## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Intrinsic Radiosensitivity Biomarkers
#### 4.1.1 Cytogenetic Markers  
Cytogenetic assays are extremely effective tools for detecting and analyzing chromosomal abnormalities in cells. Various types of chromosomal aberrations, including deletions, duplications, translocations, and inversions, can be detected by the examination of chromosomes, either at the metaphase stage or the interphase nucleus. Cytogenetic assays, such as the G2 assay, micronucleus (MN) assay, and fluorescence in situ hybridization (FISH), have been used to measure the extent of DNA damage and genetic alterations in cells after exposure to radiation and provide valuable information about an individual's susceptibility to radiation-induced cancer and the effectiveness of RT. A cell-cycle-based technique that has been utilized by several groups to investigate correlations between human chromosomal radiosensitivity and increased susceptibility to cancer is the G2 chromosomal radiosensitivity assay [37; 38; 39]. The G2 assay is a cytogenetic methodology used to evaluate cell radiosensitivity. It assesses the extent of chromosomal aberrations induced by IR at the G2 phase of the cell cycle, which can be visualized at metaphase spreads, where chromosomes are condensed, using standard cytogenetic techniques. In order to assess the conversion of unrepaired DNA lesions into chromosomal damage during the G2 to M phase transition (as chromatid breaks at metaphase), the methodology entails in vitro irradiation of peripheral blood lymphocytes in the G2 phase, followed by repair of DNA damage and a colemid block of spindle formation during mitosis. Enhanced chromosomal radiosensitivity, manifested by radiation-induced chromatid aberrations in lymphocytes, has been observed in many types of cancer and genomic instability syndromes. The results of the G2 chromosomal radiosensitivity assay performed by [PERSON]. [PERSON] et al., 2005, on 15 patients with benign prostatic hyperplasia (BPH) [40], 17 patients with prostate cancer (which have not received radio-, chemo-, or immunotherapy for at least 6 months before sampling), and 14 healthy control individuals evidenced an elevated level of radiation-induced chromatid aberrations and enhanced G2 chromosomal radiosensitivity in the first two groups mentioned above, compared with the control group. [PERSON] et al., 2005 applied the G2 assay in heparinized blood samples obtained from 27 breast cancer (BC) patients and confirmed that in a significant proportion, they exhibited elevated G2 chromosomal radiosensitivity in contrast to random controls [41]. Moreover, [PERSON] et al., 2011 observed significantly higher levels of deletion-type aberrations in ex vivo irradiated blood lymphocyte metaphases in a group of BC patients with severely marked radiation-induced change compared with the group with very little/no change after RT; the authors came to the conclusion that chromatid aberration appears to be a suitable biomarker for the prediction of radiation-induced toxicity [42]. Conversely, a comparison of 25 prostate cancer patients with severe side effects and 25 patients without severe side effects after RT, as well as 23 male healthy age-matched donors analyzed in the study performed by [PERSON] et al., 2012 [43] showed that the G2 assay cannot be characterized as a reliable tool for the identification of prostate cancer patients with a high risk for developing severe clinical side effects. This observation was sustained by the study of [PERSON] et al., 2016 [44] on patients with localized T1-3N0M0 prostate cancer who were treated with 3D conformal RT. A predictive value of the G2 assay results after irradiation of lymphocytes with 0.5 Gy could not be established, as no discrimination between urinary and bowel score change could be made. The existing G2 assay protocols for assessing individual radiosensitivity have limitations, as they can only produce consistent and meaningful results under strict technical conditions [45].