J. B P Barber

Heritability of Cellular Radiosensitivity: A Marker of Low-Penetrance Predisposition Genes in Breast Cancer?

Many inherited cancer-prone conditions show an elevated sensitivity to the induction of chromosome damage in cells exposed to ionizing radiation, indicative of defects in the processing of DNA damage. We earlier found that 40% of patients with breast cancer and 5%-10% of controls showed evidence of enhanced chromosomal radiosensitivity and that this sensitivity was not age related. We suggested that this could be a marker of cancer-predisposing genes of low penetrance. To further test this hypothesis, we have studied the heritability of radiosensitivity in families of patients with breast cancer. Of 37 first-degree relatives of 16 sensitive patients, 23 (62%) were themselves sensitive, compared with 1 (7%) of 15 first-degree relatives of four patients with normal responses. The distribution of radiosensitivities among the family members showed a trimodal distribution, suggesting the presence of a limited number of major genes determining radiosensitivity. Segregation analysis of 95 family members showed clear evidence of heritability of radiosensitivity, with a single major gene accounting for 82% of the variance between family members. The two alleles combine in an additive (codominant) manner, giving complete heterozygote expression. A better fit was obtained to a model that includes a second, rarer gene with a similar, additive effect on radiosensitivity, but the data are clearly consistent with a range of models. Novel genes involved in predisposition to breast cancer can now be sought through linkage studies using this quantitative trait.

Increased chromosomal radiosensitivity in breast cancer patients: a comparison of two assays

PURPOSE It has been shown previously that sensitivity to the induction of chromosome damage by ionizing radiation is, on average, higher in G2 or G0 lymphocytes of breast cancer patients than of normal healthy controls. The authors suggested that elevated chromosomal radiosensitivity may be a marker for breast cancer predisposition. To investigate whether the G0 micronucleus assay is a true surrogate for the more demanding G2 metaphase assay, both tests have now been performed on the same blood samples from 80 patients. METHODS For the G0 micronucleus assay, cells were exposed to 3.5 Gy 137Cs gamma-rays 6 h before mitogenic stimulation, treated with cytochalasin B at 24 h post-stimulation and harvested at 90 h. For the G2 assay, at 72 h after stimulation cells were given 0.5 Gy X-rays and harvested 90 min later. RESULTS Previous observations were confirmed, now with much larger numbers of donors, in that approximately 40% of breast cancer patients showed elevated sensitivity in the G2 assay (135 patients, 105 normals) and 25% in the G0 assay (130 patients, 68 normals). However, there was no correlation between G2 and G0 sensitivity for the 80 patients tested (r = -0.001, p = 0.99). Most of the sensitive patients were either G2 or G0 sensitive, with only 4% sensitive in both assays. CONCLUSIONS The results suggest that different mechanisms of chromosomal radiosensitivity operate in G2 and G0 cells and that, in general, each chromosomally radiosensitive patient is defective in only one such mechanism, possibly via mutation (or polymorphism) of a single gene. Such mutations may confer cancer predisposition, of low penetrance, in a substantial proportion of patients.

Radiation-induced micronucleus induction in lymphocytes identifies a high frequency of radiosensitive cases among breast cancer patients: A test for predisposition?

Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a feature of many cancer-predisposing conditions. We previously showed that 42% of an unselected series of breast cancer patients and 9% of healthy control subjects showed elevated chromosomal radiosensitivity of lymphocytes irradiated in the G2 phase of the cell cycle. We suggested that, in addition to the highly penetrant genes BRCA1 and BRCA2, which confer a very high risk of breast cancer and are carried by about 5% of all breast cancer patients, there are also low-penetrance predisposing genes carried by a much higher proportion of breast cancer patients, a view supported by recent epidemiological studies. Ideally, testing for the presence of these putative genes should involve the use of simpler methods than the G2 assay, which requires metaphase analysis of chromosome damage. Here we report on the use of a simple, rapid micronucleus assay in G0 lymphocytes exposed to high dose rate (HDR) or low dose rate gamma-irradiation, with delayed mitogenic stimulation. Good assay reproducibility was obtained, particularly with the HDR protocol, which identified 31% (12 out of 39) of breast cancer patients compared with 5% (2 out of 42) of healthy controls as having elevated radiation sensitivity. In the long term, such cytogenetic assays may have the potential for selecting women for intensive screening for breast cancer.

TGF-β1 levels in pre-treatment plasma identify breast cancer patients at risk of developing post-radiotherapy fibrosis

A serious complication of radiotherapy in the treatment of cancer patients is the late onset of fibrosis in normal tissues. Transforming growth factor β (TGF‐β) is emerging as a key mediator of the fibrotic process through its effects on stimulation of fibroblast proliferation, migration and extracellular matrix (ECM) synthesis. The fact that radiation‐induced vascular injury tends to precede the development of fibrosis has led to the suggestion that vascular damage is crucial in its pathogenesis. CD105, the specific type III vascular receptor for TGF‐β1 and ‐β3, modulates cell proliferation and ECM production in response to TGF‐β in vitro. In this study, we have quantified the levels of TGF‐β1 and soluble CD105–TGF‐β1 complex in 91 pre‐radiotherapy plasma samples from early‐stage (T1 or T2) breast cancer patients utilising an enhanced chemiluminescence ELISA system. During the follow‐up period, 24 patients had developed moderate and one severe fibrosis of the breast. The mean TGF‐β1 level in these 25 patients was 203.2 ± 37.3 pg/ml, which was significantly elevated above the level for those with no fibrosis. Furthermore, a significantly lower CD105–TGF‐β1 complex level was observed in the former compared to the latter. Spearmans correlation analysis showed that TGF‐β1 was positively correlated and the CD105–TGFβ1 complex inversely correlated with the occurrence of breast fibrosis. Using a cut‐off value of 96 pg/ml, the sensitivity and specificity of TGF‐β1 levels in predicting breast fibrosis were 76% and 74%, respectively. Our results indicate that TGF‐β1 and the receptor–ligand complex appear to be of clinical value in identifying patients at risk of developing post‐radiotherapy fibrosis. Int. J. Cancer (Pred. Oncol.) 84:155–159, 1999.

Absence of mutations in the ATM gene in breast cancer patients with severe responses to radiotherapy

The effectiveness of cancer radiotherapy is compromised by the small proportion (approximately 5%) of patients who sustain severe normal tissue damage after standard radiotherapy treatments. Predictive tests are required to identify these highly radiosensitive cases. Patients with the rare, recessively inherited, cancer-prone syndrome ataxia-telangiectasia (A-T) sustain extremely severe normal tissue necrosis after radiotherapy and their cultured cells are also highly radiosensitive. Clinically normal carriers (heterozygotes) of the A-T gene have an increased risk of breast cancer, account for approximately 4% of all breast cancer cases and show a modest increase in cellular radiosensitivity in vitro. It has been suggested that a substantial proportion of highly radiosensitive (HR) breast cancer patients may be A-T heterozygotes, and that screening for mutations in the A-T gene could be used as a predictive test. We have tested this hypothesis in a group of cancer patients who showed adverse reactions to radiotherapy. Sixteen HR breast cancer patients showing mainly acute reactions (and seven HR patients with other cancers) were tested for ATM mutations using the restriction endonuclease fingerprinting assay. No mutations typical of those found in obligate A-T heterozygotes were detected. If the estimate that 4% of breast cancer cases are A-T gene carriers is correct, then ATM mutations do not confer clinical radiosensitivity. These early results suggest that screening for ATM mutations in cancer patients may not be of value in predicting adverse reactions.

A correlation between residual radiation-induced DNA double-strand breaks in cultured fibroblasts and late radiotherapy reactions in breast cancer patients.

BACKGROUND AND PURPOSE Prediction of late normal tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Measurement of residual DNA double strand breaks using pulsed field gel electrophoresis (PFGE) shows promise in this field. The aim of this study was to test the predictive potential of PFGE in a group of retrospectively studied breast cancer patients. MATERIALS AND METHODS Thirty nine patients, treated uniformly for breast cancer 9-15 years previously, with excision of the tumour and radiotherapy to the breast and drainage areas, were assessed clinically using the LENT SOMA scale, and a 5-mm punch biopsy taken from the buttock. Fibroblast cell strains were established and used to study residual DNA double strand breaks, using PFGE. RESULTS There were significant correlations between the DNA assay results and the fibrosis score (r(s) = 0.46; P = 0.003), the combined fibrosis and retraction score (r(s) = 0.45, P = 0.004) and the overall LENT score (r(s) = 0.43; P = 0.006). Using polychotomous logistic regression, the fibroblast DNA assay result was an independent prognostic factor for fibrosis severity. CONCLUSIONS There is a relationship between residual radiation-induced DNA damage in fibroblasts and the severity of the late normal tissue damage seen in the patients from whom the cells were cultured.

Detection of individual differences in radiation-induced apoptosis of peripheral blood lymphocytes in normal individuals, ataxia telangiectasia homozygotes and heterozygotes, and breast cancer patients after radiotherapy.

Abstract Barber, J. B. P., West, C. M. L., Kiltie, A. E., Roberts, S. A. and Scott, D. Detection of Individual Differences in Radiation-Induced Apoptosis of Peripheral Blood Lymphocytes in Normal Individuals, Ataxia Telangiectasia Homozygotes and Heterozygotes, and Breast Cancer Patients after Radiotherapy. Quantification of radiation-induced apoptosis in peripheral blood lymphocytes (PBLs) has been proposed as a possible screening test for cancer-prone individuals and also for the prediction of normal tissue responses after radiotherapy. We have used the TUNEL assay (terminal transferase nick-end labeling) 24 h after irradiation with 4 Gy at high dose rate to assess interindividual differences in radiation-induced apoptosis between (1) a panel of normal individuals, (2) ataxia telangiectasia (AT) homozygotes and heterozygotes, and (3) breast cancer patients who had received radiotherapy 8–13 years ago, including a number of patients who had suffered adverse responses to radiation. With this protocol, we show clear differences in radiation-induced apoptosis between individuals, and good reproducibility in the assay. In agreement with previous reports using EBV-transformed lymphoblasts, we show a very poor induction of apoptosis in AT homozygotes and a reduced level in AT heterozygotes compared to normal individuals. A similar reduced level compared to normal individuals was seen in the breast cancer patients. Despite a wide range of values in the breast cancer patients and good reproducibility on repeat samples, there was no correlation of rates of apoptosis with the severity of breast fibrosis, retraction or telangiectasia. The reduced rate of apoptosis observed in the breast cancer cases may be associated with genetic predisposition to breast cancer; however, we conclude that assays of lymphocyte apoptosis are unlikely to be of use in predicting normal tissue tolerance to radiotherapy.

Heritability of chromosomal radiosensitivity in breast cancer patients: a pilot study with the lymphocyte micronucleus assay.

Of breast cancer patients, 30% are sensitive in a lymphocyte assay of radiation-induced chromosome damage (micronucleus induction) compared with 10% of healthy controls. Twenty-two first-degree relatives of 11 sensitive patients had an average micronucleus yield significantly higher than that of 68 controls. This suggests that radiosensitivity in this assay may be an inherited characteristic associated with predisposition to breast cancer.Of breast cancer patients, 30% are sensitive in a lymphocyte assay of radiation-induced chromosome damage (micronucleus induction) compared with 10% of healthy controls. Twenty-two first-degree relatives of 11 sensitive patients had an average micronucleus yield significantly higher than that of 68 controls. This suggests that radiosensitivity in this assay may be an inherited characteristic associated with predisposition to breast cancer.

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.