Radiation therapy can cause severe side-effects on healthy tissues of patients, which inflicts several limitations in effective cancer treatment, as described in section 2.3. Unraveling the reasons for increased radiosensitivity has been subject to intense research for decades and up to date, the molecular mechanisms that control human radiosensitivity reactions are incompletely understood. Chromosome aberrations and other cellular RS reactions following radiotherapy in cells from patients with known radiosensitivity syndromes, such as A-T, NBS or FA have been repeatedly described in the literature, rendering some of these patients untreatable by radiotherapy416-420, however, a clear correlation between other
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radiosensitivity syndromes and cellular survival could not always be established, for example in patients with Bloom’s syndrome or FA-like condition421 422. It was furthermore reported that A-T carriers displayed moderately increased cellular and chromosomal radiosensitivity, suggesting that this could relate to increased clinical RS. Therefore, sequence analysis of the ATM gene was performed in genomic DNA of cancer patients with at least RTOG grade 3 acute and/or late effects, which showed sequence variations by already known polymorphism and silent mutations in only 2 of 20 patients423. One of the two is part of this project, suggesting a different genetic profile might lead to the adverse effects423. This was further supported by a study in the year 2002, in which no evidence for ATM, NBS, MRE11A or RAD50 mutation was found in patients with RTOG 3 late reactions, of which four are also part of this study424. However, a possible association between increased chromosomal sensitivity and ATM variants in form of polymorphism and silent mutations has been described in two radiosensitive breast cancer patients, one being also part of the patient collective used in this project425.
Furthermore, a strong diversity of both acute and late reactions even after identical treatment parameters has been documented already two decades ago that could not be attributed to a certain radiosensitivity syndrome347 348 350 351 426-428. At the end of the 1990s evidence converged that individual radiosensitivity is determined by the genetic background of a patient328 and prediction of personal risk of side-effects has been considered the “Holy Grail of radiobiology”429 430. In 2003, Dikomey et al. proposed a theory why contradictory results might have been obtained so far, as the assumption, that only radiosensitive patients develop reactions might not be true431. On the contrary, they implied that all patients are at risk of developing side-effects but the time frame after treatment is determinative. This model is supported by data gained in 2001, where one group described kinetics of the risk of suffering from late side-effects following an exponential decline for all patients, whether initially characterized as radiosensitive or not374. This possible lifelong risk of developing late complications demands continuous follow-up examinations to determine which biological processes might be responsible for the time dependency of late effects’ manifestation374.
Several studies addressing the relevance of polymorphisms in candidate genes followed, and positive correlations were reported between variants in TGFB1 (which controls proliferation, differentiation, is important in wound healing and is upregulated in tumors432 433), SOD2 (Superoxide Dismutase 2, involved in ROS scavenging, apoptosis and cancer434), and the two DNA repair genes XRCC3 (X-Ray Repair Cross Complementing 3) and XRCC1 (X-Ray Repair Cross Complementing 1). Increased risk of subcutaneous fibrosis and partly also telangiectasia was reported435, however, these genes were only partly identified in other studies429,434 436-439. It soon became clear that individual radiosensitivity reflects a complex trait, and not a condition that can be broken down to one or two genes, but rather relies on their synergistic effects434. Following studies aimed at increasing statistical power, providing homogenous study parameters in terms of patient selection, cancer site, parameters of treatment and functional end points for measuring toxicity effects, which should be facilitated by establishment of the international Radiogenomics Consortium440,315.
Further studies focused on relating gene expression profiles to radiation toxicity, as it has been previously shown that gene expression patterns can be altered in A-T patients441. However, the outcome of these studies again was different. One study found a high
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reciprocal correlation of the expression of CDKN1A (Cyclin Dependent Kinase Inhibitor 1A) to reaction severity442, whereas gene expression of ubiquitin, apoptosis and stress signaling networks only related to radiosensitivity to some extent in another study443. One study found 18 genes assigned to apoptosis, extracellular matrix remodeling and cell adhesion, proliferation and scavenging of ROS. Gene expression profiles could clearly distinguish between high and low risk patients of IR-induced skin fibrosis in BC patients444, however, the previously proposed marker gene CDKN1A was not found in concordance with another study445. Transcriptomics-based studies successfully correlated early and late effects with differential gene expression in genes involving inflammatory and stress responses as well as antioxidant metabolism. However, they failed to converge to a final definition of driver genes as radiotherapy involves distinct effects in a variety of tissues that are also subject to remodeling and continuous change throughout the duration of therapy, as well as in time between follow-up investigations328,446,447. Moreover, a summary of several studies aimed at generation of RNA signatures from fibroblasts and lymphocytes to predict early and late consequences and several more genes have been proposed to play an important role in the pathogenesis of radiation toxicity. Their overall functions have been summarized to DNA damage sensors and mediators, cell cycle checkpoint control genes, apoptosis and inflammatory genes, oxidative stress mediators as well as genes involved in endothelial cell damage328 and common variants in oxidative stress response NOS3 (eNOS/Nitric Oxide Synthase 3) and DNA repair XRCC1 genes have been specifically associated with acute skin toxicity in breast cancer patients more recently448.
However, candidate gene studies altogether largely failed to identify genetic variants causing the majority of radiotoxic phenotypes and the pathogeny of the disease still seemed very multifaceted. Therefore, a GWAS (Genome-wide association study) approach was already suggested in 2009 to identify variants and the way they might interact, to enable a pretreatment profile testing of patients328 which, however, requires a very large patient group and might be difficult to realize as the minority of patients develops late reactions. It was hypothesized that, in contrast to the candidate gene approach, individual RS might be determined by multiple loci ranging from common variants with minor effects to rare variants, with tremendous effects on the RS phenotype, and that the tissue- and endpoint-specificity is of considerable importance315,439.
As gene expression studies rather concentrate on the phenotype than on the genotype, radiooncology research underwent a change of direction again, stressing importance of GWAS studies in finding common genetic variants315, which came into focus in 2010 and recommendation about gene knockout studies were made429. In 2012, progress on GWAS was published, stating the identification of 67 marker genes, which were differentially induced after IR in lymphocytes from radiosensitive patients in a multicentric study449. However, no match with previously described candidate genes was achieved and interestingly, functional radiosensitivity assays showed great variability between patients.
Moreover emphasizing the importance of the GWAS approach, 80 % correlation between radiosensitivity and acute toxicity in patients was found and genes of DNA repair, apoptosis and cell cycle regulation were again identified, which supports previous data450. This multifaceted approach confirmed the reproducibility problem, as previous data stating reduced apoptosis of lymphocytes as predictive marker of radiotoxicity451 could not be validated, which was supported by other studies452 453. As progress commenced in the field of next generation sequencing, the radiogenomics field emerged, which included
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identification of increasing numbers of polymorphisms associated with radiotoxicity and larger GWAS studies315. Recent progress in this field supported the idea that inter-individual variation between RS patients is based on common genetic variants with low penetrance. A large GWAS study in 2014 confirmed association between common genetic variants and toxicity, which however, seemed to be tumor site-specific, and found associations for individual endpoints but not as an overall toxicity measure regarding all patients454. However, none of the SNPs identified in this large study were found in previously described candidate genes for radiotherapy toxicity455. However, potent radioprotective substances could be established based on these research findings, for example inhibitors of superoxide dismutase (SOD) conferred reduction of cytokine production in some tissues, for example TGFB1, when being used in parallel to several other substances including apoptosis and senescence inhibitors456. Further progress in the field of next generation sequencing, for example by exome sequencing, has enabled identification of new players of the DNA damage response, as was shown for MTPAP (mitochondrial poly-A-polymerase) more recently457. Advances in genome editing techniques also enable a whole new area of research and this methodology already emerged into the radiosensitivity field458.
Additionally to the task of unraveling genetic drivers in individual RS, a pre-radiation test needed to be established, which allowed easy and fast feasibility according to clinical routine requirements. Quantification of DNA damage by measuring DNA strand breaks in form of micronuclei formation, chromosome aberrations, clonogenic survival and later also foci analysis are all established assay systems but time-consuming in the first place and also did not produce clearly reproducible results over the last decades. While most analyses focused on investigating differences between cells of patients with established radiosensitivity syndromes in comparison to healthy controls459, this approach was also addressed concerning radiosensitive patients with no known RS syndromes. One group found a significant correlation between clonogenic survival and excess acentric fragments with radiosensitivity in dermal fibroblasts460 and fibroblast-specific parameters have been associated with fibrosis observed in clinical procedures461 and radiation-triggered generation of TGFB1, also in lymphocytes460,462,463. However, the excess risk of fibrosis could not be reliably predicted in another study although correlation between residual DSBs and the cellular radiosensitivity of the patients was possible464. Another group proposed an assay system based on apoptosis of lymphocytes, which showed good correlation with late toxicity effects in patients with a variety of cancers465, and lymphocyte-associated assay systems were found to be a good predictor of acute reactions466. Furthermore, one group has provided evidence for an impairment of the nucleo-cytoplasmic shuttling of the ATM protein in radiosensitive cell systems467 468. Despite these progresses, however, no assay which met requirements for clinical use had been established so far339 440, although new techniques are on the rise. The visualization of DNA DSB sites through immunofluorescence analyses of repair foci is a well established radiobiological endpoint, and recent findings provide evidence for the γH2AX assay as predictive clinical ex vivo marker of individual patient radiosensitivity through blood and eyebrow hair follicle samples469 and it has also been modified to enhance practicability470. Furthermore, our group could lately show good correlation with early and residual γH2AX and 53BP1 foci in the low-, and high-dose IR context of lymphocytes and LCLs of patient cells with mutations in ATM, and triple negative breast cancer cell lines with impaired TP53/BRCA1 and NBN/BRCA1326 327. Moreover, flow-cytometry based assays for the prediction of RS for clinical routine have very recently been established471,472.
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2.5 Previous and related work on TPT1 in breast cancer and ATM signaling