DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

doi:10.1016/j.ijrobp.2009.08.052

International Journal of Radiation OncologyBiologyPhysics

Volume 78, Issue 2, 1 October 2010, Pages 359-369

https://doi.org/10.1016/j.ijrobp.2009.08.052 Get rights and content

Purpose

To evaluate, in a pilot study, the phosphorylated H2AX (γH2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy.

Methods and Materials

The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting γH2AX foci in blood lymphocytes at defined time points after irradiation of blood samples.

Results

Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities, and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function.

Conclusions

Double-strand break repair alteration in children may predispose to cancer formation and may affect children's susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.

Introduction

Over the past decades, increasingly complex multimodality treatment protocols have led to tremendous improvements in the survival of children diagnosed with cancer. All cancer therapies developed to date are associated with early and late adverse effects, also known as normal-tissue toxicities. Normal-tissue responses show considerable variability among patients, whereas treatment-associated complications are not only related to the specific therapy used but may particularly be determined by the patient's individual genetic predisposition 1, 2, 3. Most convincing evidence suggests that genetic alterations in proteins participating in the DNA damage response determine the individual risk of developing severe treatment-related side effects 4, 5, 6.

Deoxyribonucleic acid double-strand breaks (DSBs) are the most deleterious of all DNA lesions. If unrepaired, DSBs can lead to loss of chromosome segments and threaten the cell's survival. Double-strand breaks are generated naturally, for example by metabolic by-products of cellular respiration, but are also produced to a great extent when cells are exposed to DNA-damaging agents, such as ionizing radiation and certain chemotherapeutics 7, 8. Cells have evolved groups of proteins that function in signaling networks that sense DSBs, arrest the cell cycle, and activate the DNA repair pathways nonhomologous end-joining and homologous recombination(9). The ataxia–telangiectasia mutated (ATM) protein kinase is a critical component in these pathways and integrates the cellular response to DSBs by phosphorylating key proteins involved in cell-cycle regulation and DNA repair, and thus plays a central role in the maintenance of genomic integrity. Whereas carriers of monoallelic ATM mutations (ATM^+/- heterozygote) have slightly elevated cancer risks and increased radiosensitivity, the complete lack of ATM function results in the clinical syndrome ataxia–telangiectasia (ATM^-/- homozygote), characterized by progressive neuromotor dysfunction, immunodeficiency, genomic instability, predisposition to cancer, and profound hypersensitivity to ionizing radiation 10, 11, 12, 13, 14. Such highly expressing DNA repair disorders are rare, and malignancies arising in the context of cancer predisposition syndromes account for only 5–10% of human tumors (15). However, minor DSB repair deficiencies based on subtle heterogenous mutations in DNA damage response genes are expected to be more common in the human population.

In a clinical trial with children suffering pediatric malignancies, we evaluated the potential of the γH2AX foci approach to identify patients with DSB repair deficiencies, who may overreact to DNA-damaging cancer therapy. For this purpose, we applied the highly sensitive γH2AX foci approach to analyze DSB repair in blood lymphocytes after external irradiation of blood samples. This method is based on the detection of specific histone modifications, the phosphorylation of histone H2AX molecules, occurring on induction of DSBs (16). Phosphorylated H2AX, designated γH2AX, extends to megabase chromatin regions around the lesion and can be visualized by immunofluorescence microscopy as discrete nuclear foci reflecting sites of DSBs. The kinetic of γH2AX foci loss strongly correlates with the time course of DSB repair (17). In recent experimental studies, we could show that γH2AX foci analysis of blood lymphocytes provides precise information about the genetically defined DSB repair capacity, shown to be valid for different and complex organs in a given individual 18, 19. Moreover, previous clinical studies established the γH2AX foci approach as a sensitive technique to quantify the repair of radiation-induced DSBs in blood lymphocytes from individuals undergoing computed tomography examination (20). In that study, γH2AX analysis allowed us to verify a substantial DSB repair defect in a patient previously displaying severe side effects after radiotherapy, sustaining the close relationship between DSB repair deficiency and pronounced clinical radiosensitivity.

First, we analyzed blood samples of children with ataxia–telangiectasia (ATM^-/- homozygote) and their heterozygous parents (ATM^+/- heterozygote) to evaluate the feasibility of the γH2AX foci approach in the clinical setting to verify not only pronounced but also subtle, genetically defined DSB repair deficiencies. Subsequently, we investigated the DSB repair capacity of children with different solid tumors and monitored their treatment-associated normal-tissue responses. The primary objective was to evaluate the clinical potential of the γH2AX foci approach to identify patients with DSB repair deficiencies, as a screening tool in predictive testing for normal-tissue toxicities.

Section snippets

ATM^-/- homozygote and ATM^+/- heterozygote probands

Three different families with 4 ATM^-/- homozygote adolescents and 5 ATM^+/- heterozygote parents were analyzed and compared with normal ATM^+/+ individuals. The precise ATM gene mutations were previously identified and are listed in Table 1(21).

Patients

Children with histologically confirmed solid tumors who received chemotherapy and/or radiotherapy in our departments between June 2006 and June 2008 were included in this study (n = 23; listed in Table 2) (sole exception: the patient HNEE was treated at

Results

The DSB repair capacity of ATM^-/- homozygote and ATM^+/- heterozygote probands was evaluated to test the sensitivity and reliability of the γH2AX foci approach in the clinical setting to identify DSB repair deficiencies. Whole-blood as well as isolated blood lymphocytes were irradiated with 1 Gy or 2 Gy, respectively. This low dose range was chosen to avoid radiation-induced apoptotic processes in blood lymphocytes, which might falsify results 23, 24. At defined time points after irradiation,

Discussion

Major advances in pediatric cancer therapy have resulted in substantial improvements in survival. However, growing concern has emerged about severe normal-tissue toxicities associated with complex multimodality treatment strategies, compromising the clinical outcome of affected children.

In this pilot study, the highly sensitive γH2AX foci analysis was evaluated to identify patients with an impaired DSB repair capacity as the determining factor for high-grade normal-tissue toxicities. First, the

Acknowledgments

The authors thank all the patients and their parents for participating in this study; and Dr. Dirk Prawitt (Section of Medical Genetics and Molecular Medicine, Children's Hospital, Johannes-Gutenberg University of Mainz, Germany) and Prof. Detlev Schindler (Department of Human Genetics, University of Würzburg, Germany) for scientific advice.

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While some studies determined a similar number of foci 1 h after irradiation between AT patients and controls [30,47], other analyses revealed a lower induction of γH2A.X [29,48,49]. However, a slower decrease in foci number and higher residual damage 8 h and 24 h after irradiation were common hallmarks of AT cells in several studies [29,47,48,50,51]. In this study, high individual differences between AT patients in signaling and repair were observed.
Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.
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So far, however, no general correlation could be drawn between the cellular or chromosomal IR sensitivity assessed with functional bioassays and the clinical response to IR for FA patients. Previous studies on the IR response of FA cells in vitro were mostly conducted with primary fibroblasts or lymphocytes of various but often undefined FA complementation groups to investigate the repair of IR-induced DSBs using cytogenetic methods [17–20,22–26,28,30] or the detection of DSB repair foci [27,28,32–36], such as the phosphorylated histone variant H2AX (γH2AX) or 53BP1. Very few of these studies explored the repair of IR-induced DSBs in FA in a cell cycle-dependent manner, but their outcomes on the efficiency and fidelity of DSB repair remain contradicting and this clinically relevant issue has not yet been explored and clarified to its full extent.
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: Supported by Saarland University (Grant HOMFOR 2007/27) and in part by Elterninitiative Krebskranker Kinder im Saarland e.V.

: Conflict of interest: none.

View full text

Clinical InvestigationDNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

Purpose

Methods and Materials

Results

Conclusions

Introduction

Section snippets

ATM-/- homozygote and ATM+/- heterozygote probands

Patients

Results

Discussion

Acknowledgments

Radiother Oncol

Int J Radiat Oncol Biol Phys

DNA Repair (Amst)

Mutat Res

J Biol Chem

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

J Biol Chem

Lab Invest

Mol Cell

Cell

Preventing or reducing late side effects of radiation therapy: Radiobiology meets molecular pathology

Nat Rev Cancer

Toxicity from radiation therapy associated with abnormal transcriptional responses to DNA damage

Proc Natl Acad Sci U S A

Analysis of gene expression using gene sets discriminates cancer patients with and without late radiation toxicity

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Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents

Environ Mol Mutagen

Regulation of DNA double-strand break repair pathway choice

Cell Res

Molecular basis of ataxia telangiectasia and related diseases

Acta Pharmacol Sin

Atm haploinsufficiency results in increased sensitivity to sublethal doses of ionizing radiation in mice

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Ataxia-telangiectasia: From a rare disorder to a paradigm for cell signalling and cancer

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Cancer in patients with ataxia-telangiectasia and in their relatives in the Nordic countries

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DNA double-strand break repair of blood lymphocytes and normal tissues analysed in a preclinical mouse model: Implications for radiosensitivity testing

Clin Cancer Res

In vivo formation and repair of DNA double-strand breaks after computed tomography examinations

Proc Natl Acad Sci U S A

Characterization of ATM gene mutations in 66 ataxia telangiectasia families

Hum Mol Genet

German register for detection of late sequelae after radiotherapy for children and adolescents (RiSK): Present status and first results

Strahlenther Onkol

Clinical Investigation
DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

ATM^-/- homozygote and ATM^+/- heterozygote probands