Combining a noble gas with radiotherapy: glutamate receptor antagonist xenon may act as a radiosensitizer in glioblastoma.

BACKGROUND: Ionotropic glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as a radiosensitizer in GB. METHODS: We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine. RESULTS: The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Indications for the presence of glutamate receptor subunits were present in glioblastoma-derived and HeLa cells. Significantly reduced clonogenic survival by xenon was shown in U87 and U251 at irradiation doses of 4-8 Gy and 2, 6 and 8 Gy, respectively (p < 0.05). Clonogenic survival was further reduced by the addition of memantine, showing a significant effect at 2-8 Gy for both glioblastoma cell lines (p < 0.05). Xenon did not significantly reduce the surviving fraction of HeLa cells until a radiation dose of 8 Gy. CONCLUSION: The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled indications of radiosensitizing properties of xenon gas in glioblastoma.

Large-scale meta-genome-wide association study reveals common genetic factors linked to radiation-induced acute toxicities across cancer types.

BACKGROUND: This study was designed to identify common genetic susceptibility and shared genetic variants associated with acute radiation-induced toxicity across 4 cancer types (prostate, head and neck, breast, and lung). METHODS: A genome-wide association study meta-analysis was performed using 19 cohorts totaling 12 042 patients. Acute standardized total average toxicity (STATacute) was modelled using a generalized linear regression model for additive effect of genetic variants, adjusted for demographic and clinical covariates (rSTATacute). Linkage disequilibrium score regression estimated shared single-nucleotide variation (SNV-formerly SNP)-based heritability of rSTATacute in all patients and for each cancer type. RESULTS: Shared SNV-based heritability of STATacute among all cancer types was estimated at 10% (SE = 0.02) and was higher for prostate (17%, SE = 0.07), head and neck (27%, SE = 0.09), and breast (16%, SE = 0.09) cancers. We identified 130 suggestive associated SNVs with rSTATacute (5.0 × 10‒8 < P < 1.0 × 10‒5) across 25 genomic regions. rs142667902 showed the strongest association (effect allele A; effect size ‒0.17; P = 1.7 × 10‒7), which is located near DPPA4, encoding a protein involved in pluripotency in stem cells, which are essential for repair of radiation-induced tissue injury. Gene-set enrichment analysis identified 'RNA splicing via endonucleolytic cleavage and ligation' (P = 5.1 × 10‒6, P = .079 corrected) as the top gene set associated with rSTATacute among all patients. In silico gene expression analysis showed that the genes associated with rSTATacute were statistically significantly up-regulated in skin (not sun exposed P = .004 corrected; sun exposed P = .026 corrected). CONCLUSIONS: There is shared SNV-based heritability for acute radiation-induced toxicity across and within individual cancer sites. Future meta-genome-wide association studies among large radiation therapy patient cohorts are worthwhile to identify the common causal variants for acute radiotoxicity across cancer types.

The correlation between pre-treatment symptoms, acute and late toxicity and patient-reported health-related quality of life in non-small cell lung cancer patients: Results of the REQUITE study.

BACKGROUND AND PURPOSE: To investigate the association between clinician-scored toxicities and patient-reported health-related quality of life (HRQoL), in early-stage (ES-) and locally-advanced (LA-) non-small cell lung cancer (NSCLC) patients receiving loco-regional radiotherapy, included in the international real-world REQUITE study. MATERIALS AND METHODS: Clinicians scored eleven radiotherapy-related toxicities (and baseline symptoms) with the Common Terminology Criteria for Adverse Events version 4. HRQoL was assessed with the European Organization for Research and Treatment of Cancer core HRQoL questionnaire (EORTC-QLQ-C30). Statistical analyses used the mixed-model method; statistical significance was set at p = 0.01. Analyses were performed for baseline and subsequent time points up to 2 years after radiotherapy and per treatment modality, radiotherapy technique and disease stage. RESULTS: Data of 435 patients were analysed. Pre-treatment, overall symptoms, dyspnea, chest wall pain, dysphagia and cough impacted overall HRQoL and specific domains. At subsequent time points, cough and dysphagia were overtaken by pericarditis in affecting HRQoL. Toxicities during concurrent chemo-radiotherapy and 3-dimensional radiotherapy had the most impact on HRQoL. Conversely, toxicities in sequential chemo-radiotherapy and SBRT had limited impact on patients' HRQoL. Stage impacts the correlations: LA-NSCLC patients are more adversely affected by toxicity than ES-NSCLC patients, mimicking the results of radiotherapy technique and treatment modality. CONCLUSION: Pre-treatment symptoms and acute/late toxicities variously impact HRQoL of ES- and LA-NSCLC patients undergoing different treatment approaches and radiotherapy techniques. Throughout the disease, dyspnea seems crucial in this association, highlighting the additional effect of co-existing comorbidities. Our data call for optimized radiotherapy limiting toxicities that may affect patients' HRQoL.

Targeting the DNA Damage Response and DNA Repair Pathways to Enhance Radiosensitivity in Colorectal Cancer.

Radiotherapy is an important component of current treatment options for colorectal cancer (CRC). It is either applied as neoadjuvant radiotherapy to improve local disease control in rectal cancers or for the treatment of localized metastatic lesions of CRC. DNA double-strand breaks (DSBs) are the major critical lesions contributing to ionizing radiation (IR)-induced cell death. However, CRC stem cells promote radioresistance and tumor cell survival through activating cell-cycle checkpoints to trigger the DNA damage response (DDR) and DNA repair after exposure to IR. A promising strategy to overcome radioresistance is to target the DDR and DNA repair pathways with drugs that inhibit activated cell-cycle checkpoint proteins, thereby improving the sensitivity of CRC cells to radiotherapy. In this review, we focus on the preclinical studies and advances in clinical trials of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related kinase (ATR), checkpoint kinase 1 (CHK1), checkpoint kinase 2 (CHK2), WEE1 and poly (ADP-ribose) polymerase 1 (PARP1) kinase inhibitors in CRC. Importantly, we also discuss the selective radiosensitization of CRC cells provided by synthetic lethality of these inhibitors and the potential for widening the therapeutic window by targeting the DDR and DNA repair pathways in combination with radiotherapy and immunotherapy.

Development and Optimization of a Machine-Learning Prediction Model for Acute Desquamation After Breast Radiation Therapy in the Multicenter REQUITE Cohort.

Purpose: Some patients with breast cancer treated by surgery and radiation therapy experience clinically significant toxicity, which may adversely affect cosmesis and quality of life. There is a paucity of validated clinical prediction models for radiation toxicity. We used machine learning (ML) algorithms to develop and optimise a clinical prediction model for acute breast desquamation after whole breast external beam radiation therapy in the prospective multicenter REQUITE cohort study. Methods and Materials: Using demographic and treatment-related features (m = 122) from patients (n = 2058) at 26 centers, we trained 8 ML algorithms with 10-fold cross-validation in a 50:50 random-split data set with class stratification to predict acute breast desquamation. Based on performance in the validation data set, the logistic model tree, random forest, and naïve Bayes models were taken forward to cost-sensitive learning optimisation. Results: One hundred and ninety-two patients experienced acute desquamation. Resampling and cost-sensitive learning optimisation facilitated an improvement in classification performance. Based on maximising sensitivity (true positives), the "hero" model was the cost-sensitive random forest algorithm with a false-negative: false-positive misclassification penalty of 90:1 containing m = 114 predictive features. Model sensitivity and specificity were 0.77 and 0.66, respectively, with an area under the curve of 0.77 in the validation cohort. Conclusions: ML algorithms with resampling and cost-sensitive learning generated clinically valid prediction models for acute desquamation using patient demographic and treatment features. Further external validation and inclusion of genomic markers in ML prediction models are worthwhile, to identify patients at increased risk of toxicity who may benefit from supportive intervention or even a change in treatment plan.

Overview of health-related quality of life and toxicity of non-small cell lung cancer patients receiving curative-intent radiotherapy in a real-life setting (the REQUITE study).

OBJECTIVES: Radiotherapy-induced toxicity may negatively impact health-related quality of life (HRQoL). This report investigates the impact of curative-intent radiotherapy on HRQoL and toxicity in early stage and locally-advanced non-small cell lung cancer patients treated with radiotherapy or chemo-radiotherapy enrolled in the observational prospective REQUITE study. MATERIALS AND METHODS: HRQoL was assessed using the European Organisation for Research and Treatment of Cancer QLQ-C30 questionnaire up to 2 years post radiotherapy. Eleven toxicities were scored by clinicians using the Common Terminology Criteria for Adverse Events (CTCAE) version 4. Toxicity scores were calculated by subtracting baseline values. Mixed model analyses were applied to determine statistical significance (p ≤ 0.01). Meaningful clinical important differences (MCID) were determined for changes in HRQoL. Analysis was performed on the overall data, different radiotherapy techniques, multimodality treatments and disease stages. RESULTS: Data of 510 patients were analysed. There was no significant change in HRQoL or its domains, except for deterioration in cognitive functioning (p = 0.01). Radiotherapy technique had no significant impact on HRQoL. The addition of chemotherapy was significantly associated with HRQoL over time (p <.001). Overall toxicity did not significantly change over time. Acute toxicities of radiation-dermatitis (p =.003), dysphagia (p =.002) and esophagitis (p <.001) peaked at 3 months and decreased thereafter. Pneumonitis initially deteriorated but improved significantly after 12 months (p =.011). A proportion of patients experienced meaningful clinically important improvements and deteriorations in overall HRQoL and its domains. In some patients, pre-treatment symptoms improved gradually. CONCLUSIONS: While overall HRQoL and toxicity did not change over time, some patients improved, whereas others experienced acute radiotherapy-induced toxicities and deteriorated HRQoL, especially physical and cognitive functioning. Patient characteristics, more so than radiotherapy technique and treatment modality, impact post-radiotherapy toxicity and HRQoL outcomes. This stresses the importance of considering the potential impact of radiotherapy on individuals' HRQoL, symptoms and toxicity in treatment decision-making.

Selective inhibitors of bromodomain BD1 and BD2 of BET proteins modulate radiation-induced profibrotic fibroblast responses.

Radiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation-induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan-BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I-BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan-inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET-BD1 [GSK778] and iBET-BD2 [GSK046]). Here, their potential to attenuate radiation-induced fibroblast activation with low-toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I-BET151 and iBET-BD1, but not with iBET-BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I-BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET-BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor-treatment. In conclusion, iBET-BD2 efficiently suppressed radiation-induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2-selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy-induced fibrosis.

Targeting Cell Cycle Checkpoint Kinases to Overcome Intrinsic Radioresistance in Brain Tumor Cells.

Radiation therapy is an important part of the standard of care treatment of brain tumors. However, the efficacy of radiation therapy is limited by the radioresistance of tumor cells, a phenomenon held responsible for the dismal prognosis of the most aggressive brain tumor types. A promising approach to radiosensitization of tumors is the inhibition of cell cycle checkpoint control responsible for cell cycle progression and the maintenance of genomic integrity. Inhibition of the kinases involved in these control mechanisms can abolish cell cycle checkpoints and DNA damage repair and thus increase the sensitivity of tumor cells to radiation and chemotherapy. Here, we discuss preclinical progress in molecular targeting of ATM, ATR, CHK1, CHK2, and WEE1, checkpoint kinases in the treatment of brain tumors, and review current clinical phase I-II trials.

Predicting response to neoadjuvant chemoradiotherapy in rectal cancer: from biomarkers to tumor models.

Colorectal cancer (CRC) is a major contributor to cancer-associated morbidity worldwide and over one-third of CRC is located in the rectum. Neoadjuvant chemoradiotherapy (nCRT) followed by surgical resection is commonly applied to treat locally advanced rectal cancer (LARC). In this review, we summarize current and novel concepts of neoadjuvant therapy for LARC such as total neoadjuvant therapy and describe how these developments impact treatment response. Moreover, as response to nCRT is highly divergent in rectal cancers, we discuss the role of potential predictive biomarkers. We review recent advances in biomarker discovery, from a clinical as well as a histopathological and molecular perspective. Furthermore, the role of emerging predictive biomarkers derived from the tumor environment such as immune cell composition and gut microbiome is presented. Finally, we describe how different tumor models such as patient-derived cancer organoids are used to identify novel predictive biomarkers for chemoradiotherapy (CRT) in rectal cancer.

Use of angiotensin converting enzyme inhibitors is associated with reduced risk of late bladder toxicity following radiotherapy for prostate cancer.

BACKGROUND AND PURPOSE: Genome-wide association studies (GWAS) of late hematuria following prostate cancer radiotherapy identified single nucleotide polymorphisms (SNPs) near AGT, encoding angiotensinogen. We tested the hypothesis that patients taking angiotensin converting enzyme inhibitors (ACEi) have a reduced risk of late hematuria. We additionally tested genetically-defined hypertension. MATERIALS AND METHODS: Prostate cancer patients undergoing potentially-curative radiotherapy were enrolled onto two multi-center observational studies, URWCI (N = 256) and REQUITE (N = 1,437). Patients were assessed pre-radiotherapy and followed prospectively for development of toxicity for up to four years. The cumulative probability of hematuria was estimated by the Kaplan-Meier method. Multivariable grouped relative risk models assessed the effect of ACEi on time to hematuria adjusting for clinical factors and stratified by enrollment site. A polygenic risk score (PRS) for blood pressure was tested for association with hematuria in REQUITE and our Radiogenomics Consortium GWAS. RESULTS: Patients taking ACEi during radiotherapy had a reduced risk of hematuria (HR 0.51, 95%CI 0.28 to 0.94, p = 0.030) after adjusting for prior transurethral prostate and/or bladder resection, heart disease, pelvic node radiotherapy, and bladder volume receiving 70 Gy, which are associated with hematuria. A blood pressure PRS was associated with hypertension (odds ratio per standard deviation 1.38, 95%CI 1.31 to 1.46, n = 5,288, p < 0.001) but not hematuria (HR per standard deviation 0.96, 95%CI 0.87 to 1.06, n = 5,126, p = 0.41). CONCLUSIONS: Our study is the first to show a radioprotective effect of ACEi on bladder in an international, multi-site study of patients receiving pelvic radiotherapy. Mechanistic studies are needed to understand how targeting the angiotensin pathway protects the bladder.

Intracellular Delivery of Doxorubicin by Iron Oxide-Based Nano-Constructs Increases Clonogenic Inactivation of Ionizing Radiation in HeLa Cells.

In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONPCO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONPCO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONPCO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMFSF0.1 = 1.13 ± 0.05 (p = 0.01)). IONPDOX did enhance the cytotoxicity of 6 MV X-rays (DMFSF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONPCO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONPDOX induced radiosensitization compared to IONPCO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

Development of a method for generating SNP interaction-aware polygenic risk scores for radiotherapy toxicity.

AIM: To identify the effect of single nucleotide polymorphism (SNP) interactions on the risk of toxicity following radiotherapy (RT) for prostate cancer (PCa) and propose a new method for polygenic risk score incorporating SNP-SNP interactions (PRSi). MATERIALS AND METHODS: Analysis included the REQUITE PCa cohort that received external beam RT and was followed for 2 years. Late toxicity endpoints were: rectal bleeding, urinary frequency, haematuria, nocturia, decreased urinary stream. Among 43 literature-identified SNPs, the 30% most strongly associated with each toxicity were tested. SNP-SNP combinations (named SNP-allele sets) seen in ≥10% of the cohort were condensed into risk (RS) and protection (PS) scores, respectively indicating increased or decreased toxicity risk. Performance of RS and PS was evaluated by logistic regression. RS and PS were then combined into a single PRSi evaluated by area under the receiver operating characteristic curve (AUC). RESULTS: Among 1,387 analysed patients, toxicity rates were 11.7% (rectal bleeding), 4.0% (urinary frequency), 5.5% (haematuria), 7.8% (nocturia) and 17.1% (decreased urinary stream). RS and PS combined 8 to 15 different SNP-allele sets, depending on the toxicity endpoint. Distributions of PRSi differed significantly in patients with/without toxicity with AUCs ranging from 0.61 to 0.78. PRSi was better than the classical summed PRS, particularly for the urinary frequency, haematuria and decreased urinary stream endpoints. CONCLUSIONS: Our method incorporates SNP-SNP interactions when calculating PRS for radiotherapy toxicity. Our approach is better than classical summation in discriminating patients with toxicity and should enable incorporating genetic information to improve normal tissue complication probability models.

A Deep Learning Approach Validates Genetic Risk Factors for Late Toxicity After Prostate Cancer Radiotherapy in a REQUITE Multi-National Cohort.

Background: REQUITE (validating pREdictive models and biomarkers of radiotherapy toxicity to reduce side effects and improve QUalITy of lifE in cancer survivors) is an international prospective cohort study. The purpose of this project was to analyse a cohort of patients recruited into REQUITE using a deep learning algorithm to identify patient-specific features associated with the development of toxicity, and test the approach by attempting to validate previously published genetic risk factors. Methods: The study involved REQUITE prostate cancer patients treated with external beam radiotherapy who had complete 2-year follow-up. We used five separate late toxicity endpoints: ≥grade 1 late rectal bleeding, ≥grade 2 urinary frequency, ≥grade 1 haematuria, ≥ grade 2 nocturia, ≥ grade 1 decreased urinary stream. Forty-three single nucleotide polymorphisms (SNPs) already reported in the literature to be associated with the toxicity endpoints were included in the analysis. No SNP had been studied before in the REQUITE cohort. Deep Sparse AutoEncoders (DSAE) were trained to recognize features (SNPs) identifying patients with no toxicity and tested on a different independent mixed population including patients without and with toxicity. Results: One thousand, four hundred and one patients were included, and toxicity rates were: rectal bleeding 11.7%, urinary frequency 4%, haematuria 5.5%, nocturia 7.8%, decreased urinary stream 17.1%. Twenty-four of the 43 SNPs that were associated with the toxicity endpoints were validated as identifying patients with toxicity. Twenty of the 24 SNPs were associated with the same toxicity endpoint as reported in the literature: 9 SNPs for urinary symptoms and 11 SNPs for overall toxicity. The other 4 SNPs were associated with a different endpoint. Conclusion: Deep learning algorithms can validate SNPs associated with toxicity after radiotherapy for prostate cancer. The method should be studied further to identify polygenic SNP risk signatures for radiotherapy toxicity. The signatures could then be included in integrated normal tissue complication probability models and tested for their ability to personalize radiotherapy treatment planning.

Enhanced Internalization of Nanoparticles Following Ionizing Radiation Leads to Mitotic Catastrophe in MG-63 Human Osteosarcoma Cells.

This study aims to investigate whether ionizing radiation combined with doxorubicin-conjugated iron oxide nanoparticles (NP-DOX) improves the internalization and cytotoxic effects of the nano-carrier-mediated drug delivery in MG-63 human osteosarcoma cells. NP-DOX was designed and synthesized using the co-precipitation method. Highly stable and crystalline nanoparticles conjugated with DOX were internalized in MG-63 cells through macropinocytosis and located in the perinuclear area. Higher nanoparticles internalization in MG-63 cells previously exposed to 1 Gy X-rays was correlated with an early accumulation of cells in G2/M, starting at 12 h after treatment. After 48 h, the application of the combined treatment led to higher cytotoxic effects compared to the individual treatment, with a reduction in the metabolic capacity and unrepaired DNA breaks, whilst a low percent of arrested cells, contributing to the commitment of mitotic catastrophe. NP-DOX showed hemocompatibility and no systemic cytotoxicity, nor histopathological alteration of the main organs.

Radiation-induced malignancies after intensity-modulated versus conventional mediastinal radiotherapy in a small animal model.

A long-standing hypothesis in radiotherapy is that intensity-modulated radiotherapy (IMRT) increases the risk of second cancer due to low-dose exposure of large volumes of normal tissue. Therefore, young patients are still treated with conventional techniques rather than with modern IMRT. We challenged this hypothesis in first-of-its-kind experiments using an animal model. Cancer-prone Tp53+/C273X knockout rats received mediastinal irradiation with 3 × 5 or 3 × 8 Gy using volumetric-modulated arc therapy (VMAT, an advanced IMRT) or conventional anterior-posterior/posterior-anterior (AP/PA) beams using non-irradiated rats as controls (n = 15/group, ntotal = 90). Tumors were assigned to volumes receiving 90-107%, 50-90%, 5-50%, and <5% of the target dose and characterized by histology and loss-of-heterozygosity (LOH). Irradiated rats predominantly developed lymphomas and sarcomas in areas receiving 50-107% (n = 26) rather than 5-50% (n = 7) of the target dose. Latency was significantly shortened only after 3 × 8 Gy vs. controls (p < 0.0001). The frequency (14/28 vs. 19/29; p = 0.29) and latency (218 vs. 189 days; p = 0.17) of radiation-associated tumors were similar after VMAT vs. AP/PA. LOH was strongly associated with sarcoma but not with treatment. The results do not support the hypothesis that IMRT increases the risk of second cancer. Thus the current practice of withholding dose-sparing IMRT from young patients may need to be re-evaluated.

Multi-centre technical evaluation of the radiation-induced lymphocyte apoptosis assay as a predictive test for radiotherapy toxicity.

Predicting which patients will develop adverse reactions to radiotherapy is important for personalised treatment. Prediction will require an algorithm or nomogram combining clinical and biological data. The radiation-induced lymphocyte apoptosis (RILA) assay is the leading candidate as a biological predictor of radiotherapy toxicity. In this study we tested the potential of the assay for standardisation and use in multiple testing laboratories. The assay was standardised and reproducibility determined using samples from healthy volunteers assayed concurrently in three laboratories in Leicester (UK), Mannheim (Germany) and Montpellier (France). RILA assays were performed on samples taken prior to radiotherapy from 1319 cancer patients enrolled in the REQUITE project at multiple centres. The patients were being treated for breast (n = 753), prostate (n = 506) or lung (n = 60) cancer. Inter-laboratory comparisons identified several factors affecting results: storage time, incubation periods and type of foetal calf serum. Following standardisation, there was no significant difference in results between the centres. Significant differences were seen in RILA scores between cancer types (prostate > breast > lung), by smoking status (non-smokers > smokers) and co-morbidity with rheumatoid arthritis (arthritics > non-arthritics). An analysis of acute radiotherapy toxicity showed as expected that RILA assay does not predict most end-points, but unexpectedly did predict acute breast pain. This result may elucidate the mechanism by which the RILA assay predicts late radiotherapy toxicity. The work shows clinical trials involving multiple laboratory measurement of the RILA assay are feasible and the need to account for tumour type and other variables when applying to predictive models.

Association of CD4+ Radiation-Induced Lymphocyte Apoptosis with Fibrosis and Telangiectasia after Radiotherapy in 272 Breast Cancer Patients with >10-Year Follow-up.

PURPOSE: Radiation-induced lymphocyte apoptosis (RILA) has been suggested as a predictive assay for adverse late reactions after radiotherapy. Thus, low RILA values of T-lymphocyte subpopulations have been associated with increased risk for various endpoints at 2 to 3 years of follow-up. The purpose was to test if such associations persist for specific endpoints (subcutaneous fibrosis, telangiectasia) in breast cancer patients with at least 10 years of follow-up.Experimental Design: Two hundred and seventy-two female patients who had received breast-conserving therapy within the German ISE study were included (median follow-up: 11.6 years). Radiotherapy-induced side effects were scored according to the Late Effects in Normal Tissues-Subjective, Objective, Management, and Analytic (LENT-SOMA) classification system. RILA in the CD4+, CD8+, and natural killer (NK) subpopulations from peripheral blood was analyzed by flow cytometry. Multivariate predictive modeling was performed including relevant clinical risk factors. RESULTS: Low CD4+ RILA was associated with increased risk for both fibrosis (P = 0.011) and telangiectasia (P < 0.001). For fibrosis, the association was stronger outside the surgical area (Fibout; P = 0.004) than within (Fibin; P = 0.17). Predictive multivariate modeling including clinical risk factors yielded OR of 3.48 (95% confidence interval, 1.84-6.58) for any fibrosis and 8.60 (2.71-27.3) for telangiectasia. Addition of CD4+ RILA to the clinical variables improved discrimination (c statistics) from 0.62 to 0.68 for any fibrosis, 0.62 to 0.66 for Fibin, 0.61 to 0.69 for Fibout, and from 0.65 to 0.76 for telangiectasia. CD8+ and NK RILA were not significantly associated with radiotherapy-related late reactions. CONCLUSIONS: The results provide first evidence that low CD4+ RILA is associated with increased subcutaneous fibrosis and telangiectasia even after 10 years. This supports the potential usefulness for predicting individual clinical risk.

A HYPOTHESIS OF RADIORESISTANCE AND CELL-SURVIVAL CURVE SHAPE BASED ON CELL-CYCLE PROGRESSION AND DAMAGE TOLERANCE.

Exponential survival curves of early-passage human fibroblasts challenge classic biophysical models of cell inactivation. Thus, X-ray doses of 2-4 Gy inactivate normal, human skin fibroblasts in spite of negligible residual double-strand breaks. By contrast, radioresistant p53-mutant U251 glioblastoma cells proliferate in spite of residual damage. Similarly, p53 wildtype TK6 lymphoblastoid cells show exponential survival curves while the related p53-mutant WTK1 cell line continued to proliferate and showed a shouldered survival curve. Here, we propose a model in which the radioresistant shoulder region is due to tolerance to certain types or amounts of residual damage that would otherwise inactivate normal cells. Thus, the steeper initial slope and absence of a shoulder in the survival curve of normal cells may not imply a higher number of residual lesions but rather non-tolerance to these lesions.