## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Intrinsic Radiosensitivity Biomarkers
#### 4.1.2 DNA Damage Response
A measure of the DNA repair machinery efficiency in response to radiation-induced DNA damage can be provided by the co-localization of \(\gamma\)H2 AX and 53 BP1 proteins since the 53 BP1 protein recognizes the \(\gamma\)H2 AX foci and forms nuclear foci at the site of DNA damage, playing a critical role in the repair of DSBs. This co-localization assay showed promising results in the clinical study of [PERSON] et al., 2016 [63] in which the \(\gamma\)H2 AX and 53 BP1 response was investigated in ex vivo irradiated PBL from 16 patients who developed severe late radiation toxicity following RT (RTOG Grade 3-4 late toxicity), representing the OR group, and from 12 control patients (matched for sex, treatment site and intent, RT dose, use of chemotherapy, and approximate age) constituting the NOR group. By applying the co-localization assay at five different time points up to 24 h post irradiation, the authors concluded that the colocalization of \(\gamma\)H2 AX and 53 BP1 foci support the hypothesis that the RS phenotype is associated with compromised DNA repair. A lower co-localization reflects decreased cooperation of DNA repair factors, which presumably impacts the efficiency of DSB processing. One of the major strengths of the \(\gamma\)H2 AX assay is its sensitivity in detecting radiation-induced DSBs [67]. Even at low doses, \(\gamma\)H2 AX foci can be visualized and quantified using IF, hence enabling the assessment of cellular response with high precision [68]. Furthermore, as activated oncogenes induce the stalling and collapse of DNA replication forks leading to the formation of DSBs in precancerous cells, \(\gamma\)H2 AX levels may reflect genomic instability in tissues, and serve to detect precancerous lesions. Accordingly, preventive measures can be taken or treatment options can be better informed [69]. In addition, the \(\gamma\)H2 AX assay can be applied to various biological samples, including peripheral blood that is collected by a non-invasive procedure for radiosensitivity assessment [70]. Although it is accepted that -with some exceptions- almost every DSB forms \(\gamma\)H2 AX foci, whether every \(\gamma\)H2 AX focus identifies a DSB still remains controversial. A \(\gamma\)H2 AX focus may persist over time in some tumor cells after the initiating DSBs have been rejoined [71], as DSB complete repair also involves restoring the original chromatin conformation, which may also be facilitated by the presence of \(\gamma\)H2 AX foci. The \(\gamma\)H2 AX assay is limited by false positives and negatives due to variations in staining protocols and interobserver variability. Standardization of protocols and scoring criteria is essential to ensure accurate and reproducible results between laboratories. Furthermore, the assay primarily detects DNA damage and repair kinetics, overlooking other factors that influence radiosensitivity, such as the cell cycle phase. H2 AX is not only phosphorylated in response to DNA damage but also during normal replication and in response to replication stress [72]. Progression of damaged cells through the cell cycle can lead to further breakage in the S phase and, therefore, S-phase cells should be avoidedfor analysis. The assessment of radiosensitivity based solely on \(\gamma\)H2 AX foci formation may not provide a comprehensive understanding of cellular responses to radiation, and a combination of assays involving both non-proliferating and proliferating tissues might be necessary to address this issue. Regarding the 53 BP1 assay, since 53 BP1 is one of the early recruited repair proteins participating in both NHEJ and HR, it represents a promising candidate to study the architecture and dynamics of repair clusters in relation to their importance for DSB repair [73]. Moreover, despite similar labeling and microscopy parameters, 53 BP1 foci can be better separated from one another than \(\gamma\)H2 AX foci [74], leading to more reliable results. On the other hand, 53 BP1 is expressed throughout the cell cycle, leading to its diffuse localization in non-irradiated nuclei [75].