Genetic variation in normal tissue toxicity induced by ionizing radiation

Abstract

Radiotherapy is an important weapon in the treatment of cancer, but adverse reactions developing in the co-irradiated normal tissue can be a threat for patients. Early reactions might disturb the usual application schedule and limit the radiation dose. Late appearing and degenerative reactions might reduce or destroy normal tissue function. Genetic markers conferring the ability to identify hyper-sensitive patients in advance would considerably improve therapy. Association studies on genetic variation and occurrence of side effects should help to identify such markers. This survey includes published studies and novel data from our own laboratory. It illustrates the presence of candidate polymorphisms in genes involved in the cellular response to irradiation which could be used as predictive markers for radiosensitivity in breast or prostate cancer patients. For other tumor types such as head and neck cancers or brain tumors, the available data are much more limited. In any case, further validation of these markers is needed in large patient cohorts with systematically recorded data on side effects and patient characteristics. Genetic variation contributing to radiosensitivity should be screened on a broader basis using newly developed, more comprehensive approaches such as genome-wide association studies.

Introduction

Radiotherapy, either alone or in combination with surgery and chemotherapy, is one of the major modalities used in the treatment of cancer. About 50% of all cancer patients in the world receive radiotherapy at some point in their treatment (World Cancer Report [1]). The aim of radiotherapy is to effectuate local tumor control. Considerable technical efforts have been taken to improve the effective tumor dose and to decrease the amount of normal tissue included inevitably in the treatment volume. While today the majority of patients tolerate standard radiation therapy well, clinicians still observe a substantial amount of patients (up to 10%) who suffer from strong to life threatening adverse effects arising from the intrinsic sensitivity of normal tissue. Although exact definitions are still under debate, adverse reactions to radiotherapy are commonly classified in early and late effects [2]. Early or acute effects emerge during or immediately after the end of therapy, or they have not healed by 90 days after the end of therapy, while late side effects develop after latent periods of months to years. Acute effects are predominantly observed in high turnover tissues such as skin and mucosa while late effects are generally seen in low turnover tissues including lung and nervous system.

Early side effects of radiotherapy occur for example in the skin of breast cancer patients or in the intestinal or urinary bladder mucosa of prostate cancer patients. The observed reactions can be dose-limiting and may question a curative treatment intent [3], [4]. Skin reactions, for example, can range from mild erythema and desquamation up to necrosis and ulceration. In contrast to early reactions, late side effects of radiotherapy such as fibrosis, telangiectasia, and atrophy are mainly considered to be irreversible and progressive [2]. They can result in poor cosmetic outcome, pain, and limited or lost organic function. The association between the appearance of acute effects and the risk of developing late effects is not clear but has been reported in a number of tissues [5]. Our own prospective studies on more than 400 breast cancer patients receiving radiotherapy after breast-conserving surgery confirmed this observation. Patients suffering from acute skin toxicity were at a significantly higher risk to develop also telangiectasia [6], [7].

Inter-individual variability in the development of adverse reactions in normal tissue of radiotherapy patients is well-documented for both acute and late effects. There is evidence that, in addition to patient-related factors such as age and life-style factors, these patient-to-patient differences are attributable to a genetic basis. Early indications came from the observation that individuals with specific genetic disorders cannot tolerate standard radiotherapy [8]. Cells of these patients harbor defects in genes such as ATM (ataxia telangiectasia), the FANC gene family (Fanconi's anemia), NBN (Nijmegen breakage syndrome), and BLM (Bloom's syndrome). Predisposition to an adverse therapy response is dominated in such disorders by the strong effects of a few high-penetrance mutations in these susceptibility genes.

Further evidence for a genetic basis of radiosensitivity comes from well controlled animal studies which have the distinction that animals show less genetic heterogeneity and the environmental and other factors can be regulated concurrently. For example, mouse strains prone or resistant to radiation-induced fibrosis have contributed to the assumption that especially radiation-induced late side effects are genetically regulated [9]. Furthermore, radiation susceptible and resistant mouse strains have been investigated to elucidate the mechanism of early response to ionizing radiation. Lindsay et al. [10] reported on differences in expression and activity of the p53 pathway. Susceptible mice showed a more prolonged p53 response than resistant mice. Preferential upregulation of the pro-apoptotic BCL2-family member BAX, a downstream target of p53, as well as a 2-fold higher rate of apoptotic cells were found in the resistant mice in comparison to the susceptible animals. Evidence is also provided that levels of apoptosis and different patterns of p53 phosphorylation are tissue-specific, and that p53 regulation at the mRNA level is inherited [10], [11], [12].

Although these studies give useful hints at the molecular basis of radiosensitivity, the observed inter-individual variability in clinical radiosensitivity is considerable and cannot be explained by the known genetic defects only, as they are very rare and therefore of limited relevance to this common clinical phenomenon. It was therefore hypothesized that common genetic variants with modest functional effects (low penetrance variants) cause the bulk of the unexplained inter-individual variability. Radiosensitivity is thus suggested to be a complex, polygenic trait which results from the interaction of a number of genes in different cellular pathways [9], [13]. These pathways include genes related to DNA damage induction and repair, apoptosis, pro-fibrotic and inflammatory cytokines, endogenous antioxidant enzymes, as well as to general metabolism and homeostasis. A major challenge is to identify the combination of multiple low-penetrance genetic variants (single nucleotide polymorphisms, SNPs) which affect the complex cellular and clinical phenotype and may therefore be useful as potential biomarkers to predict normal tissue response after radiotherapy [14], [15].

It should be mentioned that there are other approaches, such as evaluating the transcriptional response to irradiation by mRNA microarrays, which allow identifying genes potentially involved in the development of side effects and developing predictive signatures for tumor response and normal tissue reaction [16], [17]. It is known that a variety of genes are up- or down-regulated in response to radiation representing many pathways, e.g., DNA repair, apoptosis, cell cycle control, fibrosis, cell adhesion, intracellular signaling, metabolism, and stress response. However, mRNA expression might be affected by life-style factors and the interpretation of microarray data is laborious and involves the evaluation of transcriptional response at different time points in different cell and tissue types. Thus, expression signatures might be more difficult to use as clinical markers than stable germ line variants. Therefore, we concentrated this review on the impact of genetic variants on clinical radiosensitivity.

Here, several studies have been performed during the last years. Powerful and economic genotyping techniques have been developed including high-throughput methods and methods to analyze a greater number of SNPs simultaneously. Even so called whole genome scans with up to one million SNPs are now possible. For successful application of these techniques, patient cohorts must be established which are well characterized for tumor characteristics, demographic and life-style factors and, most importantly, for side effects developed already during but also years after radiotherapy. Therefore, grading systems have been adopted for many tumor sites and irradiated tissues (e.g., Cancer Therapy Evaluation Program CTEP of the National Institute of Health, USA (http://ctep.cancer.gov/reporting/index.html), RTOG (Radiation Therapy Oncology Group) morbidity criteria [18], Late Effects of Normal Tissue-Subjective Objective Management Analytical (LENT-SOMA) scale [19]. Especially late side effects require careful recording as they might progress or appear only after a longer latency period. Grading of patients changes with time and a follow-up of patients over several years is required [2], [20]. In addition, the time point when an individual is considered as sensitive has to be indicated in a study. Therefore, evaluation of markers in clinical epidemiological studies should consider the time until appearance of side effects, e.g., using COX regression models.

However, this is a demanding, long lasting and expensive task which should profit from already established knowledge and avoid short-comings of published studies.

In this review, we summarized studies on the association of genotypes and clinical radiosensitivity and focused our evaluation on the following questions: (i) which marker genes or genetic variants appear from the existing studies, (ii) which pathways and genes, potentially involved in development of side effects, have been considered up to now, and (iii) which tumor sites and concomitant adverse effects have so far been analyzed. In addition, we will report on various SNPs in DNA repair genes analyzed in prostate cancer patients who underwent radiotherapy. We will describe their association with the development of acute adverse effects of radiotherapy in these patients. Finally, this review will contribute to establish new strategies and approaches to identify molecular markers predicting radiosensitivity of patients. These markers will help to improve and personalize radiotherapy.