## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Free DNA-Based Biomarkers
From another perspective, higher amounts of tumor cfDNA obtained after RT allowed the detection of mutations that account for the tumor drug resistance induced by previous treatment with a tyrosine kinase inhibitor [174]. Such data were difficult to obtain using the low tumor cfDNA levels in the absence of RT. Altogether, cfDNA analysis is of utmost importance for the RT precision that needs personalized irradiation dose adjustment during a fractionated scheme for maximizing therapy efficacy, while limiting radiotoxicity to levels that do not produce prolonged impairment of the life quality [181]. Moreover, the cfDNA approach is also informative on the radiation-induced resistance of tumors, requiring a dose increase in the next irradiation session or an adjunctive therapy to increase the radiosensitivity of tumor cells [182; 183; 184]. In addition, a recent study on methylated cfDNA from breast cancer patients identified dose-dependent and specific tissue responses, with right-sided-breast treated patients showing endothelial DNA implicating liver tissue as an affected tissue. Three patients in this study developed a grade 2 skin toxicity, and showed increased breast basal and luminal epithelial damage. This highlights the complexity of the RT responses and the potential of identifying specific tissue interactions in order to design radioprotective methods. For the moment, cfDNA has been used for the detection of the tumor response to RT. It is expected that in the future, extensive studies on leukocyte-derived cfDNA will be developed for assessing immune cells' response to RT as a measure of potential radiotoxic effects. In this context, the differential analysis of cfDNA deriving from tumor and hematopoietic cells is expected to provide helpful information on therapy outcome and the response of patients to therapy in terms of therapy efficacy (mirrored by changes in tumor cfDNA) and radiotoxicity (mirrored by changes in cfDNA derived from blood leukocytes and normal tissues affected by therapy). The main issue lies in distinguishing between the different forms of cfDNA according to their origin, both in basal conditions and during/after therapy. The field of translational research related to cfDNA is expected to revolutionize precision oncology. A potential technological development may be related to the analysis of DNA fragments across the whole genome [185] using the DNA evaluation of fragments for early interception (DELFI) technology. It was found that the fragmentation patterns of cfDNA across 245 healthy individuals reflected nucleosomal patterns of white blood cells, whereas the fragmentation patterns in the plasma of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancer had distinctive disease-specific fragmentation profiles. This investigational approach was able to improve diagnostic evaluation by identifying subsets of individuals more likely to have a particular type of cancer. The DELFI technology, combined with mutation-based cfDNA analysis, also shows promise for deeply monitoring the outcome of pre-operative and curative RT in oncologic patients, as well as for evaluating radiotoxicity at the level of immune cells. Challenges and Opportunities for Cancer Radiosensitivity Biomarkers in the Era of Artificial Intelligence  
For over a decade, there has been a growing interest in cancer radiosensitivity/toxicity biomarker research due to the important role of RT in cancer treatment. At the moment, there is a restricted application of the aforementioned biomarkers (Figure 1) for clinical use, with an experimental character among medical laboratories and university hospitals. This multi-sided difficulty originates from the fact that enough evidence should be provided to persuade the international radiation therapy and oncology communities to start using such biomarkers in the clinical routine.