Potential strategies to ameliorate risk of radiotherapy-induced second malignant neoplasms.

This review is aimed at the issue of radiation-induced second malignant neoplasms (SMN), which has become an important problem with the increasing success of modern cancer radiotherapy (RT). It is imperative to avoid compromising the therapeutic ratio while addressing the challenge of SMN. The dilemma is illustrated by the role of reactive oxygen species in both the mechanisms of tumor cell kill and of radiation-induced carcinogenesis. We explore the literature focusing on three potential routes of amelioration to address this challenge. An obvious approach to avoiding compromise of the tumor response is the use of radioprotectors or mitigators that are selective for normal tissues. We also explore the opportunities to avoid protection of the tumor by topical/regional radioprotection of normal tissues, although this strategy limits the scope of protection. Finally, we explore the role of the bystander/abscopal phenomenon in radiation carcinogenesis, in association with the inflammatory response. Targeted and non-targeted effects of radiation are both linked to SMN through induction of DNA damage, genome instability and mutagenesis, but differences in the mechanisms and kinetics between targeted and non-targeted effects may provide opportunities to lessen SMN. The agents that could be employed to pursue each of these strategies are briefly reviewed. In many cases, the same agent has potential utility for more than one strategy. Although the parallel problem of chemotherapy-induced SMN shares common features, this review focuses on RT associated SMN. Also, we avoid the burgeoning literature on the endeavor to suppress cancer incidence by use of antioxidants and vitamins either as dietary strategies or supplementation.

N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays.

STUDY QUESTION: Is there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays? SUMMARY ANSWER: N-cadherin isolates clonogenic, self-renewing human endometrial epithelial progenitor cells with high proliferative potential that differentiate into cytokeratin+ gland-like structures in vitro and identifies their location in some cells of gland profiles predominantly in basalis endometrium adjacent to the myometrium. WHAT IS KNOWN ALREADY: Human endometrium contains a small population of clonogenic, self-renewing epithelial cells with high proliferative potential that differentiate into large gland-like structures, but their identity and location is unknown. Stage-specific embryonic antigen-1 (SSEA-1) distinguishes the epithelium of basalis from functionalis and is a marker of human post-menopausal (Post-M) endometrial epithelium. STUDY DESIGN, SIZE, DURATION: Prospective observational study of endometrial epithelial cells obtained from hysterectomy samples taken from 50 pre-menopausal (Pre-M) and 24 Post-M women, of which 4 were from women who had taken daily estradiol valerate 2 mg/day for 8 weeks prior. PARTICIPANTS/MATERIALS, SETTING, METHODS: Gene profiling was used to identify differentially expressed surface markers between fresh EpCAM (Epithelial Cell Adhesion Molecule)-magnetic bead-selected basalis-like epithelial cells from Post-M endometrium compared with predominantly functionalis epithelial cells from Pre-M endometrium and validated by qRT-PCR. In vitro clonogenicity and self-renewal assays were used to assess the stem/progenitor cell properties of magnetic bead-sorted N-cadherin+ and N-cadherin- epithelial cells. The cellular identity, location and phenotype of N-cadherin+ cells was assessed by dual colour immunofluorescence and confocal microscopy for cytokeratin, proliferative status (Ki-67), ERα, SSEA-1, SOX9 and epithelial mesenchymal transition (EMT) markers on full thickness human endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: CDH2 (N-cadherin gene) was one of 11 surface molecules highly expressed in Post-M compared to Pre-M endometrial epithelial cells. N-cadherin+ cells comprise a median 16.7% (n = 8) and 20.2% (n = 5) of Pre-M endometrial epithelial cells by flow cytometry and magnetic bead sorting, respectively. N-cadherin+ epithelial cells from Pre-M endometrium were more clonogenic than N-cadherin- cells (n = 12, P = 0.003), underwent more population doublings (n = 7), showed greater capacity for serial cloning (n = 7) and differentiated into cytokeratin+ gland-like organoids. N-cadherin immunolocalised to the lateral and apical membrane of epithelial cells in the bases of glands in the basalis of Pre-M endometrium and Post-M gland profiles, co-expressing cytokeratin, ERα but not SSEA-1 or SOX9, which localized on gland profiles proximal to N-cadherin+ cells. N-cadherin+ cells were quiescent (Ki-67-) in the basalis and in Post-M endometrial glands and co-localized with EMT markers vimentin and E-cadherin. LARGE SCALE DATA: The raw and processed data files from the gene microarray have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus data set with accession number GSE35221. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study in human endometrium only using in vitro stem cell assays. The differential ability of N-cadherin+ and N-cadherin-cells to generate endometrial glands in vivo was not determined. A small number of uterine tissues analysed contained adenomyosis for which N-cadherin has been implicated in epithelial-EMT. WIDER IMPLICATIONS OF THE FINDINGS: A new marker enriching for human endometrial epithelial progenitor cells identifies a different and potentially more primitive cell population than SSEA-1, suggesting a potential hierarchy of epithelial differentiation in the basalis. Using N-cadherin as a marker, the molecular and cellular characteristics of epithelial progenitor cells and their role in endometrial proliferative disorders including endometriosis, adenomyosis and thin dysfunctional endometrium can be investigated. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Cancer Council Victoria grant 491079 (C.E.G.) and Australian National Health and Medical Research Council grants 1021127 (C.E.G.), 1085435 (C.E.G., J.A.D.), 145780 and 288713 (C.N.S.), RD Wright Career Development Award 465121 (C.E.G.), Senior Research Fellowship 1042298 (C.E.G.), the Victorian Government's Operational Infrastructure Support and an Australian Postgraduate Award (HPTN), and China Council Scholarship (L.X.). The authors have nothing to declare.

DNA damage and repair kinetics after microbeam radiation therapy emulation in living cells using monoenergetic synchrotron X-ray microbeams.

A novel synchrotron-based approach, known as microbeam radiation therapy (MRT), currently shows considerable promise in increased tumour control and reduced normal tissue damage compared with conventional radiotherapy. Different microbeam widths and separations were investigated using a controlled cell culture system and monoenergetic (5.35 keV) synchrotron X-rays in order to gain further insight into the underlying cellular response to MRT. DNA damage and repair was measured using fluorescent antibodies against phosphorylated histone H2AX, which also allowed us to verify the exact location of the microbeam path. Beam dimensions that reproduced promising MRT strategies were used to identify useful methods to study the underpinnings of MRT. These studies include the investigation of different spatial configurations on bystander effects. γH2AX foci number were robustly induced in directly hit cells and considerable DNA double-strand break repair occurred by 12 h post-10 Gy irradiation; however, many cells had some γH2AX foci at the 12 h time point. γH2AX foci at later time points did not directly correspond with the targeted regions suggesting cell movement or bystander effects as a potential mechanism for MRT effectiveness. Partial irradiation of single nuclei was also investigated and in most cases γH2AX foci were not observed outside the field of irradiation within 1 h after irradiation indicating very little chromatin movement in this time frame. These studies contribute to the understanding of the fundamental radiation biology relating to the MRT response, a potential new therapy for cancer patients.

Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status.

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks.

PURPOSE: The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. MATERIALS AND METHODS: We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. RESULTS: We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. CONCLUSIONS: These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

Abscopal Gene Expression in Response to Synchrotron Radiation Indicates a Role for Immunological and DNA Damage Response Genes.

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

Telomere position effect and silencing of transgenes near telomeres in the mouse.

Reversible transcriptional silencing of genes located near telomeres, termed the telomere position effect (TPE), is well characterized in Saccharomyces cerevisiae. TPE has also been observed in human tumor cell lines, but its function remains unknown. To investigate TPE in normal mammalian cells, we developed clones of mouse embryonic stem (ES) cells that contain single-copy marker genes integrated adjacent to different telomeres. Analysis of these telomeric transgenes demonstrated that they were expressed at very low levels compared to the same transgenes integrated at interstitial sites. Similar to the situation in yeast, but in contrast to studies with human tumor cell lines, TPE in mouse ES cells was not reversed with trichostatin A. Prolonged culturing without selection resulted in extensive DNA methylation and complete silencing of telomeric transgenes, which could be reversed by treatment with 5-azacytidine. Thus, complete silencing of the telomeric transgenes appears to involve a two-step process in which the initial repression is reinforced by DNA methylation. Extensive methylation of the telomeric transgenes was also observed in various tissues and embryonic fibroblasts isolated from transgenic mice. In contrast, telomeric transgenes were not silenced in ES cell lines isolated from 3-day-old preimplantation embryos, consistent with the hypothesis that TPE plays a role in the development of the embryo.

A Functional Immune System Is Required for the Systemic Genotoxic Effects of Localized Irradiation.

PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.

Potential for the G2/M arrest assay to predict patient susceptibility to severe reactions following radiotherapy.

BACKGROUND AND PURPOSE: Cell-cycle regulation and checkpoint activation are crucial factors for radiation-induced DNA damage processing. The G2/M phase arrest was assessed in lymphoblastoid cell lines and phytohemagglutinin-stimulated T-lymphocytes of different radiosensitivities to study the relationship of G2/M arrest to radiosensitivity. MATERIAL AND METHODS: G2/M arrest was analyzed after in vitro irradiation by 2 and 5 Gy of ionizing radiation up to 6 days using 17 lymphoblastoid cell lines from healthy individuals, ataxia-telangiectasia (AT) patients, Nijmegen breakage syndrome (NBS) patients and cancer patients with clinically increased radiosensitivity. In a second approach, phytohemagglutinin-stimulated T-lymphocytes from 15 healthy individuals, twelve cancer patients, and five cancer patients hypersensitive to ionizing radiation were studied. Image cytometry was performed to analyze G2/M arrest. RESULTS: Two of the three AT cell lines showed markedly increased G2/M arrest compared to controls. NBS cells were comparable to controls up to day 3, but then demonstrated a slightly increased G2/M arrest. Two of the six radiosensitive lymphoblast cell lines and the five radiosensitive cancer patients' T-lymphocytes assayed showed a reduction in G2/M arrest, while healthy individuals showed no difference from cancer patients. CONCLUSION: The interrelation between G2/M arrest and radiosensitivity is not readily apparent since a variety of radiosensitive cells from patients with radiosensitive syndromes and patients identified as radiosensitive following radiation treatment showed inconsistent G2/M arrest dynamics. Secondary effects, like loss of clonogenicity, G1/S phase arrest and failure of G2/M arrest may contribute to variation of the G2/M arrest endpoint and obscure assessment of cellular radiosensitivity using this method.

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells.

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.

Time and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity.

PURPOSE: Proper detection of DNA damage and signal transduction to other proteins following irradiation (IR) is essential for cellular integrity. The serine 15 (Ser15) on p53 is crucial for p53 stabilization and a requirement for transient and permanent cell cycle arrest. Here, we sought to determine the relationship between p53 serine 15 phosphorylation (p53-p-Ser15) on cellular sensitivity and if this modification is associated with DNA double-strand break (DSB) repair. MATERIALS AND METHODS: Eight lymphoblastoid cell lines including ataxia-telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and radiosensitive patient derived cell lines were irradiated with 1 Gy, 2 Gy and 5 Gy. Then growth inhibition, p53 induction and phosphorylation on Ser15 as assessed by immunoblotting and DNA DSB repair as assessed by constant field gel electrophoresis were examined. RESULTS: Phosphorylation of p53 at Ser15 in control cells rapidly increased, peaking at 3-6 hours and then sustained a low level of phosphorylation for up to 6 days following IR. For these cell lines, the amount of p53-p-Ser15 corresponded to the sensitivity of cells and the amount of DNA DSB. In A-T cells, p53-p-Ser15 was reduced in spite of increased DNA DSB. NBS cells had similar phosphorylation dynamics as the control cell line, which was not consistent with their increased sensitivity. Radiosensitive patients' cell lines differed only slightly from controls. CONCLUSIONS: Cells that are competent in signal transduction have p53-p-Ser15 kinetics corresponding to cellular radiosensitivity as assessed by clonogenicity and DNA DSB repair, and cells impaired in signal transduction lack this correspondence. Therefore, using p53-p-Ser15 as a general marker of radiation sensitivity has confounding factors which may impair proper radiosensitivity prediction.

Uncoupling of telomere length and radiosensitivity in mouse lymphoma cell lines of similar genetic background.

PURPOSE: To investigate the link between radiosensitivity and telomere length in murine lymphoid cell line stocks that have similar genetic backgrounds but different radiosensitivities. MATERIALS AND METHODS: We used two stocks from both the parental L5,178Y-R cell line and the repair-deficient radiosensitive subline, L5,178Y-S, to assess telomere length. We used terminal restriction fragment analysis and flow-fluorescence in situ hybridization (FISH) telomere length assessment to determine telomere lengths in the related radiosensitive and non-radiosensitive cell lines. Each cell line was further tested for retention of its original radiation response phenotype using cell growth assays after treatment with ionizing radiation. RESULTS: One stock of L5,178Y-R cells had long telomeres, whereas the other stock had short telomeres. Likewise, one stock of L5,178Y-S cells had long telomeres, whereas the other stock had short telomeres. Telomere lengths in these cell lines were relatively stable for over 80 divisions in culture. Each cell line was confirmed to have retained its original radiosensitivity phenotype. CONCLUSION: We conclude that radiosensitivity is independent of telomere length in these genetically similar cell lines.

Follistatin attenuates radiation-induced fibrosis in a murine model.

PURPOSE: Fibrosis can be a disabling, severe side effect of radiotherapy that can occur in patients, and for which there is currently no effective treatment. The activins, proteins which are members of the TGFß superfamily, have a major role in stimulating the inflammatory response and subsequent fibrosis. Follistatin is an endogenous protein that binds the activins virtually irreversibly and inhibits their actions. These studies test if follistatin can attenuate the fibrotic response using a murine model of radiation-induced fibrosis. EXPERIMENTAL DESIGN: C57BL/6 mice were subcutaneously injected with follistatin 24 hours prior to irradiation. Mice were irradiated in a 10 x 10 mm square area of the right hind leg with 35 Gy and were given follistatin 24 hours before radiation and three times a week for six months following. Leg extension was measured, and tissue was collected for histological and molecular analysis to evaluate the progression of the radiation-induced fibrosis. RESULTS: Leg extension was improved in follistatin treated mice compared to vehicle treated mice at six months after irradiation. Also, epidermal thickness and cell nucleus area of keratinocytes were decreased by the follistatin treatment compared to the cells in irradiated skin of control mice. Finally, the gene expression of transforming growth factor ß1 (Tgfb1), and smooth muscle actin (Acta2) were decreased in the irradiated skin and Acta2 and inhibin ßA subunit (Inhba) were decreased in the irradiated muscle of the follistatin treated mice. CONCLUSIONS: Follistatin attenuated the radiation-induced fibrotic response in irradiated mice. These studies provide the data to support further investigation of the use of follistatin to reduce radiation-induced fibrosis in patients undergoing radiotherapy for cancer.

Localized Synchrotron Irradiation of Mouse Skin Induces Persistent Systemic Genotoxic and Immune Responses.

The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFß1, and TGFß2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR.

Individual differences in chromosomal aberrations after in vitro irradiation of cells from healthy individuals, cancer and cancer susceptibility syndrome patients.

BACKGROUND: Radiosensitivity of normal tissue is a crucial factor of radiotherapy (RT)-related side effects. Here, we report the analysis of spontaneous and in vitro irradiation-induced chromosomal aberrations in 256,679 metaphases from 222 different individuals using three-color fluorescence in situ hybridization as a measure of radiosensitivity. MATERIALS AND METHODS: Samples were categorized into the following 6 groups: (1) healthy individuals, (2) cancer patients prior to radiotherapy, (3) RT-treated cancer patients, (4) individuals heterozygous or (5) homozygous for a mutation in the ataxia telangiectasia mutated (ATM) gene or in the Nijmegen breakage syndrome (NBS1) gene and (6) hypersensitive patients (outliers). RESULTS: A normal distribution of the number of chromosomal aberrations, measured as breaks per metaphase (B/m), was adopted for all examined groups. The mean value of the control group was 0.40B/m (SD+/-0.07). This value was lower compared to the mean breakage rate from 175 non-exposed (0.50+/-0.12B/m) and pre-exposed (0.50+/-0.16B/m) cancer patients. Nineteen of the metaphase spreads from the analyzed cancer patients had a high number of chromosomal aberrations (1.04+/-0.29B/m) and were designated as a separate hypersensitive subgroup (outliers). The aberration frequency of this group was comparable to those of ATM or NBS1 heterozygotes (0.86+/-0.26B/m). The highest incidence of aberrations was observed in ATM and NBS1 homozygous patients (2.23+/-1.03B/m). CONCLUSION: The frequency of break events in the analyzed groups resulted in a normal distribution with varying means and broadnesses defining a characteristic sensitivity pattern for each group. In the RT-relevant group of cancer patients, those patients who have cancer, about one-third of the normally distributed samples were determined to be sensitive as defined by the number of induced aberrations higher than the 99% confidence interval of the normal individual's Gaussian distribution. About 5% of these samples were outside of the 99% confidence interval for the RT-relevant group's normal distribution. These outliers with higher chromosomal breakage rates suggest a unique class of hypersensitive individuals that are susceptible to chromosomal damage and may be directly associated with an increased risk to suffer from radiotherapy-related complications.

Breakpoint locations within chromosomes 1, 2, and 4 of patients with increased radiosensitivity.

The exposure to low LET-radiation leads to a relative homogeneous distribution of initial damage at the DNA. Subsequent repair and post-repair mechanisms might lead to a selection of specific breakpoint locations along chromosomes. Cells from patients with increased radiosensitivity may have more specific breakpoints due to impaired repair mechanisms. We tested whether cells from patients with increased radiosensitivity had an increase in specific breakpoint clusters. Structural chromosomal aberrations of in vitro irradiated lymphocytes from 11 healthy individuals and another 3 patients with increased radiosensitivity were examined. The chromosome pairs 1, 2, and 4 were treated using the three-color FISH technique. The breakpoints were analyzed by means of computerized imaging software. In total, 1752 chromosomal breakpoints had been considered, 498 from healthy individuals, and 1254 from patients with increased radiosensitivity. For both groups there was a non-homogeneous breakpoint distribution along the chromosomes and a trend towards increased breaks in the telomere-proximal region. Also, both groups had distinct locations with increased breaks. No evidence for significant breakpoint patterns across all patients with increased radiosensitivity was found.

Inter-relation of apoptosis and DNA double-strand breaks in patients with multiple primary cancers.

Since the development of multiple primary cancers in an individual is considered an unlikely event, it is suspected that a defect in DNA repair or apoptosis is the underlying cause for some of these patients. Therefore, this study was based on the hypothesis that such patients have increased remaining DNA double-strand breaks (DSBs) and reduced levels of apoptosis after in vitro irradiation. To investigate these mechanisms in cancer patients, 19 with multiple primary cancers were selected out of 25 121 cancer patients. For inclusion in this study, patients had to present with first malignancy at an early age, have a positive family history of cancer and no risk factors. The exclusion criteria were recurrence of cancer or metastasis, haematological tumours and tumours possibly connected to a patient risk factor such as smoking or drinking. Their peripheral blood lymphocytes were tested for proper repair of DNA DSBs and apoptosis after in vitro irradiation. DSBs were measured using constant field gel electrophoresis at 0, 8 and 24 h after irradiation. Apoptotic rates were determined at 24, 48 and 72 h after irradiation using the TUNEL assay. We found that patients' lymphocytes had significantly more initial DNA DSBs compared with controls, but there was no difference in the number of remaining DNA DSBs. Apoptotic rates of lymphocytes were only slightly lower in patients than in controls. These findings show that there are limited differences between patients with multiple cancers and healthy individuals. However, we found a trend towards an inverse correlation between remaining DNA DSBs and apoptotic rates in patients' lymphocytes. This is indicative of DNA DSBs persisting in patients' cells, presumably leading to a higher level of stable chromosomal aberrations that may contribute to tumour formation.

Chromosomal radiosensitivity in two cell lineages derived from clinically radiosensitive cancer patients.

PURPOSE: Despite its prominent contribution to cancer cure and palliation, around 1% to 5% of cancer patients suffer serious side effects from radiotherapy. A cardinal goal in the fields of radiobiology and oncology is to predict normal tissue radiosensitivity of a cancer patient before radiotherapy. Higher tumor control rates are likely if radiotherapy individualization could be achieved by applying predictive approaches. EXPERIMENTAL DESIGN: Here, we make use of the cytokinesis block micronucleus assay to assess radiosensitivity in cell lines derived from two different cell lineages obtained from clinically radiosensitive patients. We determined the micronucleus frequency after graded doses of ionizing radiation to primary fibroblasts and lymphoblast cell lines derived from 36 highly radiosensitive cancer patients. RESULTS: Many cell lines, following exposure to ionizing radiation, from patients with severe clinical reactions to radiotherapy showed statistically significantly higher frequencies of micronuclei than those from patients who had normal reactions to radiotherapy. One individual revealed significantly higher micronucleus frequencies in both cell lineages. Interestingly, lymphoblast cell lines from one patient showed micronucleus frequencies similar to ataxia telangiectasia mutated-deficient cells. CONCLUSIONS: These results indicate that the micronucleus assay may have use for identifying predisposition to clinical radiosensitivity, at least in a subset of patients as a component of a pretreatment radiosensitivity assay for use in the clinic.

Cytogenetic instability in young patients with multiple primary cancers.

Patients younger than 45 years with multiple cancers and a family history of cancer were identified and examined for cytogenetic instability. The cohort included 50 individuals: 19 patients suffering from at least 2 independent cancers, 11 healthy control individuals, a positive control group of 5 highly radiosensitive patients (>grade 3, RTOG), and a tumor control group of 15 patients with a single tumor. Peripheral blood lymphocytes were irradiated in vitro (0.7 Gy, 2.0 Gy). Metaphase chromosomes 1, 2, and 4 were labeled by means of 3-color fluorescence in situ hybridization. Chromosomal aberrations (breaks per metaphase [B/M], complex chromosomal rearrangements [CCR/M]) were analyzed. Very high levels of chromosomal aberrations were detected in a "core group" of 5 patients. These patients displayed much higher rates of B/M and CCR/M than controls. Ten patients had moderately elevated chromosomal aberrations and 4 patients were indistinguishable from controls. We conclude that a significant proportion of young patients with multiple tumors and a family background of cancer display cytogenetic instability.

Immunological markers that predict radiation toxicity.

Radiotherapy is a major modality of cancer treatment responsible for a large proportion of cancer that is cured. Radiation exposure induces an inflammatory response which can be influenced by genetic, epigenetic, tumour, health and other factors which can lead to very different treatment outcomes between individuals. Molecules involved in the immunological response provide excellent potential biomarkers for the prediction of radiation-induced toxicity. The known molecular and cellular immunological responses in relation to radiation and the potential to improve cancer treatment are presented in this review. In particular, immunological biomarkers of radiation-induced fibrosis and pneumonitis in cancer radiotherapy patients are discussed.