## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Intrinsic Radiosensitivity Biomarkers
#### 4.1.2 DNA Damage Response
Different results were produced in a subsequent study, in which [PERSON] et al., 2016 studied 25 prostate cancer patients treated with 3D conformal RT, with minor and larger score changes (Expanded Prostate Cancer Index Composite (EPIC)) by applying the \(\gamma\)H2 AX, G2, and apoptosis assays in PBMCs. Patients have been surveyed prospectively before, on the last day of RT, and at a median time of 2 months and 16 months after RT, using a validated QoL questionnaire (EPIC). Contrary to the aforementioned studies, no correlation was found between the number of \(\gamma\)H2 AX foci and radiotoxicity at the level of PBMCs [44]. [PERSON] et al., 2010 also conducted a study based on lymphocyte cells isolated from the blood of cancer patients with and without severe reaction to RT by following gH2 AX foci kinetics after IR, which showed that there is no detectable difference between the control group and the RS group as a whole. Nevertheless, with the utilization of \(\gamma\)H2 AX foci assay they managed to identify an RS cancer patient cell line with a novel ionizing radiation-induced DNA DSB repair defect and thus concluded that in combination with other predictive assessments, the \(\gamma\)H2 AX assay may eventually facilitate the tailoring of RT regimes to individuals [59]. The differences between the results of different clinical studies might be attributed to methodological reasons. Along with \(\gamma\)H2 AX, the 53 BP1 assay is a promising tool for predicting radiosensitivity. 53 BP1 is a pivotal DDR protein that plays a critical role in maintaining genomic stability. It is recruited to DSBs and forms discrete nuclear foci that are detected by IF. 53 BP1 acts as a scaffold protein that recruits other DDR proteins to the site of DNA damage and promotes the repair of DNA breaks via the non-homologous end joining (NHEJ) pathway. The phosphorylation status of 53 BP1 is regulated by several kinases, including ATM and the DNA-PKcs protein kinase involved in NHEJ. 53 BP1 foci developed in response to radiation-induced DSBs are scored following similar procedures as the \(\gamma\)H2 AX assay. [PERSON] et al., 2011 investigated via immunofluorescence both proteins in ex vivo irradiated blood lymphocytes with 0.5 and 4 Gy, using 250 kV X-rays from BC patients who had undergone surgical excision of the primary tumor and postoperative RT to the whole breast, with late adverse effects. The authors analyzed the scores from post-surgical photographs of breasts collected before RT and at 2 years and 5 years post RT. Two groups of patients were identified. The first group apppertained to severely marked radiation-induced change (cases) and the second to very little/no change (controls) in the breast. The authors evidenced higher levels of residual DSBs in ex vivo irradiated blood lymphocytes from clinically radiosensitive BC patients at 0.5 h after the ex vivo irradiation with 0.5 Gy, or in the case of cellular samples irradiated ex vivo with 4 Gy and analyzed at 24 h. The role of DSB repair in the development of late radiation-induced damage of normal tissue was demonstrated [42]. [PERSON] et al., 2013 along with the \(\gamma\)H2 AX marker, studied the 53 BP1 protein as a marker of DSB formation in PBMCs isolated from the blood samples of 57 BC patients and came to the conclusion that the 53 BP1 assay was less sensitive than that for histone \(\gamma\)H2 AX in the case of endogenous (0 Gy) and RT-induced (0.5 Gy, 30 min) foci [61]. In a very recent research study, [PERSON] et al., 2023 analyzed peripheral blood samples from 26 RS and NOR BC patients treated with RT using the combined \(\gamma\)H2 AX/53 BP1 foci assay. A significantly higher amount of endogenous \(\gamma\)H2 AX and 53 BP1 DNA repair foci was found in peripheral blood lymphocytes (PBL) collected from RS patients prior to RT, in comparison with NOR patients. This effect was not reproduced by analyzing in vitro exposed frozen-thawed cells derived from 2 RS and 2 NOR patients before RT [62].