## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Free DNA-Based Biomarkers  
There is a tremendous need for biomarker discovery in blood for characterizing the molecular and functional profile of tumors, especially in the case of tumors that are difficult to access for biopsy (such as tumors of the central nervous system) [169]. Blood biomarkers are highly needed for non-invasive early diagnosis as well as for treatment selection and real-time monitoring, avoiding repeated invasive biopsies. A valuable source of liquid biomarkers is the cell-free DNA (cfDNA), which derives from damaged tumors and blood cells, and circulates in blood and other biological fluids, such as the cerebrospinal fluid [169; 170]. In cancer patients, higher levels of tumor cfDNA were detected compared with normal individuals, possibly due to increased tumor cell proliferation and damage or death by necrosis/apoptosis, especially during anti-cancer therapies [171]. This allows tumor cfDNA quantitation via digital polymerase chain reaction during therapy and the detection of cancer-specific or therapy-induced molecular alterations via sequencing [172] if sufficiently high amounts of tumor cfDNA are available in plasma. For example, the evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of patients with stage I and II disease (71, 59, 59, and 68%, respectively) [173] that were highly correlated with alterations detected in the tumors of these patients. It was also evident that higher amounts of preoperative tumor cfDNA were associated with disease recurrence and decreased overall survival of patients with resectable colorectal cancers. Considering that RT inflicts profound damage to the tumor and normal cells, it is expected to obtain enough cfDNA to perform a detailed analysis of tumor radiosensitivity and radiation-induced resistance, along with local and systemic alterations of normal tissues, including blood leukocytes. Various publications in the field have addressed tumor cfDNA in connection with RT-related biomarkers. It has been shown that RT can increase tumor cfDNA levels in plasma, as shown in a study on 17 stage I-II (early) and IV (advanced) non-small cell lung cancer (NSCLC) patients who underwent RT [174]. In this study, tumor cfDNA levels at 24 h after irradiation were significantly higher than in the corresponding pre-RT samples, peaking at 7 days post irradiation, while maximal tumor reduction was observed only later, at 4-8 weeks after RT. Moreover, the post-RT levels of cfDNA were lower than the corresponding pre-RT levels in stage I-II NSCLC patients, indicating a significant tumor reduction. The dynamics of tumor cfDNA released into circulation due to irradiation was investigated in tumor cell lines, showing that mitotic catastrophe dominates the early response to radiation exposure, with a peak in apoptosis and cfDNA release occurring later, at around 3-6 days after irradiation [175]. Several recent studies have demonstrated that the persistence of tumor-derived cfDNA following primary therapy can predict those patients who may benefit from adjuvant therapy or subsequently relapse with early metastatic disease [176; 177]. A recent study has shown that the cfDNA I-score, calculated based on low-depth whole-genome sequencing, is a predictor of radiation response in patients with lung, esophageal, and head and neck cancer. The score could detect the minimal residual disease after RT earlier than by using imagistic methods [178]. In addition, the analysis of tumor-associated cfDNA in 43 early-stage non-small cell lung cancer patients subjected to high-dose proton beam therapy (PBT) indicated that cfDNA detection before treatment is an effective biomarker that is able to predict out-of-field recurrence after PBT [179]. In turn, tumor-associated cfDNA levels at the mid-term of chemo-RT detected using the Cancer Personalized Profiling by deep Sequencing(CAPP-Seq) method could predict treatment outcomes in 41 patients with localized lung cancer [180].