assay

PURPOSE: The feasibility of using cryopreserved lymphocytes to detect inter-individual differences in chromosomal radiosensitivity was investigated. Typically, such studies are conducted with fresh blood samples but, in a clinical setting, when availability of samples is unpredictable, this is not always convenient. The sensitivity of 23 normal healthy donors, 11 breast cancer patients who had shown severe acute skin reactions to radiotherapy and seven ataxia telangiectasia (A-T) heterozygotes was determined. MATERIALS AND METHODS: Thawed lymphocytes were exposed to high (HDR) or low dose rate (LDR) gamma irradiation (3.5 Gy) in Go, stimulated with PHA, treated with cytochalasin-B 24 h later and then harvested at 90 h for the determination of micronucleus (MN) yields in binucleate cells. RESULTS: Each normal donor was tested one to three times. Mean MN yields were 76.1 +/- 9.3/100 cells at HDR and 44.5 +/- 5.3 at LDR, giving an LDR sparing effect of 39.6 +/- 9.3%. A relatively high proportion of tests failed to yield sufficient binucleate cells for analysis. Inter-experimental variability was also high and it was not possible to demonstrate inter-individual differences in sensitivity in spite of the use of an internal control sample from a single normal donor in each experiment. There was a small but significant increase in radiation-induced MN in the breast cancer patients compared with the normals at LDR (but not at HDR), but a complete overlap with the normal range. There was no increase in sensitivity in the A-T heterozygotes at HDR. The LDR samples failed because the LDR protocol reduced proliferation rates, and radiation-induced mitotic inhibition in this group was higher than in normals. CONCLUSIONS: In comparison with previous experience with fresh blood samples, the use of frozen lymphocytes is not as satisfactory because: (1) experimental failures are higher; (2) inter-experiment variability is higher: (3) dose-rate sparing is lower, suggesting poorer repair; and (4) the ability to discriminate between breast cancer cases and normals is probably lower.     

A semi-automated micronucleus-centromere assay to assess low-dose radiation exposure in human lymphocytes

Abstract

Purpose: The in vitro micronucleus (MN) assay is a reliable method to assess radiation-induced chromosomal damage in human peripheral blood lymphocytes. It is used to evaluate in vivo radiation over-exposure and to assess in vitro chromosomal radiosensitivity. A limitation of the MN assay is the relatively high and variable spontaneous MN frequency that restricts low-dose estimation to doses of about 0.3 gray (Gy). As radiation-induced MN mainly contain acentric fragments and spontaneous MN originate from lagging chromosomes, both MN types can be distinguished from each other by using fluorescence in situ hybridisation (FISH) with a pan-centromeric probe. The aim of this study was to investigate if the sensitivity, reliability and processing time of the MN assay can be enhanced by combining the automated MN assay with pan-centromere scoring.

Materials and methods: Blood samples from 10 healthy donors were irradiated in vitro with low doses of gamma-rays. Dose response curves were determined for fully-automated and semi-automated MN scoring and semi-automated scoring of centromere negative MN (MNCM?).

Results: A good correlation was obtained between fully-automated and semi-automated MN scoring (r² = 0.9973) and between fully automated MN scoring and semi-automated scoring of MNCM? (r² = 0.998). With the Wilcoxon test, a significant p value was obtained between 0 and 0.2 Gy for the fully-automated MN analysis, between 0 and 0.1 Gy for semi-automated MN analysis and between 0 and 0.05 Gy for semi-automated scoring of MNCM?.

Conclusion: The semi-automated micronucleus-centromere assay combines high-speed MN analysis with a more accurate assessment in the low-dose range which makes it of special interest for large-scale radiation applications.     

Individual Radiosensitivity Does not Correlate with Radiation-Induced Apoptosis in Lymphoblastoid Cell Lines or CD<Superscript>3+</Superscript> Lymphocytes

Background and Purpose: Spontaneous and radiation-induced apoptosis of lymphoblastoid cell lines (LCLs) derived from healthy donors, cancer patients and donors with radiosensitivity syndromes as well as CD³⁺ lymphocytes from patients with grade 3 late toxicity were investigated as a possible marker for the detection of individual radiosensitivity. These investigations are based on the hypothesis that hypersensitive patients have reduced levels of apoptosis after in vitro irradiation as a result of a defect in the signaling pathway. Material and Methods: Epstein-Barr virus-(EBV-)transformed LCLs derived from five healthy donors, seven patients with heterozygous or homozygous genotype for ataxia-telangiectasia or Nijmegen breakage syndrome and five patients with grade 3 late toxicity (RTOG) were investigated. In addition, CD³⁺ lymphocytes from 21 healthy individuals and 18 cancer patients including five patients with a proven cellular hypersensitivity to radiation were analyzed. Cells were irradiated in vitro with a dose of 2 and 5 Gy and were incubated for 48 h. Apoptotic rates were measured by the TUNEL assay followed by customized image analysis. Results: Four out of seven radiosensitivity syndrome patients were identified to have an increased cellular radiosensitivity as determined by reduced apoptotic rates after irradiation of their respective LCLs. Comparatively, only two of the five hypersensitive cancer patients were clearly identified by reduced apoptotic rates. Spontaneous apoptotic rates were very homogeneous among all 39 samples from controls and patients, while lymphocytes of all cancer patients showed significantly lower radiation-induced rates. Conclusion: Only a subgroup of hypersensitive patients may be identified by reduction of radiation-induced apoptotic rate. It is concluded that the hypothesis according to which hypersensitive cells have reduced levels of apoptosis is only conditionally true. The authors suggest that this assay can be used in combination with additional tests evaluating DNA double-strand break repair, cell-cycle control and chromosomal aberrations for the evaluation for individual hypersensitivity.     

Radiosensitivity in patients suffering from chronic kidney disease

Abstract

Purpose: Patients suffering from chronic kidney disease (CKD) exhibit a high incidence of cancer, as well as high levels of genetic damage. We hypothesized that these patients show genomic instability detected as an increased chromosomal radiosensitivity in front of the genetic damage induced by ionizing radiation.

Material and methods: The background levels of genetic damage and the net genetic damage after in vitro irradiation with 0.5 Gy were analyzed using the micronucleus (MN) assay in peripheral blood lymphocytes. A total number of 552 individuals (179 controls and 373 CKD patients) were included in the study.

Results: The net radiation-induced genetic damage was significantly higher in CKD patients than in controls; but no differences between those patients submitted to hemodialysis and those in pre-dialytic stages were detected. A positive correlation was observed between basal and net micronucleus frequencies in CKD patients what would indicate an underlying genetic background modulating DNA damage levels.

Conclusions: Our results indicate that CKD patients present genomic instability, measured as an increased chromosomal radiosensitivity in front of ionizing radiation.     

Lymphocyte telomere length correlates with in vitro radiosensitivity in breast cancer cases but is not predictive of acute normal tissue reactions to radiotherapy

PURPOSE: To examine the hypothesis that lymphocyte telomere length may be predictive of both breast cancer susceptibility and severity of acute reactions to radiotherapy. MATERIALS AND METHODS: Peripheral blood lymphocyte cultures from breast cancer patients (with normal or severe skin reactions to radiotherapy) and normal individuals were assessed for in vitro radiosensitivity as measured by apoptosis, cell cycle delay and cytotoxicity. Telomere lengths were determined by a flow cytometric fluorescence in situ hybridization assay (FLOW-FISH). RESULTS: Female breast cancer cases (n = 24) had reduced lymphocyte telomere lengths by comparison with healthy controls (n = 20, p < 0.04). However, the average age of healthy controls was less (45.4) than cases (53). When the control group was modified to give a better age match (51.5, n = 13) the reduced telomere length in cases was not significantly different from controls. Lymphocytes from breast cancer cases also showed reduced cell cycle delay (p < 0.001) and increased apoptosis (p < 0.01) following irradiation in vitro at 3 and 5 Gy respectively, compared to healthy controls. Statistical significance was maintained with the improved age matching of groups. Comparison of lymphocytes from breast cancer patients with normal (n = 11) and severe (n = 13) skin reactions to radiotherapy failed to identify differences in telomere length or cellular radiosensitivity in this limited sample. CONCLUSIONS: This study adds to the evidence suggesting a correlation between altered cellular radiosensitivity and breast cancer. However, in the cases investigated, telomere length does not appear to be predictive of acute skin reactions to radiotherapy.     

Association between plasma proteome profiles analysed by mass spectrometry,a lymphocyte-based DNA-break repair assay and radiotherapy-induced acute mucosal reaction in head and neck cancer patients

Purpose:?The plasma proteome was analysed as a potential source of markers of radiosensitivity in patients treated with definitive radiotherapy for head and neck cancer.

Materials and methods:?Acute mucosal reactions that developed during radiotherapy were assessed in 55 patients. Blood samples were collected from each patient before the treatment and also from 50 healthy donors. The low-molecular-weight fraction of the plasma proteome (2,000–10,000 Da range) was analysed by the Matrix-Assisted Laser Desorption Ionisation mass spectrometry. The capacity for DNA break repair was assessed by the comet assay using lymphocytes irradiated in vitro.

Results:?Spectral components registered in plasma samples were used to build classifiers that discriminated patients from healthy individuals with about 90% specificity and sensitivity (components of 4469, 6929 and 8937 Da were the most essential for cancer classification). Four spectral components were identified (2219, 2454, 3431 and 5308 Da) whose abundances correlated with a maximal intensity of the acute reaction. Several spectral components whose abundances correlated with the rate of DNA repair in irradiated lymphocytes were also detected. Additionally, a more rapid escalation of an acute reaction was correlated with a higher level of unrepaired damage assessed by the comet assay.

Conclusions:?The plasma proteome could be considered as a potential source of predictive markers of acute reaction in patients with head and neck cancer treated with radiotherapy.