Association of single nucleotide polymorphisms in the genes ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with risk of severe erythema after breast conserving radiotherapy

Annette Raabe 0 Katharina Derda 0 Sebastian Reuther 0 Silke Szymczak Kerstin Borgmann 0 Ulrike Hoeller Andreas Ziegler Cordula Petersen 0 Ekkehard Dikomey 0 0 Department of Radiotherapy and Radiooncology, Laboratory of Radiobiology & Experimental Radiooncology, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf , Martinistr 52, D-20246 Hamburg , Germany Purpose: To examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position 509), XPD (codon 751), and XRCC1 (codon 399) with the risk of severe erythema after breast conserving radiotherapy. Methods and materials: Retrospective analysis of 83 breast cancer patients treated with breast conserving radiotherapy. A total dose of 50.4 Gy was administered, applying 1.8 Gy/fraction within 42 days. Erythema was evaluated according to the Radiation Therapy Oncology Group (RTOG) score. DNA was extracted from blood samples and polymorphisms were determined using either the Polymerase Chain Reaction based Restriction-Fragment-Length-Polymorphism (PCR-RFL) technique or Matrix-Assisted-Laser-Desorption/Ionization -Time-Of-Flight-Mass-Spectrometry (MALDI-TOF). Relative excess heterozygosity (REH) was investigated to check compatibility of genotype frequencies with Hardy-Weinberg equilibrium (HWE). In addition, p-values from the standard exact HWE lack of fit test were calculated using 100,000 permutations. HWE analyses were performed using R. Results: Fifty-six percent (46/83) of all patients developed erythema of grade 2 or 3, with this risk being higher for patients with large breast volume (odds ratio, OR = 2.55, 95% confidence interval, CI: 1.03-6.31, p = 0.041). No significant association between SNPs and risk of erythema was found when all patients were considered. However, in patients with small breast volume the TGFB1 SNP was associated with erythema (p = 0.028), whereas the SNP in XPD showed an association in patients with large breast volume (p = 0.046). A risk score based on all risk alleles was neither significant in all patients nor in patients with small or large breast volume. Risk alleles of most SNPs were different compared to a previously identified risk profile for fibrosis. Conclusions: The genetic risk profile for erythema appears to be different for patients with small and larger breast volume. This risk profile seems to be specific for erythema as compared to a risk profile for fibrosis. - Introduction The treatment of malignant tumours by radiotherapy (RT) is limited by the need to avoid unacceptable normal tissue toxicity. Despite advances in RT-technique, treatment modalities as well as therapeutic strategies, normal tissue damage is still a limiting factor in radiotherapy. In this context, late complications are especially important because they are generally progressive and appear to be associated with a lifelong risk [1]. In contrast, acute normal tissue toxicity is generally a transient phenomenon, with symptoms settling within months after treatment. However, these effects are not less clinically relevant, especially when accelerated fractionation schedules or adjuvant radiochemotherapy treatment are used [2-4] with new substances often increasing normal tissue toxicity, e.g. Cetuximab. Both acute and late normal tissue effects are known to vary considerably, ranging from negligible to severe, even between patients treated with identical schedules. It has been suggested that these variations in clinical radiosensitivity mainly result from differences in genetically determined radiosensitivity, as only 30% of this variation can be attributed to changes in treatment related parameters [5,6]. In addition to clinical and genetic parameters for radiation response, a number of patient-related confounding factors exist influencing adverse effects definitely, some of which probably have yet to be identified. Based on this background, pronounced scientific interest is currently being directed towards the use of genetic markers such as single nucleotide polymorphisms (SNPs) as parameters for the individual risk of experiencing radiation-induced normal tissue toxicity. Studies on the impact of SNPs are either performed following the candidate gene approach or employing genome-wide association (GWA) analysis. So far, numerous studies have been performed, but the results obtained are heterogeneous and often conflicting; for reviews, see [7,8]. Even for the C-509 T polymorphism in the transforming growth factor 1 (TGFB1) gene, which represents one of the most studied SNPs, there are several reports [9-13] showing that this polymorphism promotes chronic inflammatory and fibrotic reactions, but there are also data suggesting a lack of association or even the opposite effect [14-17]. In addition to the identification of those SNPs which are relevant for normal tissue toxicity, their functional consequences concerning the involved molecular pathways and their mechanisms of action are currently of great scientific interest. A respective consortium addressing this topic has recently been established [18]. The discrepancies in the currently available data may be attributed to the fact that with a frequency of about one SNP every 160 to 180 bp [19-21], the vast majority of SNPs are assumed to have no or a small effect on the respective protein or functional pathway. respectively. Therefore individual genetic characteristics must be determined by the combination of several SNPs each one associated with a small effect. This was indicated by previous studies [7,22] which demonstrated a significant association with normal tissue toxicity only if several - each of one only weakly associated SNPs - were combined to a risk score. Such risk scores can easily be created by adding the number of risk alleles per patients and correlating the resulting numerical value with the severity of the normal tissue toxicity. It is discussed that a risk profile, based on a combination of SNPs in genes which are involved in relevant pathways may vary for the type of normal tissue toxicity scored [7], i.e. different endpoints are characterized by different mosaic-like displays of certain SNPs. This concept implies that it might be more informative to analyse a certain combination of SNPs in independent studies rather than to change this combination within different studies. To date, such analyses have only been performed by Andreassen et al. [10,14,23] using certain combinations of SNPs all including SNPs in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM genes and studying their association with skin fibrosis in post-mastectomy radiotherapy patients. While the agreement between the first two studies analysing two different cohorts was fairly good [10,14], the third study on a larger cohort of patients [14] failed to confirm the association with radiation induced, indicating that future studies should consider other combination of SNPs [7]. In our studies, we concentrat (...truncated)