For over a century, radiotherapy (RT) has remained at the forefront of cancer patient treatment. It contributes to ~40% of curative cancer treatments, alone or in combination with chemotherapy, and tends to be less morbid than surgery ^[1]. The continued and rapid progress in the field of RT includes radiation type and delivery schemes that provide a conformal dose distribution in the tumor niche, leading to the maximum possible efficacy in cancer cell killing. At the same time, emphasis is given to preserving the functional and structural integrity of neighboring healthy tissues. Some advanced RT techniques such as intensity-modulated radiation therapy (IMRT), stereotactic radiosurgery (SRS), and proton/carbon ion therapy (hadron therapy) greatly improve clinical outcomes and provide better control of cancer progression via precise delivery of radiation doses to the tumor tissue. New modalities, such as FLASH proton radiotherapy, where high doses are delivered in an ultra-high dose rate fashion, are currently under clinical trials [2,3]^[2][3]. Importantly, in the last few years, the interest in RT modalities has been renewed due to the ability of RT to promote systemic immune responses that can overcome resistance to immunotherapy. Combinations of RT with immunotherapeutic agents, such as immune checkpoint inhibitors, are increasingly favored for overcoming the barriers and synergizing the efficacy of each intervention alone ^[4].

While RT effectively targets and destroys cancer cells, it can also result in toxicity and adverse effects at the level of healthy tissues in the vicinity of the irradiated area, as well as at the systemic level. Along with interfering with the basic biological properties of cancer cells (such as growth signal autonomy, unlimited replicative potential, and evasion from growth inhibitory signals along with angiogenesis, invasion, and metastasis), ionizing radiation can often inflict life-threatening damage to normal cells. Common toxicities associated with RT include acute and chronic radiation dermatitis (erythema, dryness, skin desquamation and telangiectasias, fibrosis, and ulceration, respectively), mucositis, fatigue, diarrhea, dysphagia, and radiation-induced fibrosis among others depending on the site of exposure. Cell injury due to IR comprises a spectrum of sequential events and biological processes involving intricate cellular morphologic, molecular, and functional changes that are triggered by IR within minutes to hours after irradiation (early toxicity), related to oxidative stress, DNA damage, and cell death, as well as inflammation and cell proliferation ^[5]. During the first few minutes after exposure, part of the tumor cell is committed to cell death (apoptosis), while part attempts to repair the damaged DNA by developing a robust stress response. IR-induced cell alterations progress over weeks, months, and even years (late toxicity), leading to late tumor and organ dysfunction (atrophy and fibrosis) and therapy-induced secondary malignancies in some cases. The mechanisms underlying radiotoxicity in human tissues involve increased cell death and inflammation, as well as defective tissue regeneration ^[6].

Patient radiosensitivity is emerging as a multi-dimensional problem [7,8]^[7][8], ranging from the microworld of the cell and its environment to the macroworld of patients and their families, including time scale (from minutes to years) and medical physics parameters (dose and RT modalities). The adverse effects exhibit complex patterns across multiple tissues and have unclear boundaries, rather than being a mere and distinct classification to radiosensitive versus radioresistant individuals. The occurrence of radiogenic side effects is a strong limitation of therapeutic radiation, requiring the adjustment of the effective individual dose for the prolongation of the patient’s life. Additionally, late effects of RT include radiation-induced secondary malignancies ^[9]. In view of these clinical complications, cut-offs need to be defined that can guide medical decisions and reliably stratify patients who can tolerate a standardized treatment RT scheme, exhibiting no or mild side effects, from those who show individualized over-sensitivity responses, entailing a combination of adverse effects that counteract therapeutic benefits ^[10].

2. The Importance of Biomarkers in Monitoring RT Toxicity

A molecular biomarker is any molecular indicator of normal and pathogenic biological processes or the pharmacologic responses to therapeutic interventions [35]^[11]. Biomarkers improve prediction, characterization, and treatment of human diseases. In the oncology field, biomarkers guide informed decisions of clinicians regarding chemo- and radiotherapeutic strategies, relative to the risk of potential side effects and tumor recurrence. Biomarkers are measured, if possible, in readily accessible biological samples before, during, and after RT. Medical imaging with new combined techniques and higher definition, together with assays in biological fluids, constitute the current main strategy in the clinical evaluation of RT-induced radiosensitivity and toxicity. In radiation response investigations, blood and its components, serum/plasma and cells, mostly lymphocytes, are considered a readily accessible source of biomarkers to study IR toxicity. An ideal blood biomarker would fulfill the following criteria: (a) precise targeting of a biological process related to the radiosensitivity of the peripheral immune system; (b) specificity for a certain type of cancer with alterations in the systemic immune response; (c) tumor reactivity to RT mirrored in peripheral blood; (d) measurement accuracy in blood samples and validation of the detection method according to international standards; (e) distinct delimitation of the biomarker levels between patients with a certain type and stage of cancer and patients with systemic inflammatory processes; (f) independence from diverse environmental factors (e.g., dietary habits, alcohol intake, etc.) or treatments unrelated to disease therapy; (g) detection of measurable changes during disease evolution and/or therapy; (h) reliability of laboratory measurements and generation of results and; (i) undetectability, if possible, when patients attain remission. The advancement of our understanding of genomic and epigenomic processes, including mutations, gene expression changes, and post-translational modifications, has shifted radiobiology research to gene and protein biomarkers that are indicative of the radiation-induced responses in normal and cancer cells. Deep-sequencing approaches confirmed that complex DNA damage and repair mechanisms, along with chromosomal aberrations and IR-induced cell apoptosis in various irradiated tissue samples can be considered predictors of radiosensitivity and post-irradiation toxicity. Protein biomarkers can also be used for the estimation of the received dose and response of the irradiated biological system (cell/tissue), the assessment of radiosensitivity of different human cell lines, the investigation of susceptibility of an irradiated individual as a whole organism, the detection of early signs arising from radiation-induced disease, and the evaluation of late health effects that are present a long period of time after exposure before the initiation of an associated disease. At the same time, progress in the field of biomarkers that can be widely used in the clinic is rather slow. This is because for a biomarker to qualify as indicative of a clinical outcome, the variation in its values must be strongly correlated with the corresponding changes in disease status.

2.1. Cell-Intrinsic Radiosensitivity Biomarkers

Over the years, a variety of assays and endpoints have been developed to measure radiosensitivity, each with its advantages and disadvantages. The clonogenic assay has been widely used and is considered the gold standard for assessing cellular radiosensitivity in vitro [36]^[12]. However, it is time-consuming and requires a high level of technical expertise.

2.1.1. 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]^[13][14][15]. 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 colcemid 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 Howe O. L. et al., 2005, on 15 patients with benign prostatic hyperplasia (BPH) [40]^[16], 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. Howe 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]^[17]. Moreover, Chua 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]^[18]. 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 Brzozowska et al., 2012 [43]^[19] 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 Pinkawa et al., 2016 [44]^[20] 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]^[21]. The variability in the yields of radiation-induced chromatid breaks in various samples from the same individual is a well-known issue [46]^[22]. 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]^[23]. To overcome the above-mentioned limitations, a standardized G2 assay using an ATM/ATR inhibitor as an internal control was established by Pantelias and Terzoudi 2011 [48]^[24], 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]^[25]. 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, Baert 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 γ-rays, while no increased radiosensitivity was observed in non-carrier relatives of BRCA1 and BRCA2 families [50]^[26]. 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]^[27]. 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]^[28], 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]^[29]. Dunst 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]^[30]. The latter four patients showed increased clinical radiosensitivity, experiencing severe acute reactions and exhibiting chromosomal damage in lymphocytes after irradiation. Particularly, significant differences were found in radiation-induced chromosomal damage, measured as breaks per 1000 mitoses ranging from 70 to 556 after 0.7 Gy and from 420 to 1210 after 2 Gy. Thus, the authors concluded that this technique may be useful to detect patients with severely enhanced radiosensitivity. Huber et al., 2011 quantified chromosomal aberrations by means of FISH using specific probes for chromosomes 1, 4, and 12 in blood samples from 47 BC patients collected 6 weeks post RT. Meanwhile, clinical side effects of RT were evaluated weekly during the therapy, and the scoring was carried out according to the Common Toxicity Criteria (NCI-CTC scale; scale digits 0, 1, 2, 3, 4). The study’s findings were increased chromosomal aberrations in patients with more severe side effects, a correlation between the frequency of a specific aberration (painted chromosomes bearing one centromere with a color junction type t(Ba)), and the occurrence of skin side effects. Based on the frequency of t(Ba) aberrations, the authors identified patients with short latency (early side effects) when no correlation was found for other aberrations (painted chromosomes bearing one centromere with a color junction type t(Ab), dicentrics(dic), color junctions(cj)) with side effects or latency. Results have indicated that the estimation of chromosomal radiosensitivity at the translocation level in peripheral blood lymphocytes can be proposed as a predictive assay for the detection of radiosensitive individuals [55]^[31]. The 2, 11, and 17 paint probes tested in localized prostate cancer patients treated with 3D conformal RT, with (PS) and without (P0) side effects, as well as in a healthy group, led to negative results in the study of Schmitz et al., 2013 [56]^[32] as no pronounced differences in chromosomal radiosensitivity levels between donor groups, after a 2 Gy-irradiation, were mentioned. A dose-dependent increase in aberrations for all analyzed chromosomes, with smaller chromosomes 11 and 17 being less frequently involved in aberrations compared with the larger chromosome 2, was found. Nonetheless, the FISH assay did not manage to demonstrate significant differences in chromosomal radiosensitivity between donor groups when comparing the chromosomes with each other. However, the dose-dependent increase in aberrations suggests that higher radiation doses may be needed to reveal such differences. FISH has some limitations compared with the G2 and MN assay, as it is a laborious technique that requires specialized equipment, while the preparation of FISH probes, hybridization, and image analysis processes can be time-consuming, limiting its widespread implementation and applicability in routine clinical practice. In turn, the utilization of the FISH assay for assessing radiosensitivity offers several advantages, including the detection of chromosomal aberrations with high sensitivity, specificity, and accuracy. These strengths make it a valuable tool in detailing an individual’s response to IR toward the development of personalized RT strategies.

2.1.2. DNA Damage Response

Numerous studies have examined DNA damage response (DDR-related) proteins as radiosensitivity biomarkers. These proteins are indicative of DNA damage and play a critical role in repairing the DNA damage caused by IR, and alterations in their expression levels have been clearly associated with radiosensitivity. In this respect, the histone H2AX as a radiosensitivity biomarker in various types of cancer, including breast, prostate, and head and neck tumors, was investigated. H2AX is a key protein that signals DNA damage (mainly double-strand breaks, DSBs) by its phosphorylation at serine139. This process is known as γH2AX formation, and practically results in the appearance of γH2AX foci at sites of DNA damage [64]^[33]. γH2AX foci help to recruit and coordinate the DDR, and, for this reason, are broadly utilized as a marker for the extent and location of DNA damage, allowing for the quantification and visualization of DNA repair events via immunofluorescence (IF) [65]^[34]. The main steps of the γH2AX IF assay protocol involve fixation, permeabilizing, and blocking of the cells. The primary antibody against γH2AX is then added, followed by a fluorescently conjugated secondary antibody and nuclear counterstaining while washing steps are taking place between labeling [66]^[35]. The final samples are analyzed via fluorescence microscopy. Moreover, the γH2AX IF assay can be combined with other techniques such as Western blotting or flow cytometry for quantitative analysis for measuring DNA damage. Fleckenstein et al., 2011 examined peripheral lymphocytes of 31 patients with resected head and neck cancer, undergoing adjuvant RT or radio-chemotherapy (RCT), using the γH2AΧ assay to evaluate the in vivo impact of individual DNA double-strand break (DSB) repair mechanisms on the incidence of severe oral mucositis. γH2AX foci were quantified before and at 0.5 h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and were found to correlate well with DSB repair. An important finding of this study was that, at 24 h after RT, patients with a proportion of unrepaired DSBs higher than the mean value plus one standard deviation had an increased incidence of severe oral mucositis. The study concludes that the in vivo DSB repair by evaluating γH2AX foci loss is reliable in clinical practice, allowing the identification of cancer patients with impaired DSB repair. In turn, the incidence of oral mucositis has not been found to be closely correlated with DSB repair under the evaluated conditions [57]^[36]. In a prospective study, Mumbrekar et al., 2014 analyzed DNA DSBs and repair via microscopic γH2AX foci analysis in peripheral lymphocytes from 80 post-chemotherapy breast cancer patients with histologically confirmed cancer, without distant metastasis and without prior RT, and in 38 healthy female donors without recent diagnostic radiation exposure or any chronic diseases. The aim of the study was the examination of the usefulness of γH2AX foci analysis expressed as a percentage of residual damage (PRD), which referred to measurements at 3 and 6 h after in vitro irradiation of peripheral lymphocytes with 2 Gy of 120 kV X rays for predicting adverse reactions of IR at the level of normal skin. For confirming the in vitro results in a clinical setting, breast cancer patients subjected to RT were categorized as over-responders (ORs) and non-over-responders (NORs) with respect to the extent of acute adverse reactions of clinical irradiation in the skin, according to the Radiation Therapy Oncology Group (RTOG) criteria. A statistical difference between the healthy and the NOR groups, as well as between the healthy and the OR groups, has been found. The authors concluded that the slow rate of foci disappearance with respect to the initial DNA damage may be the cause of clinically radiosensitive phenotypes [58]^[37]. Accordingly, the H2AX assay is adequate to quantify PRD while considering the degree of the initial DNA damage and is accordingly an informative predictive assay for monitoring RT. Similar results were obtained by Djuzenova et al., 2013. In tThis study, the γH2AΧ assay was performed in peripheral blood mononuclear cells (PBMCs) isolated from the blood samples of 57 breast cancer (BC) patients, collected before and during clinical irradiation (72 h after five radiation fractions), against 12 clinically healthy donors. In this clinical study, six retrospectively identified BC patients with an early adverse skin reaction to RT, developed at the level of the irradiation field, were used as a reference for clinical radiosensitivity according to the RTOG score, and showed striking differences in the results of the γH2AX assay compared with healthy individuals. The data obtained using the γH2AX assay identified BC patients with normal clinical reactions to RT based on the induced and residual DNA damage, but not on the background DNA damage. It was concluded that the γH2AX assay may have the potential for screening individualized radiosensitivity of BC patients, as the mean number of γH2AX foci after five clinical fractions was significantly higher than before RT, especially in clinically radiosensitive patients [61]^[38]. Different results were produced in a subsequent study, in which Pinkawa 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 γH2AX, 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 γH2AX foci and radiotoxicity at the level of PBMCs [44]^[20]. Vasireddy 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 gH2AX 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 γH2AX 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 γH2AΧ assay may eventually facilitate the tailoring of RT regimes to individuals [59]^[39]. The differences between the results of different clinical studies might be attributed to methodological reasons. Along with γH2AX, the 53BP1 assay is a promising tool for predicting radiosensitivity. 53BP1 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. 53BP1 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 53BP1 is regulated by several kinases, including ATM and the DNA-PKcs protein kinase involved in NHEJ. 53BP1 foci developed in response to radiation-induced DSBs are scored following similar procedures as the γH2AΧ assay. Chua 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 appertained 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]^[18]. Djuzenova et al., 2013 along with the γH2AΧ marker, studied the 53BP1 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 53BP1 assay was less sensitive than that for histone γH2AX in the case of endogenous (0 Gy) and RT-induced (0.5 Gy, 30 min) foci [61]^[38]. In a very recent research study, Durdik et al., 2023 analyzed peripheral blood samples from 26 RS and NOR BC patients treated with RT using the combined γH2AX/53BP1 foci assay. A significantly higher amount of endogenous γH2AX and 53BP1 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]^[40]. A measure of the DNA repair machinery efficiency in response to radiation-induced DNA damage can be provided by the co-localization of γH2AX and 53BP1 proteins since the 53BP1 protein recognizes the γH2AX 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 Lobachevsky et al., 2016 [63]^[41] in which the γH2AX and 53BP1 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 γH2AX and 53BP1 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 γH2AX assay is its sensitivity in detecting radiation-induced DSBs [67]^[42]. Even at low doses, γH2AX foci can be visualized and quantified using IF, hence enabling the assessment of cellular response with high precision [68]^[43]. Furthermore, as activated oncogenes induce the stalling and collapse of DNA replication forks leading to the formation of DSBs in precancerous cells, γH2AX 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]^[44]. In addition, the γH2AX assay can be applied to various biological samples, including peripheral blood that is collected by a non-invasive procedure for radiosensitivity assessment [70]^[45]. Although it is accepted that -with some exceptions- almost every DSB forms γH2AX foci, whether every γH2AX focus identifies a DSB still remains controversial. A γH2AX focus may persist over time in some tumor cells after the initiating DSBs have been rejoined [71]^[46], as DSB complete repair also involves restoring the original chromatin conformation, which may also be facilitated by the presence of γH2AX foci. The γH2AX 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. H2AX is not only phosphorylated in response to DNA damage but also during normal replication and in response to replication stress [72]^[47]. Progression of damaged cells through the cell cycle can lead to further breakage in the S phase and, therefore, S-phase cells should be avoided for analysis. The assessment of radiosensitivity based solely on γH2AX 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 53BP1 assay, since 53BP1 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]^[48]. Moreover, despite similar labeling and microscopy parameters, 53BP1 foci can be better separated from one another than γH2AX foci [74]^[49], leading to more reliable results. On the other hand, 53BP1 is expressed throughout the cell cycle, leading to its diffuse localization in non-irradiated nuclei [75]^[50]. Last but not least, 53BP1 is an early indicator of the starting activity of NHEJ/HR repair machinery. Hence, it is reasonable to investigate the behavior of 53BP1 during DNA repair independently [76]^[51]. Immunofluorescence (IF) is commonly used to visualize γH2AX foci and 53BPI, but this technique has some limitations related to a reduced level of quantification and subjectivity in interpretation. Another limitation of IF is that blood cells used for assessing the radiosensitivity of normal tissues are non-adherent cells that must be experimentally manipulated for attaching to slides so as to perform IF. Being dedicated to cells in suspension, flow cytometry appears to be more appropriate to analyze the response of blood leukocytes to IR. In standard flow cytometry, cells are labeled in suspension with specific functional and phenotypic fluorescent indicators and are analyzed one by one from the point of view of morphology (size and granularity) and surface/intracellular fluorescence intensity. The results are finally processed as a distribution of fluorescence intensity per cell, and the mean fluorescence and various other parameters of this distribution are finally calculated using dedicated software. Besides the analysis of cells in suspension, flow cytometry has several other advantages: (i) it allows gating and differential analysis of particular cell populations or sub-populations within a multi-cellular sample, (ii) it provides comprehensive multi-parametric information on single cells in suspension within a complex cellular sample, combining functional, phenotypic, and molecular parameters; for instance, in the radiosensitivity context, DNA damage evaluation (dsDNA breaks evaluated via flow cytometry with the fluorescent TUNEL and γH2AX foci or other specific assays) can be combined with investigations on leukocyte viability [77]^[52], apoptosis (annexin V-propidium iodide assay), senescence (β-galactosidase assay), leukocyte activation (MHCII, CD69), proliferation (CFDA-SE assay), and cell cycle (Vybrant orange stains) [78]^[53]. The currently available flow cytometers allow simultaneous measurement of more than eight parameters in the very same sample. Recently, the advent of high-dimensional time-of-flight mass cytometry (CyTOF) using antibodies labeled with metals and not with fluorophores enables broad dimensional and unbiased examination of blood cells [79]^[54], allowing simultaneous interrogation of more than 40 parameters in the same cellular sample. Clustering techniques were developed to reduce the dimensionality of the data in order to create a visual representation in a reduced number of dimensions. Altogether, this new investigational approach is highly important for deep biomarker screening in blood exposed to various challenges (such as IR), particularly when sample sizes are limited (i.e., immune cells in tumors). From another perspective, the new imaging flow cytometry platforms [80]^[55], which combine statistical power and high fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy, provide a broader image of the IR impact on blood leukocytes.

2.1.3. Other Biomarkers of the DDR Pathway

Promising biomarkers for assessing radiosensitivity in various cancer types are also the MRN complex and the ATM (ataxia–telangiectasiamutated) protein. The MRN complex, consisting of Mre11, Rad50, and Nbs1/Xrs2 proteins, is an important player in DNA repair and maintenance of genome stability. This complex acts as a sensor and mediator of DNA damage and is involved in multiple DNA repair mechanisms, including HR and NHEJ [81]^[56]. While the MRN complex has a crucial role in DNA damage sensing and repair processes, ATM is a key regulator of the cellular response to DSBs. Upon detection of DSBs, ATM is activated by the MRN complex, undergoing autophosphorylation and activation and leading to a signaling cascade initiation that regulates various cellular processes (including DNA repair, cell cycle checkpoint activation, and apoptosis) [82]^[57]. Several studies have demonstrated the correlation between a dysfunctional MRN complex, especially the MRE11 protein, and increased radiosensitivity, suggesting its potential utility as a predictive biomarker for RT outcomes for better treatment decisions. The MRN complex and the ATM expression are commonly measured by IF for visualizing their localization and dynamics in response to DNA damage. Seminal contributions have been made by Söderlund et al., 2007 by analyzing the tumor samples from 224 premenopausal women with early breast cancer. The study pointed out that high expression of the MRN complex, but not of the ATM, is necessary for tumor cell killing by RT. Therefore, a reduced expression of the MRN complex predicts a poor therapeutic effect of RT in patients with early breast cancer [83]^[58]. On the contrary, Yan et al., 2020 have shown that the methylation status of the ATM promoter in hepatocellular carcinoma (HCC) has a predictive value for RT outcome, as assessed by performing PCR (qPCR) and immunohistochemistry (IHC) in 50 paired HCC and adjacent normal tissues, and 68 locally advanced HCC biopsy tissues [84]^[59]. Yuan et al., 2012 performed an immunochemical analysis of breast cancer tissues obtained from 254 surgically treated female patients with confirmed pathology of invasive ductal carcinoma and showed that high MRE11 expression in breast cancer cells was associated with malignant behavior, higher recurrence rates, lymph node metastasis, resistance to RT and chemotherapy, and decreased patient survival [85]^[60]. The study by Ho et al., 2018 having analyzed surgical specimens collected from 265 patients treated with CRT or neoadjuvant RT followed by surgery for rectal cancer, concluded that high tissue expression levels of the three MRN complex proteins are prognostic indicators in rectal cancer and in response to preoperative therapy [86]^[61]. Therefore, the incorporation of the MRN complex and the ATM protein as biomarkers for the radiosensitivity evaluation holds great promise for improving personalized cancer treatment and for optimizing RT procedures and outcomes. As the MRN complex is essential for DSB repair and telomere maintenance, it is postulated that defective function and low expression of its components lead to DNA damage and malignant transformation. Also, owing to its ability to repair DSBs, it is likely that the levels of MRN expression influence the response of cancer cells to chemotherapy and RT in terms of apoptosis. Generally, conflicting data were obtained in different studies; some studies have associated high expression of MRN and its components with poorer outcome and treatment resistance to chemo-radiation [87,88,89]^[62][63][64], while others have found the opposite [90]^[65]. In parallel to MRE11, ATM overexpression has a significant relationship with poor disease-free survival in rectal cancer and lymph node positivity [86]^[61], suggesting that the observed poor overall survival was likely due to the aggressiveness and metastatic properties of the tumor; the latter’s progression could be reasonably attributed to the increased MRE11 levels that stabilize RAD50 and NBS1 and recruit ATM [91]^[66]. Because of the importance of the MRN complex and the ATM protein in DDR, they both remain promising biomarkers for predicting tumor response to RT.

2.2. Apoptosis Biomarkers

Cell death may occur by various mechanisms such as apoptosis, necrosis, mitotic catastrophe, ferroptosis, senescence, and autophagy [92,93,94]^[67][68][69]. Apoptosis as a programmed cell death usually occurs in multicellular organisms under normal conditions to maintain homeostasis by eliminating damaged cells. It is mediated by intrinsic and extrinsic mechanisms that activate those members of the proteases family that lead to the initiation and amplification of proteolytic cascades: the intrinsic and extrinsic ones. Extrinsic apoptosis is triggered by external signal proteins that bind to specific receptors of the cell surface and lead to the activation of the apoptotic process. Intrinsic apoptosis involves the release of mitochondrial proteins into the cytosol, which activates a proteolytic cascade. In other cases, the two pathways work harmoniously together to activate a member of the pro-apoptotic Bcl2 proteins for killing the cell. It is of note that when the apoptosis rate among cells deviates from the norm, either excessively or insufficiently, and when there is an inaccurate timing and location of apoptotic death, tumorigenesis and autoimmune mechanisms may be triggered [95]^[70]. Since apoptosis is characterized as asynchronous in cells and the persistence of apoptotic cells is relatively short, the in vivo detection of apoptosis biomarkers is complicated. Some of the most studied cells in relation to RT-induced apoptosis are endothelial cells, lymphocytes, and bone marrow progenitor cells [96]^[71]. An acknowledged biomarker related to apoptosis is caspase-3, which actively participates in apoptosis as a primary executioner of this process. Caspase-3 initiates DNA fragmentation through the proteolytical inactivation of DEF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases, in turn, the active complex DEF40/CAD (caspase-activated DNase), the endonuclease associated with the inhibitor [97]^[72]. Pathways leading to the caspase-3 activation are shown to be either dependent or independent of the cytochrome c release and the caspase-9 synergy [98]^[73]. Santos et al., 2017 reported a dose-dependent increase in active caspase-3 expression levels in human blood lymphocytes irradiated with 1, 2, and 4 Gy gamma radiation, which was investigated after different times of in vitro incubation (24, 48, and 72 h). Accordingly, caspase-3 might be used for detecting differences in the radiation sensitivity of patients before undergoing RT [99]^[74], as also reported in several other studies [100,101,102]^[75][76][77]. A well-documented study by Yang et al., 2005 with irradiated MCF-7 breast cancer cells indicates that caspase-3 plays a critical role in RT-induced apoptosis [103]^[78]. It has also been reported that caspase-3 is necessary for the radiation-induced apoptosis of human B-lymphocytes and lymphoblastoid cells [104,105]^[79][80]. The study of Cao et al., 2011 making use of the radiosensitive CD4⁺CD25⁺ T regulatory cells (Treg) from patients with hepatocellular carcinoma, reveals high expression of radiation-induced active caspase-3 [106]^[81]. Similar results were independently obtained with ¹³⁷Cs-irradiation of T cells by Nguyen et al., 2020, who observed an increase in caspase-3 levels due to irradiation [107]^[82]. Annexin V has also been shown as a useful biomarker of radiation-induced apoptosis in human T-lymphocytes and as an indicator of radiation toxicity. This anticoagulant protein may detect apoptotic cells exhibiting on their surface negatively charged phospholipids like phosphatidylserine. Several studies emphasized annexin V as a promising candidate biomarker for evaluating radiation toxicity in oncologic patients [108,109]^[83][84].