The Role of Predictive Biomarkers in Modern Prostate Cancer Radiotherapy: A Literature Review on Personalised Treatment Strategies and the Prediction of Adverse Effects

Academic Editor: Joon-Yong Chung Received: 2 May 2025 Revised: 18 June 2025 Accepted: 30 June 2025 Published: 2 July 2025 Citation: Stani´c, J.; Šovi´c, I.; Jovanovic, L.; Mati´c, I.Z.; Niki´c, P.; Nikitovi´c, M. The Role of Predictive Biomarkers in Modern Prostate Cancer Radiotherapy: A Literature Review on Personalised Treatment Strategies and the Prediction of Adverse Effects. Life 2025,15, 1062. https://doi.org/ 10.3390/life15071062 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Review The Role of Predictive Biomarkers in Modern Prostate Cancer Radiotherapy: A Literature Review on Personalised Treatment Strategies and the Prediction of Adverse Effects Jelena Stani´c 1,2,*, Ivana Šovi´c 3, Luka Jovanovic 1, Ivana Z. Mati´c 4, Predrag Niki´c 2,5 and Marina Nikitovi´c 1,2 1Department of Radiation Oncology, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 2Faculty of Medicine, University of Belgrade, Dr. Suboti´ca Sr. 8, 11000 Belgrade, Serbia 3Department of Radiological Diagnostics, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 4Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 5Clinic of Urology, University Clinical Center of Serbia, 11000 Belgrade, Serbia *Correspondence: [email protected]; Tel.: +381-61-188-43-32 Abstract Prostate cancer is one of the most prevalent malignancies in men, posing a significant public health challenge due to its high incidence and long-term treatment-related toxicities. Long-lived patients often experience prolonged side effects that can severely diminish their quality of life. Despite advancements in radiotherapy techniques like IMRT and VMAT, some patients still experience acute and late side effects. Current treatment protocols do not account for individual variability in normal-tissue radiosensitivity, highlighting the need for predictive tools and a personalised treatment approach. Genetic factors and molecular regulators like microRNAs (miRNAs) contribute to these variations by influencing DNA repair, inflammation, and apoptosis. This review explores potential biomarkers of radiotoxicity, focusing on immune-related factors such as IL-6 and TGFβ 1, SNPs influencing radiosensitivity, miRNAs involved in radiation responses, and functional assays including the radiation-induced lymphocyte apoptosis (RILA) test. These approaches offer promising tools for identifying radiosensitive patients and enabling risk-adapted radiotherapy. Keywords: prostate cancer; radiotoxicity; radiosensitivity biomarkers; personalised treatment 1. Introduction Prostate cancer is the second most common malignant tumour, after lung cancer, and the fifth leading cause of cancer death in men worldwide. At some point in their lives, around one in eight men may receive a diagnosis of this cancer. Data suggest that prostate cancer imposes a substantial public health burden due to the significant proportion of older men in the general population. The incidence of prostate cancer is three times greater in developed countries than in developing countries [1–3]. In Serbia, there has been a continuous increase in cancer incidence and mortality, with 41,578 new cancer cases and 19,350 new cancer-related deaths in 2022. Prostate cancer was the fifth most common cancer both in terms of incidence and prevalence, and it ranks third in terms of incidence and mortality among Serbian men. This unfavourable epidemiological situation, along with the rising number of new prostate cancer patients, can be attributed to the lack of a national screening programme, limited media efforts to raise awareness among Life 2025,15, 1062 https://doi.org/10.3390/life15071062 Life 2025,15, 1062 2 of 21 men, and an overall lack of sufficient public information [ 4 ]. The differences in prostate cancer incidence versus mortality are largely influenced by the extent of PSA screening, which predominantly detects cancer at a localised stage when treatment outcomes are more favourable [5]. In general, when diagnosed on time, most patients with prostate cancer have a long life expectancy after diagnosis. Furthermore, improved therapeutic alternatives for localised prostate cancer have led to higher cure rates, with various treatment options achieving similar outcomes but different toxicity profiles. As a result, some patients may never experience health issues or die from complications directly related to the progression of their disease. However, many patients do live longer with complications from their treatment, which greatly affects their quality of life. Determining the incidence and severity of the toxicity of each therapeutic modality is critical for making decisions about appropriate treatment. Considering all that is mentioned above, long-term radiation-induced toxicity is emerging as a critical concern that cannot be overlooked [6,7]. 2. The Modern Radiotherapy of Prostate Cancer Prostate cancer is best treated by a multidisciplinary team of specialists, consisting of urologists, radiation oncologists, and medical oncologists, who collaborate to provide comprehensive care. The treatment modality is decided based on the stage of the disease, histopathological characteristics of tumours, survival benefits, and possible side effects of each of the therapeutic options. However, regardless of the stage of the disease, a patient’s socioeconomic status, personal preferences, and clinical practice patterns at various medical centres represent important factors in selecting the appropriate therapy [ 6 , 8 ]. Current treatment recommendations for prostate cancer, according to the NCCN guidelines, are summarised in Table 1and emphasise a risk-adapted approach, ranging from active surveillance in low-risk cases to more-aggressive treatments such as surgery, radiation therapy, and androgen deprivation therapy in intermediateand high-risk patients [9]. Table 1. Prostate cancer treatment recommendations based on risk group (adapted from NCCN Guidelines, Version 1.2025) [9]. Risk Group TNM Stage PSA (ng/mL) Gleason Score/Grade Group Treatment Options Very low T1c <10 GS ≤6 (Grade Group 1), <3 positive biopsy cores, PSA density < 0.15 - Active surveillance (preferred) - Radical prostatectomy (selected cases) - Brachytherapy (rare) Low T1-T2a <10 GS ≤6 (Grade Group 1) - Active surveillance (preferred) - Radical prostatectomy - EBRT or brachytherapy Favourable intermediate T1-T2b 10–20 GS 3 + 4 (Grade Group 2) OR <50% biopsy cores positive - Radical prostatectomy ±lymph node dissection - EBRT ±short-term ADT (4–6 mo) - Brachytherapy ±EBRT Unfavourable intermediate T2c or ≥ 50% cores + or GS 4 + 3 (Grade Group 3) or PSA 10–20 10–20 GS 4 + 3 (Grade Group 3) or multiple intermediate factors - Radical prostatectomy ±pelvic LN dissection - EBRT + short-term ADT - EBRT + brachytherapy boost High T3a or PSA > 20 or GS 8 (Grade Group 4) >20 GS 8 (Grade Group 4) - EBRT + long-term ADT (2–3 years) - EBRT + brachytherapy + ADT - Radical prostatectomy (select cases, as part of multimodal treatment) Life 2025,15, 1062 3 of 21 Table 1. Cont. Risk Group TNM Stage PSA (ng/mL) Gleason Score/Grade Group Treatment Options Very high T3b-T4 or GS 9–10 (Grade Group 5) or >4 biopsy cores with Grade Group 4 or 5 Any GS 9–10 (Grade Group 5) - EBRT + long-term ADT ±abiraterone - EBRT + brachytherapy + ADT - Radical prostatectomy (select cases) Regional (N1) Any T, N1, M0 Any Any - EBRT + long-term ADT - ADT alone (non-curative setting) - Consider abiraterone + ADT Metastatic (M1) Any T, any N, M1 Any Any - ADT + novel hormonal therapy (abiraterone, enzalutamide, and apalutamide) -±Docetaxel (in high-volume disease) - Bone-protecting agents (zoledronic acid or denosumab) - Palliative radiotherapy-Lu-177 PSMA, PARP inhibitors (selected mCRPC) GS: Gleason score; EBRT: external beam radiation therapy; ADT: androgen deprivation therapy; LN: lymph node; and mCRPC: metastatic castration-resistant prostate cancer. It is important to emphasise that in the era of personalised oncology therapy, the introduction of precise diagnostic technologies, along with the increasing availability of safe and effective localised nonor minimally invasive treatment options, has led to a rise in the incidence of and clinical interest in oligometastatic prostate cancer. The definition of oligometastatic prostate cancer is somewhat inconsistent; it ranges from the presence of a single metastasis to between three and five metastases. However, most prospective studies use the definition of three or fewer metastatic lesions with predefined locations [ 10 ]. Moreover, the treatment approach for these patients is increasingly shifting towards a more-aggressive strategy. Numerous retrospective studies have demonstrated that in the metastatic stage of the disease, procedures such as radical prostatectomy and the local radiotherapy of metastases can be performed with minimal risk of toxicity, and may improve therapeutic outcomes. The management of oligometastatic prostate cancer, characterised by a limited number of metastatic sites, remains challenging, with cytoreductive surgery and radiotherapy being the main treatment options. Recent research suggests that cytoreductive surgery may offer better cancer-specific and overall survival compared to radiotherapy, although progressionfree survival appears to be similar between the two. Both treatments have manageable side effects, supporting their use in clinical practice. These findings highlight the importance of personalised treatment decisions and the need for further studies to refine therapy for this patient group [11]. Radiotherapy, whether used alone or in conjunction with systemic therapy and surgery, plays a crucial role in the management of prostate cancer, reducing the risk of local recurrence and improving overall survival. It is utilised in the treatment of nearly 50% of prostate cancer patients, and approximately 40% of long-term cancer survivors have received radiotherapy at some stage of their treatment journey [ 12 , 13 ]. Depending on the treatment goal, radiation therapy can be classified as radical, postoperative (adjuvant or salvage), or palliative. In terms of its delivery, radiation therapy for prostate cancer can be administered as external beam radiation therapy (EBRT) or as brachytherapy (BT). In radical treatment, Life 2025,15, 1062 4 of 21 radiotherapy is delivered in one, two, or three phases depending on the target volume: prostate only (one phase), prostate and seminal vesicles (two phases), or prostate, seminal vesicles, and regional lymph nodes (three phases). Conventional fractionation (1.8–2 Gy per fraction) is used, with total doses ranging from 72 up to 80 Gy, based on institutional protocols. In postoperative settings, radiotherapy is usually delivered in a single phase with 64–66 Gy to the prostate bed or up to 70 Gy for macroscopic disease. If prophylactic nodal irradiation is indicated, treatment may be delivered in two phases [ 6 , 8 , 9 ]. External beam radiation therapy is currently one of the most-effective treatments for localised prostate cancer, being used in almost one-third of men with this diagnosis. When planning radiotherapy, the risk of radiation-induced damage to nearby healthy tissues determines the dose limits. The aim is to deliver the highest possible dose to the tumour while minimising exposure to surrounding normal tissue. Modern, highly precise techniques like intensitymodulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) are considered the gold standard for treating patients with prostate cancer. These advanced radiotherapy techniques enable the precise delivery of high radiation doses, up to 80 Gy, while minimising exposure to surrounding healthy tissue (Figures 1and 2) [14–16]. Figure 1. Radical prostate cancer radiotherapy using the VMAT technique: the clinical target volume (CTV), marked in red, was delineated using co-registered magnetic resonance imaging (MRI), ensuring precise mapping and accurate treatment delivery (material from the Institute for Oncology and Radiology of Serbia). Although surrounding organs at risk, especially the bladder and rectum, are better protected during radiation therapy, some radiation still affects healthy tissue. In prostate cancer radiotherapy, the most clinically significant toxicities are those that lead to treatment interruptions, thereby prolonging the overall course of therapy and adversely affecting patients’ quality of life. Gastrointestinal (GI) toxicity arises from damage to the rectum and bowel, while genitourinary (GU) toxicity results from injury to the urethra, bladder, or prostate. Gastrointestinal symptoms can range from mild issues such as increased bowel frequency to more-severe complications like rectal bleeding, anal pain, or fistula formation. Genitourinary toxicity may present as frequent urination, haematuria, dysuria, urinary incontinence, or urethral stricture [6–8]. Life 2025,15, 1062 5 of 21 Figure 2. The medical linear accelerator (LINAC) for IMRT and VMAT radiotherapy (material from the Institute for Oncology and Radiology of Serbia). Consequently, 5–10% of patients may experience severe side effects, resulting in complications such as fibrosis, necrosis, atrophy, and vascular damage. Additionally, among the serious long-term adverse effects of radiotherapy are secondary malignancies. Although secondary cancers are uncommon after radiotherapy for pelvic malignancies such as prostate cancer, they still represent a potentially serious late adverse effect, even in the era of advanced radiotherapy techniques [ 17 , 18 ]. Recent studies on radiation toxicity have identified multiple factors that may influence the risk of complications following radiotherapy. These include the specific radiotherapy technique used, patient age, smoking and alcohol consumption habits, and clinical parameters, such as disease stage, a history of transurethral resection of the prostate, prior abdominal or pelvic surgeries, and the presence of comorbidities [ 19 – 25 ]. Despite an improved understanding of these contributing factors, individual radiosensitivity remains unpredictable. Current radiotherapy protocols do not account for this variability, highlighting the need for reliable biomarkers to assess individual radiosensitivity prior to treatment [18]. 3. Individual Radiosensitivity: The Role of Biomarkers and Predictive Assays in Personalising Radiotherapy for Prostate Cancer Patients In the era of personalised medicine, one of the main challenges in radiation oncology is the discovery of biomarkers that reflect individual sensitivity to radiation. Since it is estimated that 80% of individual differences in radiotherapy toxicity to normal tissues are attributed to a combination of genetic and epigenetic factors, there is an increasing need for a specific test that can accurately reflect individual radiosensitivity and predict the likelihood of radiotherapy-related toxicity [ 26 ]. These biomarkers are essential for determining radiosensitivity prior to treatment and enabling individualised risk assessment, which could in turn help optimise treatment planning. For example, it would allow for higher tumour doses in patients with radiation-resistant normal tissue, while those at greater risk of severe radiation toxicity could be considered for alternative approaches. These Life 2025,15, 1062 6 of 21 may include modifying the radiotherapy regimen (e.g., alternative fractionation schemes), switching to a different treatment modality (such as surgery instead of radiotherapy), or introducing pharmacological therapy early to manage symptoms [27,28]. Cellular damage caused by ionising radiation triggers the activation of proteins involved in DNA repair, cell death, inflammation, and other pathophysiologic responses, which contribute to the development of radiotherapy side effects [ 29 ]. These effects are classified as acute, subacute, or late: acute effects appear within 1–2 weeks of treatment due to inflammation or the loss of rapidly dividing cells, while late effects, such as fibrosis, vascular injury, or even secondary cancers, may appear months or years later as a result of slower tissue responses and residual DNA damage [28,29]. Potential biomarkers are measurable biological indicators that reflect normal physiological processes, pathological changes, or responses to environmental or therapeutic exposures [ 29 ]. In the context of radiotherapy, biomarkers often detected in blood or saliva can reveal molecular events triggered by ionising radiation, such as DNA damage, inflammation, or cell death pathways. These biological factors are valuable for understanding individual radiosensitivity, optimising treatment plans, and minimising toxicity. Predictive assays are functional tests, often performed in vitro , that evaluate how a patient’s cells respond to ionising radiation. These assays commonly assess endpoints such as clonogenic survival, DNA damage and repair capacity, chromosomal aberrations, or radiation-induced apoptosis. Their primary objective is to predict the risk of normal-tissue toxicity and aid in the personalisation of radiotherapy. By assessing the dynamic cellular response to radiation, predictive assays offer direct insight into an individual’s susceptibility to radiation-induced tissue damage [27–29]. 3.1. The Role of Cytokines as Potential Biomarkers for Radiation Toxicity Cytokines play a central role in the molecular response to ionising radiation, acting as key mediators of inflammation and tissue damage. Inflammatory processes are increasingly recognised as key modulators of malignant tumour responses to radiotherapy [ 30 ]. These immunoregulatory proteins, including interferons (IFNs), interleukins (ILs), and growth factors, facilitate communication between cells and can trigger autocrine, paracrine, or endocrine responses [ 30 ]. Radiation-induced tissue injury leads to changes in cytokine expression, which closely correlate with both acute and late toxicities through the activation of inflammatory signalling pathways. Circulating cytokine levels therefore reflect the extent of cellular and tissue damage caused by radiation. Because of this, cytokines are increasingly recognised as valuable biomarkers for evaluating normal surrounding tissue reactions to radiotherapy. The interplay between proand anti-inflammatory cytokines plays a key role in shaping therapeutic response, influencing the severity of radiation-induced side effects and the potential for tumour resistance [ 31 ]. In prostate cancer, in particular, altered cytokine profiles have been linked to radiation-induced toxicity, highlighting their potential for guiding personalised treatment approaches [30,31]. Early studies were the first to highlight the role of cytokines in radiation-induced toxicity, showing in preclinical and clinical lung models that levels of IL-1, transforming growth factor (TGF)- β , and tumour necrosis factor (TNF)- α increased immediately after radiation exposure, while persistently elevated TGFβ was associated with a higher risk of pulmonary fibrosis [ 32 ]. Previous studies have reported elevated circulating levels of proinflammatory cytokines, including IFNγ , IL-6, TNFα , and IL-4, during the course of radiotherapy [ 32 ]. Furthermore, in patients with prostate cancer, radiotherapy has been shown to induce an inflammatory response, characterised by increased serum concentrations of IL-6, IL-8, TNFα , and TGFβ [ 33 ]. The study conducted by Christensen et al. on a cohort of 42 patients with prostate cancer treated with IMRT [ 30 ] demonstrated a significant Life 2025,15, 1062 7 of 21 rise in serum levels of IFNγ and IL-6 during radiotherapy in prostate cancer patients. Additionally, elevated levels of IL-1 and IL-2 were linked to an increased risk of developing genitourinary and gastrointestinal radiotoxicity. Their observations suggest that IL-6, IFNγ , IL-1, and IL-2 may serve as key cytokine markers associated with radiation responses, particularly in the development of acute gastrointestinal and genitourinary toxicity. It has also been shown that there are differences in cytokine levels between patients with prostate cancer and those with benign prostate lesions or healthy individuals, as well as changes in cytokine levels following radiotherapy and/or androgen therapy [ 34 , 35 ]. These findings provide a foundation for future prospective radiotherapy trials aimed at validating the predictive value of these cytokines, with the potential to inform and personalise patient management during treatment. Changes in proinflammatory cytokine levels have been linked not only to radiationinduced toxicity but also to the tumour response to radiotherapy. More-aggressive tumour behaviour and treatment resistance have been linked to elevated IL-6 levels. Targeting IL-6 therapeutically may improve treatment results and increase tumour sensitivity to radiation [ 36 ]. Patients receiving radiation therapy for many different types of cancers have been found to have a higher proportion of regulatory T cells (Tregs), and this increase is associated with a poor response to treatment [ 37 ]. However, the complex role of proinflammatory cytokines in radiation therapy-induced toxicity has not yet been fully understood. In a study by Stanojkovic et al. [ 38 ], the evaluation of potential cytokine signatures in prostate cancer patients revealed significantly elevated circulating levels of IL-6 and IFNγ in those receiving definitive radiotherapy compared to patients treated postoperatively. This difference may reflect the higher radiation dose used in the definitive group (72 Gy vs. 66 Gy in the postoperative and salvage groups, respectively), variations in irradiated volumes, or factors such as the presence of the prostate and its response to irradiation. In a cohort of 44 patients evaluated at six time points, both univariate and multivariate analyses, adjusted for time point and treatment type, demonstrated a significant association between increased IL-6 levels during radiotherapy and higher grades of acute genitourinary toxicity. The findings also indicate that temporal changes in cytokine levels during treatment, rather than their absolute values, may serve as more-reliable indicators of radiotoxicity. A recent study on prostate cancer patients undergoing radiotherapy found a significant association between diabetes mellitus and increased acute genitourinary toxicity [ 39 ]. In addition, prostate cancer patients who were smokers reported higher maximum fatigue levels than patients who were non-smokers [ 39 ]. Interleukin-6 levels rose significantly after the 25th fraction of radiotherapy, and both IL-6 and TGFβ 1 levels, measured before radiotherapy and after the 25th radiotherapy fraction, were positively correlated with genitourinary toxicity grades. Additionally, IL-6 and TGFβ 1 concentrations after the 25th fraction were associated with fatigue scores [ 39 ]. It has also been shown that previous surgical interventions may contribute to the development of radiation toxicity at sites distant from the surgical incision, primarily through the action of TGFβ 1 [ 38 ]. Although all patients had the same prostate cancer diagnosis, differences in comorbidities and lifestyle factors likely influenced their cytokine profiles. A study by Singh et al. on a cohort of 18 patients with prostate cancer treated with IMRT demonstrated an increase in IL-6 and TNFα concentrations measured at the end of and 3 months after radiotherapy with an increase in the grade of acute genitourinary and gastrointestinal radiotoxicity, while TGFβ concentrations decreased with an increase in the grade of acute genitourinary and gastrointestinal radiotoxicity [40]. Many studies identified IL-6 as one of the most important cytokines for predicting normal-tissue radiotoxicity effects, not only in prostate cancer but also across various other malignancies treated with radiotherapy. Integrating radiotherapy-related factors, clinical Life 2025,15, 1062 8 of 21 indicators, individual patient features, and circulating cytokine profiles, notably IL-6 and TGFβ 1, into machine-learning-based predictive models may be beneficial for anticipating unfavourable normal tissue responses to radiation in prostate cancer patients [ 38 , 39 , 41 ]. The radiation-induced cytokine profiles are specific for each cancer patient since many individual, biological, clinical, and treatment-related factors may create this pattern [ 31 ]. The prospective role of cytokines in the prediction of normal-tissue reactions to radiotherapy for prostate cancer needs to be further investigated on larger homogenous cohorts of patients with prostate cancer, treated with the same type of radiation therapy, with the same radiation dose schedules, dose-volume groups, and clinicopathological features, and using the same criteria for the assessment of acute and late radiation toxicity. The roles of selected cytokines and their associations with radiotherapy-induced toxicity in prostate cancer patients are summarised in Table 2. Table 2. Summary of key cytokines involved in radiotherapy-induced toxicity in prostate cancer. Cytokine Role Association with Adverse Radiotherapy Effects TGF-β1 (Transforming Growth Factor Beta 1) Modulates immune responses and fibrosis Elevated TGF-β1 levels after radiotherapy can contribute to fibrosis and toxicity in prostate cancer patients [33,39] TNF-α(Tumour Necrosis Factor Alpha) Proinflammatory cytokine, regulates apoptosis High TNF-αlevels after radiotherapy may be linked to increased inflammation and risk of late toxicities [33] IL-6 (Interleukin-6) Mediates inflammatory responses and tissue repair Elevated IL-6 can be a marker of poor prognoses, contributing to inflammation and fibrosis post-RT [30,36] IL-1β(Interleukin-1 Beta) Promotes inflammation and tissue damage Associated with radiation-induced fibrosis and increased late toxicities in prostate cancer radiotherapy [39] IFN-γ(Interferon Gamma) Enhances immune response and regulates apoptosis Higher IFN-γlevels may correlate with increased lymphocyte apoptosis and lower late toxicity risk [30,38] IL-2 (Interleukin-2) Stimulates T-cell activation and proliferation Plays a key role in immune recovery after radiotherapy; altered levels may influence long-term toxicities [30] IL-8 (Interleukin-8) Chemotactic cytokine that attracts neutrophils Elevated IL-8 levels after radiotherapy may contribute to inflammation and exacerbate toxicities [33] Life 2025,15, 1062 9 of 21 3.2. The RILA Assay: A Tool for Stratifying Patients by Radiosensitivity to Anticipate Radiation-Induced Toxicity Various functional assays can be used to assess the radiosensitivity of patients’ cells, such as lymphocytes or fibroblasts, in order to predict the risk of radiation-induced damage to surrounding healthy tissue. It has been shown that the response of patients’ lymphocytes to radiation determined by functional tests is related to the radiosensitivity of normal tissue [ 27 ]. Local radiotherapy has a direct cytotoxic effect on circulating lymphocytes as blood flows through the radiation field. After exposure to ionising radiation, lymphocytes die by apoptosis. However, different subpopulations of lymphocytes, such as CD4 + T lymphocytes, CD8 + T lymphocytes, B lymphocytes, and NK cells, respond differently to radiation. Studies on lymphocyte depletion in patients consistently indicate that B cells are the most radiosensitive, followed by T cells and NK cells. Among T cells, CD8 + T cells exhibit greater radiosensitivity compared to CD4 + T cells [ 42 ]. Numerous studies have shown that the level of apoptosis induced by the ex vivo irradiation of patients’ peripheral blood lymphocytes before radiotherapy is associated with the risk of radiation-induced damage to normal tissues [ 43 , 44 ]. Specifically, there is a negative correlation between radiation-induced apoptosis of T lymphocytes and the development of late toxicity; patients with lower levels of T cell apoptosis tend to experience more-severe side effects compared to those with higher levels of apoptosis [26,43,44]. A key advancement in this area came in 1995, when researchers in Switzerland, led by Prof. Ozsahin and Prof. Crompton, developed a rapid assay to assess intrinsic radiosensitivity by measuring the radiation-induced apoptosis of CD4 + and CD8 + T lymphocytes. This assay evaluated the quantity of peripheral blood lymphocytes dying by apoptosis following exposure to ionising radiation at a dose of 8 Gy [ 43 ]. Then, in 2005, came the first prospective study using the radiation-induced lymphocyte apoptosis (RILA) assay and involved 399 patients with various cancers (primarily breast, head and neck, genitourinary, and gastrointestinal) who underwent curative radiotherapy [ 44 ]. T-lymphocyte apoptosis was assessed before treatment, and patients were followed for acute and late toxicity. After 30 months, no correlation was found between radiation-induced apoptosis in T-lymphocytes and early toxicity or survival. Their findings also showed that CD8 + T lymphocytes underwent higher levels of apoptosis compared to CD4 + T cells following radiation exposure. Importantly, they identified a strong inverse correlation between the RILA value and the occurrence of late radiation toxicity: patients with higher RILA scores, indicating greater apoptosis, experienced fewer grade 2 and 3 late toxicities, while those with lower apoptosis levels were more likely to suffer from severe side effects. Overall, RILA values > 16% were significantly associated with a very low risk of grade ≥ 2 late toxicity. Conversely, RILA values below 10% were strongly associated with severe late complications. This assay highlighted the potential of T lymphocyte-based tests as predictive tools for individual normal-tissue radiosensitivity [ 44 ]. This was further confirmed in the phase II multicenter CO-HO-RT trial, which included 150 breast cancer patients. The high RILA scores were again linked to a lower incidence of grade 2 or higher toxicities [ 45 ]. The clinical potential of RILA for the prediction of the risk of radiation-induced breast fibrosis was confirmed in a multicenter French trial involving over 500 breast cancer patients [46]. Foro et al. [ 47 ] confirmed a significant correlation between the radiation-induced apoptosis of CD4 + T lymphocytes and late genitourinary toxicity in 214 prostate cancer patients treated with radiotherapy. A higher RILA score was notably associated with a lower risk of late toxicity. As a result, research that addressed the issue of the prevalence and frequency of radiation toxicity in patients with prostate cancer discovered that the radiation-induced apoptosis of T cells can be utilised to predict gastrointestinal or genitourinary toxicity. The study conducted on a cohort of 50 prostate cancer patients treated with 3D conformal Life 2025,15, 1062 16 of 21 The added value of this framework lies in the biological complementarity of its components: germline genetic variants (SNPs), intrinsic cellular radiosensitivity (RILA), and treatment-induced inflammation (cytokines). Future studies should focus on prospectively validating such integrative models and incorporating advanced predictive models (e.g., machine learning) to refine prediction accuracy. Establishing standardised thresholds and harmonising assay methodologies across centres will also be crucial for successful clinical translation in addition to reporting unique radiotoxicity effects. Each of the proposed potential biological predictors of normal-tissue radiosensitivity could be rapidly evaluated using blood samples: patient-derived lymphocytes or serum and plasma, as a starting biological sample. SNP genotyping, flow cytometry-based RILA, and the determination of cytokine levels by commercially available and reliable ELISA tests are low-cost, cost-effective, and can be easily incorporated into clinical settings after robust research studies and clinical validation. 5. Conclusions Advances in the treatment of localised prostate cancer have improved survival, shifting the focus to minimising long-term side effects that can significantly impact quality of life. Despite the use of modern radiotherapy techniques, some patients continue to experience severe toxicities, highlighting the need for predictive biomarkers to guide personalised care. Current protocols do not take into account individual differences in radiosensitivity. Indeed, a primary goal of translational research in radiotherapy is the identification and validation of biomarkers that can be used to assess the risk of acute and late toxicities, as well as to predict an individual’s sensitivity to radiation. Integrating individual, clinical, and biological characteristics that are unique to each patient into treatment planning ultimately allows clinicians to tailor radiotherapy regimens, thereby balancing the risks and benefits for each patient. The RILA assay has emerged as a valuable, non-invasive tool for predicting individual radiosensitivity. When integrated into clinical models along with other patient-specific factors, RILA helps tailor treatment strategies that support dose escalation in patients at low risk of toxicity and guide alternative approaches for those at higher risk. Cytokines, such as IL-6, TNFα , and TGFβ , are also gaining attention as biomarkers of inflammation and radiation-induced tissue damage. Together, these biomarkers offer promising avenues for optimising treatment plans, reducing complications, and improving outcomes. Genetic variations, such as SNPs, likely play a significant role in individual differences in normal-tissue sensitivity to radiation, emphasising the need for personalised approaches in radiotherapy. MicroRNAs significantly influence prostate cancer radiotherapy by modulating radiosensitivity, resistance, and toxicity. Their stability and specific expression patterns make them valuable non-invasive biomarkers for predicting treatment outcomes and side effects. Ongoing research will further define their role in advancing personalised radiotherapy. Developing machine learning models to predict both acute and late normal-tissue reactions to radiotherapy in patients with various cancers is a vital step toward personalised radiotherapy. Such predictive models should integrate multiple biological factors representing different mechanisms of radiosensitivity: individual patient characteristics, clinical and treatment-related factors, RILA assay results, and a combination of potential biomarkers, such as SNPs in specific genes, gene or miRNA expression profiles, and circulating cytokine patterns. By combining these data, patients can be stratified into subgroups with varying risks of adverse effects, allowing radiotherapy to be tailored and optimised for each subgroup. Life 2025,15, 1062 17 of 21 Author Contributions: Conceptualization and drafting: J.S.; writing and editing: I.Š.; critical review and editing: L.J.; drafting and editing: I.Z.M.; discussion and manuscript revision: P.N.; critical editing, drafting, and revising: M.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Ethical review and approval were waived for this study since it is a systematic review of published literature. Informed Consent Statement: Patient consent was waived because no patients or members of the public were involved in the design, conduct of this study, or reporting of this research. Data Availability Statement: All data generated as part of this study are included in the article. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Siegel, R.L.; Giaquinto, A.N.; Jemal, A. Cancer Statistics, 2024. CA A Cancer J. Clin. 2024,74, 12–49. [CrossRef] [PubMed] 2. National Cancer Institute. Cancer Stat Facts: Prostate Cancer. Available online: https://seer.cancer.gov/statfacts/html/prost.html (accessed on 27 June 2024). 3. GLOBOCAN. Population Fact Sheet. Chrome Extension. 2020. Available online: https://backlinko.com/chrome-users (accessed on 29 June 2025). 4. Batut, Institute of Public Health of Serbia. 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