A potential biomarker of radiosensitivity in metastatic hormone sensitive prostate cancer patients treated with combination external beam radiotherapy and radium-223.

PURPOSE: The ADRRAD trial reported the safety and feasibility of the combination of external beam radiotherapy and radium-223 in the treatment of de novo bone metastatic prostate. This study aimed to determine if any biomarkers predictive of response to these treatments could be identified. EXPERIMENTAL DESIGN: 30 patients with newly diagnosed bone metastatic hormone sensitive prostate cancer were recruited to the ADRRAD trial. Blood samples were taken pre-treatment, before cycles 2 to 6 of radium-223, and 8 weeks and 6 months after treatment. Mononuclear cells were isolated and DNA damage was assessed at all timepoints. RESULTS: DNA damage was increased in all patients during treatment, with bigger increases in foci observed in patients who relapsed late compared to those who relapsed early. Increases in DNA damage during the radium-223 only cycles of treatment were specifically related to response in these patients. Analysis of hematology counts also showed bigger decreases in red blood cell and hemoglobin counts in patients who experienced later biochemical relapse. CONCLUSIONS: While some patients responded to this combination treatment, others relapsed within one year of treatment initiation. This study identifies a biomarker based approach that may be useful in predicting which patients will respond to treatment, by monitoring both increases in DNA damage above baseline levels in circulating lymphocytes and decreases in red blood cell and hemoglobin counts during treatment.

Novel unconventional radiotherapy techniques: Current status and future perspectives - Report from the 2nd international radiation oncology online seminar.

•Improvement of therapeutic ratio by novel unconventional radiotherapy approaches.•Immunomodulation using high-dose spatially fractionated radiotherapy.•Boosting radiation anti-tumor effects by adding an immune-mediated cell killing.

High-dose femtosecond-scale gamma-ray beams for radiobiological applications.

Objective. In the irradiation of living tissue, the fundamental physical processes involved in radical production typically occur on a timescale of a few femtoseconds. A detailed understanding of these phenomena has thus far been limited by the relatively long duration of the radiation sources employed, extending well beyond the timescales for radical generation and evolution.Approach. Here, we propose a femtosecond-scale photon source, based on inverse Compton scattering of laser-plasma accelerated electron beams in the field of a second scattering laser pulse.Main results. Detailed numerical modelling indicates that existing laser facilities can provide ultra-short and high-flux MeV-scale photon beams, able to deposit doses tuneable from a fraction of Gy up to a few Gy per pulse, resulting in dose rates exceeding 1013Gy/s.Significance. We envisage that such a source will represent a unique tool for time-resolved radiobiological experiments, with the prospect of further advancing radio-therapeutic techniques.

A Brief Overview of the Preclinical and Clinical Radiobiology of Microbeam Radiotherapy.

Microbeam radiotherapy (MRT) is the delivery of spatially fractionated beams that have the potential to offer significant improvements in the therapeutic ratio due to the delivery of micron-sized high dose and dose rate beams. They build on longstanding clinical experience of GRID radiotherapy and more recently lattice-based approaches. Here we briefly overview the preclinical evidence for MRT efficacy and highlight the challenges for bringing this to clinical utility. The biological mechanisms underpinning MRT efficacy are still unclear, but involve vascular, bystander, stem cell and potentially immune responses. There is probably significant overlap in the mechanisms underpinning MRT responses and FLASH radiotherapy that needs to be further defined.

Robustness of differential gene expression analysis of RNA-seq.

RNA-sequencing (RNA-seq) is a relatively new technology that lacks standardisation. RNA-seq can be used for Differential Gene Expression (DGE) analysis, however, no consensus exists as to which methodology ensures robust and reproducible results. Indeed, it is broadly acknowledged that DGE methods provide disparate results. Despite obstacles, RNA-seq assays are in advanced development for clinical use but further optimisation will be needed. Herein, five DGE models (DESeq2, voom + limma, edgeR, EBSeq, NOISeq) for gene-level detection were investigated for robustness to sequencing alterations using a controlled analysis of fixed count matrices. Two breast cancer datasets were analysed with full and reduced sample sizes. DGE model robustness was compared between filtering regimes and for different expression levels (high, low) using unbiased metrics. Test sensitivity estimated as relative False Discovery Rate (FDR), concordance between model outputs and comparisons of a 'population' of slopes of relative FDRs across different library sizes, generated using linear regressions, were examined. Patterns of relative DGE model robustness proved dataset-agnostic and reliable for drawing conclusions when sample sizes were sufficiently large. Overall, the non-parametric method NOISeq was the most robust followed by edgeR, voom, EBSeq and DESeq2. Our rigorous appraisal provides information for method selection for molecular diagnostics. Metrics may prove useful towards improving the standardisation of RNA-seq for precision medicine.

DNA DSB Repair Dynamics following Irradiation with Laser-Driven Protons at Ultra-High Dose Rates.

Protontherapy has emerged as more effective in the treatment of certain tumors than photon based therapies. However, significant capital and operational costs make protontherapy less accessible. This has stimulated interest in alternative proton delivery approaches, and in this context the use of laser-based technologies for the generation of ultra-high dose rate ion beams has been proposed as a prospective route. A better understanding of the radiobiological effects at ultra-high dose-rates is important for any future clinical adoption of this technology. In this study, we irradiated human skin fibroblasts-AG01522B cells with laser-accelerated protons at a dose rate of 109 Gy/s, generated using the Gemini laser system at the Rutherford Appleton Laboratory, UK. We studied DNA double strand break (DSB) repair kinetics using the p53 binding protein-1(53BP1) foci formation assay and observed a close similarity in the 53BP1 foci repair kinetics in the cells irradiated with 225 kVp X-rays and ultra- high dose rate protons for the initial time points. At the microdosimetric scale, foci per cell per track values showed a good correlation between the laser and cyclotron-accelerated protons indicating similarity in the DNA DSB induction and repair, independent of the time duration over which the dose was delivered.

Clinically Actionable Insights into Initial and Matched Recurrent Glioblastomas to Inform Novel Treatment Approaches.

Glioblastoma is the most common primary adult brain tumour, and despite optimal treatment, the median survival is 12-15 months. Patients with matched recurrent glioblastomas were investigated to try to find actionable mutations. Tumours were profiled using a validated DNA-based gene panel. Copy number variations (CNVs) and single nucleotide variants (SNVs) were examined, and potentially pathogenic variants and clinically actionable mutations were identified. The results revealed that glioblastomas were IDH-wildtype (IDH WT; n = 38) and IDH-mutant (IDH MUT; n = 3). SNVs in TSC2, MSH6, TP53, CREBBP, and IDH1 were variants of unknown significance (VUS) that were predicted to be pathogenic in both subtypes. IDH WT tumours had SNVs that impacted RTK/Ras/PI(3)K, p53, WNT, SHH, NOTCH, Rb, and G-protein pathways. Many tumours had BRCA1/2 (18%) variants, including confirmed somatic mutations in haemangioblastoma. IDH WT recurrent tumours had fewer pathways impacted (RTK/Ras/PI(3)K, p53, WNT, and G-protein) and CNV gains (BRCA2, GNAS, and EGFR) and losses (TERT and SMARCA4). IDH MUT tumours had SNVs that impacted RTK/Ras/PI(3)K, p53, and WNT pathways. VUS in KLK1 was possibly pathogenic in IDH MUT. Recurrent tumours also had fewer pathways (p53, WNT, and G-protein) impacted by genetic alterations. Public datasets (TCGA and GDC) confirmed the clinical significance of findings in both subtypes. Overall in this cohort, potentially actionable variation was most often identified in EGFR, PTEN, BRCA1/2, and ATM. This study underlines the need for detailed molecular profiling to identify individual GBM patients who may be eligible for novel treatment approaches. This information is also crucial for patient recruitment to clinical trials.

The Radiobiology of Proton Therapy: Challenges and Opportunities Around Relative Biological Effectiveness.

With the current UK expansion of proton therapy there is a great opportunity for clinical oncologists to develop a translational interest in the associated scientific base and clinical results. In particular, the underpinning controversy regarding the conversion of photon dose to proton dose by the relative biological effectiveness (RBE) must be understood, including its important implications. At the present time, the proton prescribed dose includes an RBE of 1.1 regardless of tissue, tumour and dose fractionation. A body of data has emerged against this pragmatic approach, including a critique of the existing evidence base, due to choice of dose, use of only acute-reacting in vivo assays, analysis methods and the reference radiations used to determine the RBE. Modelling systems, based on the best available scientific evidence, and which include the clinically useful biological effective dose (BED) concept, have also been developed to estimate proton RBEs for different dose and linear energy transfer (LET) values. The latter reflect ionisation density, which progressively increases along each proton track. Late-reacting tissues, such as the brain, where α/ß = 2 Gy, show a higher RBE than 1.1 at a low dose per fraction (1.2-1.8 Gy) at LET values used to cover conventional target volumes and can be much higher. RBE changes with tissue depth seem to vary depending on the method of beam delivery used. To reduce unexpected toxicity, which does occasionally follow proton therapy, a more rational approach to RBE allocation, using a variable RBE that depends on dose per fraction and the tissue and tumour radiobiological characteristics such as α/ß, is proposed.

Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy.

Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Multidisciplinary European Low Dose Initiative (MELODI): strategic research agenda for low dose radiation risk research.

MELODI (Multidisciplinary European Low Dose Initiative) is a European radiation protection research platform with focus on research on health risks after exposure to low-dose ionising radiation. It was founded in 2010 and currently includes 44 members from 18 countries. A major activity of MELODI is the continuous development of a long-term European Strategic Research Agenda (SRA) on low-dose risk for radiation protection. The SRA is intended to identify priorities for national and European radiation protection research programs as a basis for the preparation of competitive calls at the European level. Among those key priorities is the improvement of health risk estimates for exposures close to the dose limits for workers and to reference levels for the population in emergency situations. Another activity of MELODI is to ensure the availability of European key infrastructures for research activities, and the long-term maintenance of competences in radiation research via an integrated European approach for training and education. The MELODI SRA identifies three key research topics in low dose or low dose-rate radiation risk research: (1) dose and dose rate dependence of cancer risk, (2) radiation-induced non-cancer effects and (3) individual radiation sensitivity. The research required to improve the evidence base for each of the three key topics relates to three research lines: (1) research to improve understanding of the mechanisms contributing to radiogenic diseases, (2) epidemiological research to improve health risk evaluation of radiation exposure and (3) research to address the effects and risks associated with internal exposures, differing radiation qualities and inhomogeneous exposures. The full SRA and associated documents can be downloaded from the MELODI website ( http://www.melodi-online.eu/sra.html ).

Time-resolved dosimetric verification of respiratory-gated radiotherapy exposures using a high-resolution 2D ionisation chamber array.

The aim of this work was to track and verify the delivery of respiratory-gated irradiations, performed with three versions of TrueBeam linac, using a novel phantom arrangement that combined the OCTAVIUS(®) SRS 1000 array with a moving platform. The platform was programmed to generate sinusoidal motion of the array. This motion was tracked using the real-time position management (RPM) system and four amplitude gating options were employed to interrupt MV beam delivery when the platform was not located within set limits. Time-resolved spatial information extracted from analysis of x-ray fluences measured by the array was compared to the programmed motion of the platform and to the trace recorded by the RPM system during the delivery of the x-ray field. Temporal data recorded by the phantom and the RPM system were validated against trajectory log files, recorded by the linac during the irradiation, as well as oscilloscope waveforms recorded from the linac target signal. Gamma analysis was employed to compare time-integrated 2D x-ray dose fluences with theoretical fluences derived from the probability density function for each of the gating settings applied, where gamma criteria of 2%/2 mm, 1%/1 mm and 0.5%/0.5 mm were used to evaluate the limitations of the RPM system. Excellent agreement was observed in the analysis of spatial information extracted from the SRS 1000 array measurements. Comparisons of the average platform position with the expected position indicated absolute deviations of <0.5 mm for all four gating settings. Differences were observed when comparing time-resolved beam-on data stored in the RPM files and trajectory logs to the true target signal waveforms. Trajectory log files underestimated the cycle time between consecutive beam-on windows by 10.0 ± 0.8 ms. All measured fluences achieved 100% pass-rates using gamma criteria of 2%/2 mm and 50% of the fluences achieved pass-rates >90% when criteria of 0.5%/0.5 mm were used. Results using this novel phantom arrangement indicate that the RPM system is capable of accurately gating x-ray exposure during the delivery of a fixed-field treatment beam.

Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation.

Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation.

Radiation responses of stem cells: targeted and non-targeted effects.

Stem cells are fundamental to the development of any tissue or organism via their ability to self-renew, which is aided by their unlimited proliferative capacity and their ability to produce fully differentiated offspring, often from multiple lineages. Stems cells are long lived and have the potential to accumulate mutations, including in response to radiation exposure. It is thought that stem cells have the potential to be induced into a cancer stem cell phenotype and that these may play an important role in resistance to radiotherapy. For radiation-induced carcinogenesis, the role of targeted and non-targeted effects is unclear with tissue or origin being important. Studies of genomic instability and bystander responses have shown consistent effects in haematopoietic models. Several models of radiation have predicted that stem cells play an important role in tumour initiation and that bystander responses could play a role in proliferation and self-renewal.

Small animal image-guided radiotherapy: status, considerations and potential for translational impact.

Radiation biology is being transformed by the implementation of small animal image-guided precision radiotherapy into pre-clinical research programmes worldwide. We report on the current status and developments of the small animal radiotherapy field, suggest criteria for the design and execution of effective studies and contend that this powerful emerging technology, used in combination with relevant small animal models, holds much promise for translational impact in radiation oncology.

The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy.

A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.

Investigation into the radiobiological consequences of pre-treatment verification imaging with megavoltage X-rays in radiotherapy.

OBJECTIVE: The aim of this study was to investigate the effect of pre-treatment verification imaging with megavoltage X-rays on cancer and normal cell survival in vitro and to compare the findings with theoretically modelled data. Since the dose received from pre-treatment imaging can be significant, the incorporation of this dose at the planning stage of treatment has been suggested. METHODS: The impact of imaging dose incorporation on cell survival was investigated by clonogenic assay of irradiated DU-145 prostate cancer, H460 non-small-cell lung cancer and AGO-1522b normal tissue fibroblast cells. Clinically relevant imaging-to-treatment times of 7.5 and 15 min were chosen for this study. The theoretical magnitude of the loss of radiobiological efficacy due to sublethal damage repair was investigated using the Lea-Catcheside dose protraction factor model. RESULTS: For the cell lines investigated, the experimental data showed that imaging dose incorporation had no significant impact on cell survival. These findings were in close agreement with theoretical results. CONCLUSION: For the conditions investigated, the results suggest that allowance for the imaging dose at the planning stage of treatment should not adversely affect treatment efficacy. ADVANCES IN KNOWLEDGE: There is a paucity of data in the literature on imaging effects in radiotherapy. This article presents a systematic study of imaging dose effects on cancer and normal cell survival, providing both theoretical and experimental evidence for clinically relevant imaging doses and imaging-to-treatment times. The data provide a firm foundation for further study into this highly relevant area of research.

Investigating the potential impact of four-dimensional computed tomography (4DCT) on toxicity, outcomes and dose escalation for radical lung cancer radiotherapy.

AIMS: To investigate the potential dosimetric and clinical benefits predicted by using four-dimensional computed tomography (4DCT) compared with 3DCT in the planning of radical radiotherapy for non-small cell lung cancer. MATERIALS AND METHODS: Twenty patients were planned using free breathing 4DCT then retrospectively delineated on three-dimensional helical scan sets (3DCT). Beam arrangement and total dose (55 Gy in 20 fractions) were matched for 3D and 4D plans. Plans were compared for differences in planning target volume (PTV) geometrics and normal tissue complication probability (NTCP) for organs at risk using dose volume histograms. Tumour control probability and NTCP were modelled using the Lyman-Kutcher-Burman (LKB) model. This was compared with a predictive clinical algorithm (Maastro), which is based on patient characteristics, including: age, performance status, smoking history, lung function, tumour staging and concomitant chemotherapy, to predict survival and toxicity outcomes. Potential therapeutic gains were investigated by applying isotoxic dose escalation to both plans using constraints for mean lung dose (18 Gy), oesophageal maximum (70 Gy) and spinal cord maximum (48 Gy). RESULTS: 4DCT based plans had lower PTV volumes, a lower dose to organs at risk and lower predicted NTCP rates on LKB modelling (P < 0.006). The clinical algorithm showed no difference for predicted 2-year survival and dyspnoea rates between the groups, but did predict for lower oesophageal toxicity with 4DCT plans (P = 0.001). There was no correlation between LKB modelling and the clinical algorithm for lung toxicity or survival. Dose escalation was possible in 15/20 cases, with a mean increase in dose by a factor of 1.19 (10.45 Gy) using 4DCT compared with 3DCT plans. CONCLUSIONS: 4DCT can theoretically improve therapeutic ratio and dose escalation based on dosimetric parameters and mathematical modelling. However, when individual characteristics are incorporated, this gain may be less evident in terms of survival and dyspnoea rates. 4DCT allows potential for isotoxic dose escalation, which may lead to improved local control and better overall survival.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen.

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-ß-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.