## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Intrinsic Radiosensitivity Biomarkers
#### 4.1.1 Cytogenetic Markers
The variability in the yields of radiation-induced chromatid breaks in various samples from the same individual is a well-known issue [46]. Another significant concern related to the appropriateness of the G2 assay for identifying radiosensitive patients is the significant overlap in the yields of G2 chromatid breaks in cancer patients and in healthy individuals [47]. To overcome the above-mentioned limitations, a standardized G2 assay using an ATM/ATR inhibitor as an internal control was established by [PERSON] and [PERSON] 2011 [48], minimizing inter-laboratory and intra-experimental variations and estimating the G2 chromosomal radiosensitivity at the individual level. An alternative method to estimate an individual's radiosensitivity at the cytogenetic level is the G2 cytokinesis-block micronucleus (MN) assay. This assay has been used for almost 40 years and has become one of the most popular methods to assess the genotoxicity of different chemical and physical factors, including radiation-induced DNA damage [49]. Micronuclei are small nuclei formed when chromosomes or chromosome fragments are not incorporated into the daughter nuclei during cell division. Micronuclei can be detected in cells that have completed nuclear division; they are identified by their binucleated appearance after inhibiting the formation of the actin microfilament ring that is required for cytokinesis by adding Cytochalasin-B in the cell culture. Performing the G2 MN assay in the blood samples from 18 BRCA2-mutation carriers and 17 individuals from both BRCA1 and BRCA2 families not showing the familial mutation, [PERSON] et al., 2017 found a higher radiosensitivity in healthy BRCA2-mutation carriers compared with healthy volunteers after exposure of PBL to a dose of 2 Gy \(\gamma\)-rays, while no increased radiosensitivity was observed in non-carrier relatives of BRCA1 and BRCA2 families [50]. The radiosensitivity at the individual level proved to be mild or more severe to a higher proportion in BRCA2-mutation carriers (50%) compared with healthy volunteers (17%) and non-carriers (24%). These results indicate that the MN assay constitutes a valuable tool to identify radiosensitivity in BRCA2-mutation carriers exposed to IR either for diagnostic or therapeutic purposes. The MN assay is internationally validated due to the ease of scoring and its applicability in different cell types [51]. Despite the clear advantages of the MN assay, some limitations must be considered when performing the test. The MN assay requires expertise in sample preparation, slide preparation, and scoring of micronuclei. However, the scoring criteria and methodology can vary between laboratories and even within the same laboratory, leading to inconsistent results, hence limiting data comparison across studies or institutions. Furthermore, this assay exhibits significant inter-individual variability in control-unexposed donors and their induction can be influenced by various factors other than radiation exposure (such as chemicals, genotoxic agents, and certain physiological conditions) [52], which can affect the specificity of the MN assay as a sole indicator of radiosensitivity due to confounding factors. Another potent assay utilized for the assessment of individual radiosensitivity the is FISH assay through which certain nucleic acid sequences can be detected and localized. FISH enables the detection of cytogenetic changes, such as translocations or deletions, by using fluorescence microscopy or flow cytometry [53]. [PERSON] et al., 1995 performed the FISH assay in lymphocytes from 16 different types of cancer patients (12 patients were considered as having normal tolerance to radiotherapy while 4 were considered as having a potentially increased radiosensitivity) [54]. The latter four patients showed increased clinical radiosensitivity, experiencing severe acute reactions and exhibiting chromosomal damage in lymphocytes after irradiation.