Radiosensitivity In Breast Cancer Patients Research Articles

2823: Variability in chromosomal radiosensitivity of breast cancer patients before and after radiotherapy

2823: Variability in chromosomal radiosensitivity of breast cancer patients before and after radiotherapy

Assessment of Individual Radiosensitivity in Breast Cancer Patients Using a Combination of Biomolecular Markers

About 5% of patients undergoing radiotherapy (RT) develop RT-related side effects. To assess individual radiosensitivity, we collected peripheral blood from breast cancer patients before, during and after the RT, and γH2AX/53BP1 foci, apoptosis, chromosomal aberrations (CAs) and micronuclei (MN) were analyzed and correlated with the healthy tissue side effects assessed by the RTOG/EORTC criteria. The results showed a significantly higher level of γH2AX/53BP1 foci before the RT in radiosensitive (RS) patients in comparison to normal responding patients (NOR). Analysis of apoptosis did not reveal any correlation with side effects. CA and MN assays displayed an increase in genomic instability during and after RT and a higher frequency of MN in the lymphocytes of RS patients. We also studied time kinetics of γH2AX/53BP1 foci and apoptosis after in vitro irradiation of lymphocytes. Higher levels of primary 53BP1 and co-localizing γH2AX/53BP1 foci were detected in cells from RS patients as compared to NOR patients, while no difference in the residual foci or apoptotic response was found. The data suggested impaired DNA damage response in cells from RS patients. We suggest γH2AX/53BP1 foci and MN as potential biomarkers of individual radiosensitivity, but they need to be evaluated with a larger cohort of patients for clinics.

Role of hypoxia-inducible factor-1α and survivin in enhancing radiosensitivity of breast cancer

ObjectiveRadioresistance is associated with poor outcome in breast cancer patients. Hypoxia-inducible factor-1α (HIF-1α) and survivin are critical factors associated with radio-resistance. The aim of this study was to investigate the mechanisms of targeting HIF-1α and survivin in breast MDA-MB-231 cells and to understand their value in improving radiosensitivity in the patients. MethodsThe expression of HIF-1α and survivin in breast cancer patients was analysed by immunohistochemistry (IHC). Small interfering RNA (siRNA) was designed to target HIF-1α and survivin in cells, and the effects on proliferation, apoptosis, and radiosensitivity were measured. ResultsHIF-1α and survivin were expressed at higher levels in tumours than in normal tissues (P < 0.05). The expression of HIF-1α and survivin in recurrent tissues after radiotherapy tended to be elevated compared with tissues resected at the initial time of patients' diagnosis but there was no significant difference (P ≥ 0.05). The proliferation and apoptosis rates of cells in the RNA-interfered groups were significantly different from the blank control and NC-siRNA groups (P < 0.05). The proliferation rate of cells in each group did not change significantly with different doses of radiation (P ≥ 0.05), but the apoptosis rate in the RNA-interfered groups increased compared to the blank control and NC-siRNA groups (P < 0.05). Simultaneous targeting of HIF-1α and survivin had a stronger effect on cancer cell proliferation, apoptosis and radiosensitivity of cancer cells than individual interference of HIF-1α or survivin alone (P < 0.05). ConclusionCombinatorial therapeutic intervention of HIF-1α and survivin may be a potential strategy to improve the radiosensitivity of breast cancer patients.

Developing ZNF Gene Signatures Predicting Radiosensitivity of Patients with Breast Cancer.

Adjuvant radiotherapy is one of the main treatment methods for breast cancer, but its clinical benefit depends largely on the characteristics of the patient. This study aimed to explore the relationship between the expression of zinc finger (ZNF) gene family proteins and the radiosensitivity of breast cancer patients. Clinical and gene expression data on a total of 976 breast cancer samples were obtained from The Cancer Genome Atlas (TCGA) database. ZNF gene expression was dichotomized into groups with a higher or lower level than the median level of expression. Univariate and multivariate Cox regression analyses were used to evaluate the relationship between ZNF gene expression levels and radiosensitivity. The Molecular Taxonomy Data of the International Federation of Breast Cancer (METABRIC) database was used for validation. The results revealed that 4 ZNF genes were possible radiosensitivity markers. High expression of ZNF644 and low expression levels of the other 3 genes (ZNF341, ZNF541, and ZNF653) were related to the radiosensitivity of breast cancer. Hierarchical cluster, Cox, and CoxBoost analysis based on these 4 ZNF genes indicated that patients with a favorable 4-gene signature had better overall survival on radiotherapy. Thus, this 4-gene signature may have value for selecting those patients most likely to benefit from radiotherapy. ZNF gene clusters could act as radiosensitivity signatures for breast cancer patients and may be involved in determining the radiosensitivity of cancer.

Single nucleotide polymorphisms in ATM, TNF-α and IL6 genes and risk of radiotoxicity in breast cancer patients

Although oncological therapies have improved in the last decades, breast cancer (BC) remains a serious health problem worldwide. Radiotherapy (RT) is one of the most frequently used treatments for cancer aimed at eliminating tumor cells. However, it can also alter the surrounding normal tissue, especially the skin, and patient reactions may vary as a result of extrinsic and intrinsic factors. We evaluated the association of gene polymorphisms ATM Asp1853Asn, IL-6 G-174C and TNF-α G-308A involved in central phenotype pathways and development of individual radiosensitivity in BC patients with an exacerbated response to RT. Although univariate analysis results did not show a significant association with this trait, the interaction analysis between polymorphisms showed an increased risk of patients presenting wild-type TNF-α G-308A genotype and mutant IL-6 G-174C genotype, and heterozygous TNF-α G-308A genotype and heterozygous IL-6G-174C genotype. On the other hand, our results showed that breast size and patient age influenced the determination of RT-associated effects. Considering that the trait is multifactorial, other significant elements for the determination of individual radiosensitivity should be considered, together with the establishment of specific polymorphic variants.

Association Between Genetic Polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT Genes and Radiosensitivity in Breast Cancer Patients

Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). Individual genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR=53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR=38.26; 95% CI, 1.19-1232.52). To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be involved in cellular response to radiotherapy. Genetic polymorphisms may be promising candidates for predicting acute radiosensitivity, but further studies are necessary to confirm our findings.

Increased Radiosensitivity as an Indicator of Genes Conferring Breast Cancer Susceptibility

This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity. Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17. An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20-50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045). The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.

The use of IL-2 cultures to measure chromosomal radiosensitivity in breast cancer patients

Enhanced chromosomal radiosensitivity in breast cancer patients has been demonstrated in several studies. To investigate the chromosomal radiosensitivity of lymphocytes in breast cancer patients the G2 and micronucleus (MN) assays are often used. In these assays blood samples are exposed to ionizing radiation and the number of radiation-induced micronuclei or chromatid breaks are scored. In most studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the G2 and MN assays were performed on freshly drawn blood. The disadvantage of working with fresh blood samples is that in most cases only one blood sample can be obtained and that the assay cannot be easily repeated without further blood sampling. To allow repeated testing we propose the use of long-term cultures of T lymphocytes (IL-2 cultures). In this study we therefore investigated whether the radiation-induced MN response in IL-2 cultures was the same as in concordant whole blood cultures. For this study the MN assay (2 Gy) was performed on IL-2 cultures of 11 sensitive breast cancer patients and 20 healthy women. The results demonstrate that the enhanced chromosomal radiosensitivity observed in whole blood cultures of breast cancer patients is not present in IL-2 cultures derived from the same blood samples. Therefore, care has to be taken when IL-2 cultures are used to assess chromosomal radiosensitivity in breast cancer patients.

The use of EBV-transformed cell lines of breast cancer patients to measure chromosomal radiosensitivity.

To investigate the chromosomal radiosensitivity of lymphocytes in cancer patients the micronucleus (MN) assay is often used and performed on freshly drawn peripheral blood lymphocytes. The use of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines may have a lot of advantages (e.g. large pool of cells) compared with fresh blood samples. In this study we have investigated whether the response of EBV-transformed lymphoblastoid cell lines to irradiation in the G1/S/G2 phases of the cell cycle is the same as in concordant whole blood cultures where primary lymphocytes were irradiated in the G0 phase of the cell cycle. For this study the MN assay (2 Gy) was performed on EBV-transformed cell lines of breast cancer patients and a group of healthy women. Those breast cancer patients were selected who showed an elevated chromosomal radiosensitivity in fresh blood samples in a previous study. The results demonstrated that the enhanced chromosomal radiosensitivity observed in fresh blood cultures of breast cancer patients is not present in EBV-transformed cell lines derived from the same blood samples. Therefore, care must be taken when EBV cell lines are used to assess chromosomal radiosensitivity in breast cancer patients.

Clinical radiosensitivity in breast cancer patients carrying pathogenic ATM gene mutations: no observation of increased radiation-induced acute or late effects

Background and purpose: Reports on increased late subcutaneous toxicity after radiation therapy (RT) in breast cancer patients carrying ATM gene mutations has raised concerns about RT as part of the management in these patients. The impact of ataxia–telangiectasia (A–T) heterozygosity on clinical radiosensitivity remains a matter of debate while clinical data are scarce. Patients and methods: Between September 1995 and December 2002 genomic DNA samples were collected from 1100 unselected breast cancer patients receiving adjuvant radiotherapy at our department. Using mutation-specific assays, we screened for frequent ATM gene mutations. Analysis of acute and late radiation-related toxicity for skin and subcutaneous normal tissue was performed in patients identified as A–T heterozygotes applying common toxicity criteria (CTC) and LENT/SOMA (late effects on normal tissue/subjective objective management analytic) scoring criteria, respectively. Results: Eleven patients were identified to be heterozygous for a pathogenic ATM gene mutation. Ten patients had received at least one course of RT. Median follow-up after completion of radiotherapy was 5.1 years (range 1.7–7.2). There was no evidence of increased radiation-induced acute or late skin or subcutaneous reactions in patients treated with linac-based RT. One patient failed distantly and was subsequently irradiated at four different sites for bone and brain metastases. Local relapse occurred in the single patient who had declined adjuvant RT following breast conservative therapy. Conclusions: Our results do not provide evidence for a relative contraindication to adjuvant RT in A–T heterozygous breast cancer patients. Due to their increased cellular radiosensitivity, these patients may differentially benefit from RT and qualify for dose and volume reduction trials.

No evidence of increased radiation-induced late effects in breast cancer patients carrying ATM gene mutations

Purpose/Objective: The possible impact of ATM heterozygosity for clinical radiosensitivity in breast cancer patients remains a matter of debate while clinical data are scarce. A report on increased late subcutaneous toxicity after radiation therapy (RT) in breast cancer patients carrying ATM gene mutations has raised concerns about RT as part of the management in these patients (Iannuzzi et al. IJROBP 2002;52:606–613).

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.

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 3.5Gy 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 0.5Gy 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.

Testing for mutations of the ataxia telangiectasia gene in radiosensitive breast cancer patients

Background and purpose: To determine if clinical radiosensitivity in breast cancer patients is related to mutations of the ataxia telangiectasia gene (ATM). Materials and methods: Fifteen patients who had developed a severe late reaction to a standard radiotherapy schedule were examined for evidence of increased in vitro radiosensitivity using the MTT assay. Mutation analysis was performed using a protein truncation assay. Results: No mutations were detected in the 15 patients despite evidence of increased in vitro radiosensitivity. Conclusions: Testing for the ATM gene is unlikely to be useful for predicting clinical response to radiotherapy in breast cancer patients.

Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer

Purpose: To test whether the intrinsic radiosensitivity of skin fibroblasts from breast cancer patients correlates with the degree of breast fibrosis after breast conserving therapy. Methods: In a systematic study design, 79 patients were selected from an earlier study group of 385 patients based on observed fibrosis and seven identified clinical risk factors for fibrosis development. In vitro radiosensitivity of patients' dermal fibroblasts was determined by clonogenic assay of early passage cultures. Survival was determined after irradiation at 0, 2 and 4 Gy, given in two fractions of 2 Gy with a 6 h interval. Results: There was a significant inter-patient variation for SF2 values (coefficient of variation 40%). The ratio of SF2 values for fibroblasts from patients with breast fibrosis versus those without was 0.80 (95% CI: 0.60-1.07). This was a statistically non-significant trend (p 0.13). The same ratio for a derived value for SF2 ((SF2 SF4)/2) was 0.88 (p 0.19). Conclusions: A significant variation in intrinsic radiosensitivity of breast cancer patients' dermal fibroblasts was observed. However, the degree of radiosensitivity did not show a significant correlation with fibrosis development. This indicates that the use of fibroblast radiosensitivity will have a limited usefulness for predicting fibrosis following breast irradiation.

Normal tissue radiosensitivity in breast cancer patients

To investigate whether in vitro radiosensitivity of lymphocytes derived from a blood sample will predict late effects from radiotherapy in breast cancer patients. Blood samples were collected from consenting patients who had received radiotherapy for breast cancer. Lymphocytes were extracted and transformed by the Epstein-Barr virus. The resulting lymphoblastoid cell lines (LCLs) were assessed for in vitro radiosensitivity using a tetrazolium-based colorimetric assay (MTT). Survival curves were generated using doses of 0.5 to 2 Gy. For each analysis an LCL derived from an individual with the radiosensitive condition ataxia-telangiectasia (AT) was run as control. Some patients also consented to give skin biopsies from which fibroblast cultures were derived. Clonogenic assays were performed to generate survival curves using doses in the range of 0.5 to 4 Gy. Comparison was made with the data obtained from LCLs. Late effects of radiotherapy were assessed using the Radiation Therapy Oncology Group (RTOG) scoring scheme and compared with the in vitro radiosensitivity data. LCLs from 56 breast cancer patients were assessed for in vitro radiosensitivity. Surviving fraction to 2 Gy (SF2) generated from survival curves ranged from 0.04-0.35 with coefficient of variation for the whole group of 41%. None of the LCLs equalled the sensitivity of the AT line, but 16% showed equal or greater sensitivity to a line derived from an AT heterozygote. Comparison of LCL and fibroblast radiosensitivity showed reasonable correlation for 12 paired samples (r = 0.64). The majority of patients showed no or minimal effects after radiotherapy (Grade 0, 1 effects) but seven developed a Grade 2 reaction and four a Grade 3 or 4 reaction. Patients with a Grade 2-4 reaction were found to be more sensitive in vitro than those with a Grade 0-1 reaction (p < 0.02). The use of the MTT assay to assess LCL radiosensitivity has demonstrated considerable heterogeneity amongst the breast cancer population. The presence of a proportion of patients showing in vitro sensitivity but normal clinical response to radiotherapy would limit the usefulness of the assay for predictive purposes.