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Introduction: TACT, an investigator-led study in 4162 women with node positive (N+ve) or high risk node negative (N-ve) early breast cancer (EBC), is the largest taxane trial unconfounded by treatment (trt) duration. At principal analysis, with 5 years follow-up (fup), no evidence of improved disease-free survival (DFS) was observed by switching to 4 cycles of docetaxel (D) after 4 cycles of FEC (Ellis, Lancet 2009). Results were provocative in suggesting differential effects according to ER & HER2 status. Longer fup provides opportunity to detect emergence of late trt effects overall & within phenotypic subgroups & explore patterns of recurrence, by tumor characteristics. Patients & methods: TACT recruited women with histologically confirmed completely resected invasive EBC from 104 centers (UK (103), Belgium (1)) between 02/2001 & 07/2003 Centers chose FEC (600/60/600 mg/m² q3wk x 8) or E-CMF (E 100mg/m² q3wk x 4 →CMF 100mg/m² PO d1 -14 or 600mg/m² IV d1 &8/40/600 mg/m² q4wk x 4) as their control, reflecting standard UK practice. Patients (pts) were randomized to FEC-D (FEC q3wk x 4 → D 100 mg/m² q3wk x 4) or control. 2523 pts were from FEC centers (FEC = 1265: FEC-D = 1258) & 1639 from E-CMF centers (E-CMF = 824; FEC-D = 815). Endocrine therapy was given for 5 years. Few pts received HER2 directed therapy; 589 pts had unknown HER2 status. Median fup is now 97.5 months; this analysis updates DFS & overall survival in the ITT population. It also explores patterns of relapse by phenotypic & clinical characteristics. Analyses of trt effect are stratified by ER status due to issues of non-proportionality of hazard associated with length of fup. Results: DFS events have been reported for 1329 pts (FEC-D=640, Control=689) giving an unadjusted hazard ratio (HR) & 95%CI (stratified by control regimen & ER status) of 0.93 (0.83, 1.03) overall; p = 0.16 in favor of FEC-D & for ER+ve/HER2-ve of 0.99 (0.84, 1.17), for ER+ve/HER2+ve) 0.97 (0.73, 1.30), for ER-ve/HER2+ve 0.74 (0.53, 1.03), & ER-ve/HER2-ve 0.93 (0.73, 1.17). 1017 patients have died (FEC-D=500, Control=517); unadjusted HR=0.98 (95%CI: 0.86, 1.10); p = 0.69 with intercurrent deaths (prior to distant relapse) reported for 80 pts (FEC-D=40, Control=40). Annual event rates show different pattern of disease relapse by phenotypic subgroup Graphical representation will further explore these patterns & associated sites of relapse. Discussion: With a median fup of >8 years no clear benefit has emerged for D over standard anthracyclines within the TACT pt group. Differential effects associated with different patterns of relapse remain of interest. TACT precedes use of antiHER2 therapy which is known to have impacted on early relapse risk in HER2+ve pts. The high relapse risk observed for pts with ER-ve/HER2-ve disease remains a current clinical challenge. [ABSTRACT FROM AUTHOR]

Urinary markers of bone resorption, pyridinoline and deoxypyridinoline were measured before and at 4 weeks after radiotherapy for metastatic bone pain. An association was shown between relief of metastatic skeletal pain by radiotherapy and low marker concentrations before and after treatment, lending support to the hypothesis that relief of metastatic bone pain by radiotherapy relates to an effect on bone, rather than tumour physiology. [ABSTRACT FROM AUTHOR]

Background International standard adjuvant radiotherapy regimens following primary surgery for early breast cancer have historically delivered a high total dose (50Gy) in 25 small daily doses (fractions) over 5 weeks, however randomised trials, including START, indicate that a lower total dose delivered in fewer, larger fractions (Fr) is likely to be at least as safe and effective (START Trialists' Group, Lancet 2008 & Lancet Oncol 2008). With patients remaining at risk of local relapse for many years, information on long-term outcomes is needed to provide confidence in clinical practice. Here, we report 10-year follow-up of the UK START Trials testing 13- and 15-Fr regimens in terms of local cancer control and late adverse effects. Methods Between 1999 and 2002, 4451 women with completely excised invasive breast cancer (T1-3, N0-1, M0) were randomised after primary surgery to comparisons of 50Gy in 25Fr over 5 weeks vs 41⋅6Gy or 39Gy in 13Fr over 5 weeks (START A), or 50Gy in 25Fr over 5 weeks vs 40Gy in 15Fr over 3 weeks (START B). Women were eligible if aged over 18 years and did not have an immediate surgical reconstruction. Protocol-specified principal endpoints were local-regional (LR) tumour relapse and late normal tissue effects. Analysis was by intention to treat. Findings Median follow-up in survivors is now 9.3 years in START A and 9.9 years in START B, with 139 LR relapses in START A and 95 in START B. In START A, the 10-year rate of LR relapse was 7.4% (95%CI 5.5-10.0) after 50Gy, 6.3% (95%CI 4.7-8.5) after 41⋅6Gy and 8.8% (95%CI 6.7-11.4) after 39Gy. In START B, the 10-year rate of LR relapse was 5.5% (95%CI 4.2-7.2) after 50Gy and 4.3% (95%CI 3.2-5.9) after 40Gy. Clinician assessments suggested lower 10-year rates of any moderate/marked late normal tissue effects after 39Gy (43.9%; 95%CI 39.3-48.7) and similar rates after 41.6Gy (49.5%; 95%CI 44.9-54.3) compared with 50Gy (50.4%; 95%CI 45.8-55.3) in START A and lower rates after 40Gy in START B (37.9%; 95%CI 34.5-41.5) compared with 50Gy (45.3%; 95%CI 41.7-49.0). From a planned meta-analysis of START A and the START pilot trial (Owen et al, Lancet Oncol 2006), the adjusted estimate of α/β value for tumour control was 3.5Gy (95% CI 1.2-5.7) and for late change in photographic breast appearance was 3.1Gy (95% CI 2.0-4.2). Interpretation Long-term follow-up conf irms that breast cancer and the surrounding dose-limiting healthy tissues respond similarly to radiotherapy fraction size and thus that appropriately-dosed hypofractionated radiotherapy is safe and effective in treatment of patients with early breast cancer. 41⋅6Gy in 13Fr and 40Gy in 15Fr each appear comparable to 50Gy in 25Fr in terms of local-regional tumour control and late normal tissue effects. These results support the continued use of 40Gy in 15Fr as standard of care (UK NICE Guidance 2009) for women requiring adjuvant radiotherapy for early breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: There is a strong inverse association between the proliferation indices of early- and late-responding normal tissues and their sensitivity to radiotherapy fraction size. The aim of this study is to test for association between Ki67 index and the fractionation sensitivity of breast cancer. The hypothesis is that tumours with high Ki67 indices are relatively insensitive to fraction size and be over-represented in tumours relapsing after hypofractionated, radiotherapy. Methods: Between 1986 and 2003, the START pilot and START A trials tested 2 test dose levels of a 13-fraction regimen (3.0 or 3.3 Gy & 3.0 or 3.2 Gy fractions, respectively) in 5 weeks against 25 fractions of 2.0 Gy following primary surgery for early breast cancer. Primary tumour blocks of patients with local tumour relapse were collected for immunohistochemistry (IHC) for Ki67 (manual counting in whole sections), HER2, ER & PR (Allred quick scores in tissue sections). Ki67 was assumed to be log normally distributed with a standard deviation of 0.5-0.9. Assuming evaluable blocks for 240 patients, a standardised detectable difference of 0.45 (90%+ power, 5% significance level) corresponded to a detectable difference in geometric means of 5-10%. Results: From a total of 3646 patients entered into the START pilot and START A trials, 261 local tumour relapses were recorded at a median follow up of 8.4 years (range 0.9-17.5) and 7.2 (range 0.7-11.9) years respectively. Blocks from 213 patients were recovered, of which 176 were evaluable by IHC. There was no significant difference in proliferation between tumours relapsing after conventional and hypofractionated radiotherapy, with mean Ki67 scores of 7.63 (95%CI: 5.06-11.5) and 5.33 (95CI%: 3.86-7.35), respectively. Mean Ki67 scores in 48 primary triple negative tumours (TNT) that relapsed locally were 15.74 (95%CI: 8.53-29.06) after conventional and 7.52 (95%CI: 3.51-16.1) after hypofractionated radiotherapy, but TNT were equally represented (28%) in both groups. Conclusion: An association between proliferative indices and fractionation sensitivity in breast tumours has not been demonstrated. If confirmed, it suggests that genetic and epigenetic features unrelated to proliferation are sources of inter-tumour variation in fractionation sensitivity. [ABSTRACT FROM AUTHOR]

Background: KORTUC (0.5% hydrogen peroxide (H 2 O 2 ) in 1% sodium-hyaluronate) releases cytotoxic levels of H 2 O 2 in tissues after intratumoural injection. High levels of tumour control after radiotherapy plus KORTUC are reported in breast cancer patients. Here, we use human xenograft models to test the hypothesis that oxygen microbubbles released post-KORTUC are effective in modifying the hypoxic tumour microenvironment., Methods and Materials: Pimonidazole and Image-iT™ Red (live hypoxia marker) were utilised to assess dose-dependent changes in hypoxia post-H 2 O 2 in HCT116 and LICR-LON-HN5 spheroids. Using a dual 2-nitroimidazole-marker technique and phospho-ATM we evaluated changes in hypoxia and reactive oxygen species (ROS) respectively, in HCT116 and LICR-LON-HN5 xenografts following intratumoural KORTUC., Results: A significant reduction in Image-iT™ Red fluorescence was observed in spheroids 1 h post-H 2 O 2 at ≥1.2 mM, maintained at 24 h. Ultrasound demonstrated sustained release of oxygen microbubbles within tumours, 1 h post-KORTUC. Hypoxia markers demonstrated significant tissue reoxygenation in both models post-KORTUC and significantly increased phospho-ATM foci reflecting increased ROS production., Conclusion: Intratumoural KORTUC represents a novel oxygen delivery method, which can be exploited to enhance radiation response. If efficacy is confirmed in the ongoing phase 2 breast trial it could improve treatment of several tumour types where hypoxia is known to affect radiotherapy outcomes., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2024.)

There is a sound empirical basis for hypofractionation in radiotherapy for breast cancer. This article reviews the radiobiological implications of hypofractionation in breast cancer derived from a series of clinical trials that began when 50 Gy in 25 fractions over 5 weeks was commonplace. These trials led first to 40 Gy in 15 fractions over 3 weeks and, subsequently, to 26 Gy in five fractions over 1 week being adopted as standards of care for many patients prescribed whole- or partial-breast radiotherapy after primary surgery., (Copyright © 2022. Published by Elsevier Ltd.)

Hypofractionated radical radiotherapy is now an accepted standard of care for tumour sites such as prostate and breast cancer. Much research effort is being directed towards more profoundly hypofractionated (ultrahypofractionated) schedules, with some reaching UK standard of care (e.g. adjuvant breast). Hypofractionation exerts varying influences on each of the major clinical end points of radiotherapy studies: acute toxicity, late toxicity and local control. This review will discuss these effects from the viewpoint of the traditional 5 Rs of radiobiology, before considering non-canonical radiobiological effects that may be relevant to ultrahypofractionated radiotherapy. The principles outlined here may assist the reader in their interpretation of the wealth of clinical data presented in the tumour site-specific articles in this special issue., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Preclinical data suggest that combined gamma-tocotrienol with pentoxifylline ameliorates radiotherapy-induced gastrointestinal damage., Aim: To test whether gastrointestinal symptoms arising after radiotherapy, and persisting after maximal medical therapy, can be improved using Tocovid SupraBio 200 mg and pentoxifylline 400 mg orally twice daily for one year. Patients stratified by severity of symptoms, and randomised to active treatment or matched placebo were assessed after 12 months. The primary end point was improvement in gastrointestinal symptoms measured using the Inflammatory Bowel Disease Questionnaire, bowel subset score. Changes in bio-markers of fibrosis were assessed., Results: 62 patients, median age 66, 34(55%) treated for prostate, 21(34%) gynaecological, 6(10%) anal and one(1%) rectal cancer were recruited; 40(65%) randomised to treatment, 22(35%) to placebo, 39 months (median) after radiotherapy completion. Gamma tocotrienol was not detected in serum in 41% of treated patients, despite good compliance with study medication. Treatment was completed in 28(70%) and 17(77%) patients in the treatment and placebo groups respectively. No improvement in symptom scores nor in quality of life was identified. Thirteen serious adverse events occurred. A transient ischaemic attack, was possibly related to pentoxifylline, others were assessed as unlikely to be related to treatment. Levels of EGF, PDGF and FGF were significantly reduced and consistent trends in reduced inflammation were seen during treatment but were not sustained once treatment ended., Summary: This single centre study closed prematurely and therefore data interpretation is of necessity limited. No clinical benefit was demonstrated. However, biochemical data suggest that this intervention does have anti-inflammatory and anti-fibrotic effects., Competing Interests: Conflict of Interest No author has any conflict of Interest to declare., (Copyright © 2022. Published by Elsevier B.V.)

Introduction: The phase 3 FAST-Forward trial reported outcomes for 26 and 27 Gy schedules delivered in 5 fractions over 1 week versus 40 Gy in 15 fractions over 3 weeks in 4000 patients. We discuss concerns raised by the radiotherapy community in relation to implementing this schedule., Ipsilateral Breast Tumour Relapse (ibtr): Published estimated 5-year IBTR with 95% CI after 40 Gy in 15 fractions was 2.1% (95% CI 1.4-3.1), 1.7% (1.2-1.6) after 27 Gy and 1.4% (0.2-2.2) after 26 Gy, emphatically showing non-inferiority of the 5-fraction regimens. Subgroup analyses comparing IBTR in 26 Gy versus 40 Gy show no evidence of differential effect regarding age, grade, pathological tumour size, nodal status, tumour bed boost, adjuvant chemotherapy, HER2 status and triple negative status. The number of events in these analyses is small and results should be interpreted with caution. There was only 1 IBTR event post-mastectomy., Normal Tissue Effects: The 26 Gy schedule, on the basis of similar NTE to 40 Gy in 15 fractions, is the recommended regimen for clinical implementation. There is a low absolute rate of moderate/marked NTE, these are predominantly moderate not severe change. Subgroup analyses comparing clinician-assessed moderate or marked adverse effect for 26 Gy versus 40 Gy show no evidence of differential effects according to age, breast size, surgical deficit, tumour bed boost, or adjuvant chemotherapy., Radiobiological Considerations: The design of the FAST-Forward trial does not control for time-related effects, and the ability to interpret clinical outcomes in terms of underlying biology is limited. There could conceivably be a time-effect for tumour control. A slight reduction in α/β estimate for the late normal tissue effects of test regimens might be a chance effect, but if real could reflect fewer consequential late effects due to lower rates of moist desquamation., Conclusion: The 26 Gy 5-fraction daily regimen for breast radiotherapy can be implemented now., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Microalgae-based high-density fuels offer an efficient and environmental pathway towards decarbonization of the transport sector and could be produced as part of a globally distributed network without competing with food systems for arable land. Variations in climatic and economic conditions significantly impact the economic feasibility and productivity of such fuel systems, requiring harmonized technoeconomic assessments to identify important conditions required for commercial scale up., Methods: Here, our previously validated Techno-economic and Lifecycle Analysis (TELCA) platform was extended to provide a direct performance comparison of microalgae diesel production at 12 international locations with variable climatic and economic settings. For each location, historical weather data, and jurisdiction-specific policy and economic inputs were used to simulate algal productivity, evaporation rates, harvest regime, CapEx and OpEx, interest and tax under location-specific operational parameters optimized for Minimum Diesel Selling Price (MDSP, US$ L -1 ). The economic feasibility, production capacity and CO 2-eq emissions of a defined 500 ha algae-based diesel production facility is reported for each., Results: Under a for-profit business model, 10 of the 12 locations achieved a minimum diesel selling price (MDSP) under US$ 1.85 L -1 / US$ 6.99 gal -1 . At a fixed theoretical MDSP of US$ 2 L -1 (US$ 7.57 gal -1 ) these locations could achieve a profitable Internal Rate of Return (IRR) of 9.5-22.1%. Under a public utility model (0% profit, 0% tax) eight locations delivered cost-competitive renewable diesel at an MDSP of < US$ 1.24 L -1 (US$ 4.69 gal -1 ). The CO 2-eq emissions of microalgae diesel were about one-third of fossil-based diesel., Conclusions: The public utility approach could reduce the fuel price toward cost-competitiveness, providing a key step on the path to a profitable fully commercial renewable fuel industry by attracting the investment needed to advance technology and commercial biorefinery co-production options. Governments' adoption of such an approach could accelerate decarbonization, improve fuel security, and help support a local COVID-19 economic recovery. This study highlights the benefits and limitations of different factors at each location (e.g., climate, labour costs, policy, C-credits) in terms of the development of the technology-providing insights on how governments, investors and industry can drive the technology forward.

Purpose: Hydrogen peroxide (H 2 O 2 ) plays a vital role in normal cellular processes but at supraphysiological concentrations causes oxidative stress and cytotoxicity, a property that is potentially exploitable for the treatment of cancer in combination with radiation therapy (RT). We report the first phase 1 trial testing the safety and tolerability of intratumoral H 2 O 2 + external beam RT as a novel combination in patients with breast cancer and exploratory plasma marker analyses investigating possible mechanisms of action., Methods and Materials: Twelve patients with breast tumors ≥3 cm (surgically or medically inoperable) received intratumoral H 2 O 2 with either 36 Gy in 6 twice-weekly fractions (n = 6) or 49.5 Gy in 18 daily fractions (n = 6) to the whole breast ± locoregional lymph nodes in a single-center, nonrandomized study. H 2 O 2 was mixed in 1% sodium hyaluronate gel (final H 2 O 2 concentration 0.5%) before administration to slow drug release and minimize local discomfort. The mixture was injected intratumorally under ultrasound guidance twice weekly 1 hour before RT. The primary endpoint was patient-reported maximum intratumoral pain intensity before and 24 hours postinjection. Secondary endpoints included grade ≥3 skin toxicity and tumor response by ultrasound. Blood samples were collected before, during, and at the end of treatment for cell-death and immune marker analysis., Results: Compliance with H 2 O 2 and RT was 100%. Five of 12 patients reported moderate pain after injection (grade 2 Common Terminology Criteria for Adverse Events v4.02) with median duration 60 minutes (interquartile range, 20-120 minutes). Skin toxicity was comparable to RT alone, with maintained partial/complete tumor response relative to baseline in 11 of 12 patients at last follow-up (median 12 months). Blood marker analysis highlighted significant associations of TRAIL, IL-1β, IL-4, and MIP-1α with tumor response., Conclusions: Intratumoral H 2 O 2 with RT is well tolerated with no additional toxicity compared with RT alone. If efficacy is confirmed in a randomized phase 2 trial, the approach has potential as a cost-effective radiation response enhancer in multiple cancer types in which locoregional control after RT alone remains poor., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Photosynthetic microalgae are unicellular plants, many of which are rich in protein, lipids, and bioactives and form an important part of the base of the natural aquatic food chain. Population growth, demand for high-quality protein, and depletion of wild fishstocks are forecast to increase aquacultural fish demand by 37% between 2016 and 2030. This review highlights the role of microalgae and recent advances that can support a sustainable 'circular' aquaculture industry. Microalgae-based feed supplements and recombinant therapeutic production offer significant opportunities to improve animal health, disease resistance, and yields. Critically, microalgae in biofloc, 'green water', nutrient remediation, and integrated multitrophic aquaculture technologies offer innovative solutions for economic and environmentally sustainable development in line with key UN Sustainability Goals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Centre for the Clinical Application of Particles' Laser-hybrid Accelerator for Radiobiological Applications (LhARA) facility is being studied and requires simulation of novel accelerator components (such as the Gabor lens capture system), detector simulation and simulation of the ion beam interaction with cells. The first stage of LhARA will provide protons up to 15 MeV for in vitro studies. The second stage of LhARA will use a fixed-field accelerator to increase the energy of the particles to allow in vivo studies with protons and in vitro studies with heavier ions. BDSIM, a Geant4 based accelerator simulation tool, has been used to perform particle tracking simulations to verify the beam optics design done by BeamOptics and these show good agreement. Design parameters were defined based on an EPOCH simulation of the laser source and a series of mono-energetic input beams were generated from this by BDSIM. The tracking results show the large angular spread of the input beam (0.2 rad) can be transported with a transmission of almost 100% whilst keeping divergence at the end station very low (<0.1 mrad). The legacy of LhARA will be the demonstration of technologies that could drive a step-change in the provision of proton and light ion therapy (i.e. a laser source coupled to a Gabor lens capture and a fixed-field accelerator), and a system capable of delivering a comprehensive set of experimental data that can be used to enhance the clinical application of proton and light ion therapy., (Copyright © 2019 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Conventional fractionation for half a century has been justified on the basis that 2.0 Gy fractions spare dose-limiting late-responding normal tissues to a greater degree than cancerous tissues. Early indications that breast cancer responds more strongly to fraction size than many other common cancers were followed several decades of investigation, but there is now reliable Level I evidence that this is the case. Four randomised trials testing fraction sizes in the range 2.7-3.3 Gy have reported 10-year follow up in almost 8000 patients, and they provide robust estimates of α/β in the range of 3 Gy. The implication is that there are no advantages in terms of safety or effectiveness of persisting with 2.0 Gy fractions in patients with breast cancer. 15- or 16-fraction schedules are replacing the conventional 25-fraction regimen as a standard of care for adjuvant therapy in an increasing number of countries. A number of concerns relating to the appropriateness of hypofractionation in patient subgroups, including those treated post-mastectomy, advanced local-regional disease and/or to lymphatic pathways are addressed. Meanwhile, hypofractionation can be exploited to modulate dose intensity across the breast according to relapse risk by varying fraction size across the treatment volume. The lower limits of hypofractionation are currently being explored, one approach testing a 5-fraction schedule of local-regional radiotherapy delivered in 1 week.

Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and systems development is focused on cost reduction to open up future economic opportunities for food, fuel and freshwater production. Light is a key environmental driver for photosynthesis and optimising light capture is therefore critical for low cost, high efficiency systems. Here a novel high-throughput screen that simulates fluctuating light regimes in mass cultures is presented. The data was used to model photosynthetic efficiency (PE µ , mol photon -1 m 2 ) and chlorophyll fluorescence of two green algae, Chlamydomonas reinhardtii and Chlorella sp. Response surface methodology defined the effect of three key variables: density factor (D f , 'culture density'), cycle time (t c , 'mixing rate'), and maximum incident irradiance (I max ). Both species exhibited a large rise in PE µ with decreasing I max and a minimal effect of t c (between 3-20 s). However, the optimal D f of 0.4 for Chlamydomonas and 0.8 for Chlorella suggested strong preferences for dilute and dense cultures respectively. Chlorella had a two-fold higher optimised PE µ than Chlamydomonas, despite its higher light sensitivity. These results demonstrate species-specific light preferences within the green algae clade. Our high-throughput screen enables rapid strain selection and process optimisation.

Background: Bio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood., Methods: Exome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively., Results: Overlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson's correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94., Conclusions: There is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material.

Aims: To evaluate the feasibility and heart-sparing ability of the voluntary breath-hold (VBH) technique in a multicentre setting., Materials and Methods: Patients were recruited from 10 UK centres. Following surgery for early left breast cancer, patients with any heart inside the 50% isodose from a standard free-breathing tangential field treatment plan underwent a second planning computed tomography (CT) scan using the VBH technique. A separate treatment plan was prepared on the VBH CT scan and used for treatment. The mean heart, left anterior descending coronary artery (LAD) and lung doses were calculated. Daily electronic portal imaging (EPI) was carried out and scanning/treatment times were recorded. The primary end point was the percentage of patients achieving a reduction in mean heart dose with VBH. Population systematic (Σ) and random errors (σ) were estimated. Within-patient comparisons between techniques used Wilcoxon signed-rank tests., Results: In total, 101 patients were recruited during 2014. Primary end point data were available for 93 patients, 88 (95%) of whom achieved a reduction in mean heart dose with VBH. Mean cardiac doses (Gy) for free-breathing and VBH techniques, respectively, were: heart 1.8 and 1.1, LAD 12.1 and 5.4, maximum LAD 35.4 and 24.1 (all P<0.001). Population EPI-based displacement data showed Σ =+1.3-1.9 mm and σ=1.4-1.8 mm. Median CT and treatment session times were 21 and 22 min, respectively., Conclusions: The VBH technique is confirmed as effective in sparing heart tissue and is feasible in a multicentre setting., (Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Background and Purpose: FAST-Forward is a phase 3 clinical trial testing a 1-week course of whole breast radiotherapy against the UK standard 3-week regimen after primary surgery for early breast cancer. Two acute skin toxicity substudies were undertaken to test the safety of the test schedules with respect to early skin reactions., Material and Methods: Patients were randomly allocated to 40Gy/15 fractions (F)/3-weeks, 27Gy/5F/1-week or 26Gy/5F/1-week. Acute breast skin reactions were graded using RTOG (first substudy) and CTCAE criteria v4.03 (second substudy) weekly during treatment and for 4weeks after treatment ended. Primary endpoint was the proportion of patients within each treatment group with grade ⩾3 toxicity (RTOG and CTCAE, respectively) at any time from the start of radiotherapy to 4weeks after completion., Results: 190 and 162 patients were recruited. In the first substudy, evaluable patients with grade 3 RTOG toxicity were: 40Gy/15F 6/44 (13.6%); 27Gy/5F 5/51 (9.8%); 26Gy/5F 3/52 (5.8%). In the second substudy, evaluable patients with grade 3 CTCAE toxicity were: 40Gy/15F 0/43; 27Gy/5F 1/41 (2.4%); 26Gy/5F 0/53., Conclusions: Acute breast skin reactions with two 1-week schedules of whole breast radiotherapy under test in FAST-Forward were mild., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Aims: In radiotherapy trials, normal tissue effects (NTE) are important end points and it is pertinent to ask whether patient-reported outcome measures (PROMs) could replace clinical and/or photographic assessments. Data from the Standardisation of Breast Radiotherapy (START) trials are examined., Materials and Methods: NTEs in the treated breast were recorded by (i) annual clinical assessments, (ii) photographs at 2 and 5 years, (iii) PROMs at 6 months, 1, 2 and 5 years after radiotherapy. Hazard ratios for the radiotherapy schedules were compared. Measures of agreement of assessments at 2 and 5 years tested concordance., Results: PROMs were available at 2 and/or 5 years for 1939 women, of whom 1870 had clinical and 1444 had photographic assessments. All methods were sensitive to the dose difference between schedules. Patients reported a higher prevalence for all NTE end points than clinicians or photographs (P < 0.001 for most NTEs). Concordance was generally poor; weighted kappa at 2 years ranged from 0.05 (telangiectasia) to 0.21 (shrinkage and oedema). The percentage agreement was lowest between PROMs and photographic assessments of change in breast appearance (38%)., Conclusions: All three methods produced similar conclusions for the comparison of trial schedules, despite low concordance between the methods on an individual patient basis. Careful consideration should be given to the different contributions of the measures of NTE in future radiotherapy trials., (Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Aim: To correlate residual double strand breaks (DSB) 24h after 4Gy test doses to skin in vivo and to lymphocytes in vitro with adverse effects of earlier breast radiotherapy (RT)., Patients and Methods: Patients given whole breast RT ⩾5years earlier were identified on the basis of moderate/marked or minimal/no adverse effects despite the absence ('RT-Sensitive', RT-S) or presence ('RT-Resistant', RT-R) of variables predisposing to late adverse effects. Residual DSB were quantified in skin 24h after a 4Gy test dose in 20 RT-S and 15 RT-R patients. Residual DSB were quantified in lymphocytes irradiated with 4Gy in vitro in 30/35 patients., Results: Mean foci per dermal fibroblast were 3.29 (RT-S) vs 2.80 (RT-R) (p=0.137); 3.28 (RT-S) vs 2.60 (RT-R) in endothelium (p=0.158); 2.50 (RT-S) vs 2.41 (RT-R) in suprabasal keratinocytes (p=0.633); 2.70 (RT-S) vs 2.35 (RT-R) in basal epidermis (p=0.419); 12.1 (RT-S) vs 10.3 (RT-R) in lymphocytes (p=0.0052)., Conclusions: Residual DSB in skin following a 4Gy dose were not significantly associated with risk of late adverse effects of breast radiotherapy, although exploratory analyses suggested an association in severely affected individuals. By contrast, a significant association was detected based on the in vitro response of lymphocytes., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Late normal tissue toxicity varies widely between patients and limits breast radiotherapy dose. Here we aimed to determine its relationship to DNA damage responses of fibroblast cultures from individual patients. Thirty-five breast cancer patients, with minimal or marked breast changes after breast-conserving therapy consented to receive a 4 Gy test irradiation to a small skin field of the left buttock and have punch biopsies taken from irradiated and unirradiated skin. Early-passage fibroblast cultures were established by outgrowth and irradiated in vitro with 0 or 4 Gy. 53BP1 foci, p53 and p21/CDKN1A were detected by immunofluorescence microscopy. Residual 53BP1 foci counts 24 h after in vitro irradiation were significantly higher in fibroblasts from RT-sensitive versus RT-resistant patients. Furthermore, significantly larger fractions of p53- but not p21/CDKN1A-positive fibroblasts were found in cultures from RT-sensitive patients without in vitro irradiation, and 2 h and 6 d post-irradiation. Exploratory analysis showed a stronger p53 response 2 h after irradiation of fibroblasts established from patients with severe reaction. These results associate the radiation response of fibroblasts with late reaction of the breast after RT and suggest a correlation with severity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Purpose: It has been hypothesized that increased predisposition to breast cancer may correlate with radiosensitivity, and thus increased risk of toxicity following breast irradiation. This study investigated the relationship between common breast cancer risk variants and radiotherapy toxicity., Experimental Design: SNP genotypes were determined in female breast cancer patients from the RAPPER (Radiogenomics: Assessment of polymorphisms for predicting the effects of radiotherapy) study using the Illumina CytoSNP12 genome-wide array. A further 15,582,449 genotypes were imputed using the 1000 Genomes Project reference panel. Patient (n = 1,160) polygenic risk scores were generated by summing risk-allele dosages, both unweighted and weighted by published effect sizes for breast cancer risk. Regression models were used to test associations of individual variants and polygenic risk scores with acute and late toxicity phenotypes (telangiectasia, breast edema, photographically assessed shrinkage, induration, pigmentation, breast pain, breast sensitivity, and overall toxicity)., Results: Genotypes of 90 confirmed breast cancer risk variants were accurately determined and polygenic risk scores were approximately normally distributed. Variant rs6964587 was associated with increased breast edema 5 years following radiotherapy (Beta, 0.22; 95% confidence interval, 0.09-0.34; P = 7 × 10(-4)). No other associations were found between individual variants or the unweighted (P > 0.17) or weighted (P > 0.13) polygenic risk score and radiotherapy toxicity. This study had >87% power to detect an association between the polygenic risk score (relative risk > 1.1) and toxicity., Conclusions: Cancer patients with a high polygenic predisposition to breast cancer do not have an increased risk of radiotherapy toxicity up to 5 years following radiotherapy but individual variants may increase risk., (©2015 American Association for Cancer Research.)

Background: Hyperbaric oxygen has been used as a therapy for patients experiencing chronic intestinal syndromes after pelvic radiotherapy for decades, yet the evidence to support the use of this therapy is based almost exclusively on non-randomised studies. We aimed to provide conclusive results for the clinical benefits of hyperbaric oxygen in patients with chronic bowel dysfunction after radiotherapy for pelvic malignancies., Methods: HOT2 was a double-blind, sham-controlled, phase 3 randomised study of patients (≥18 years) with chronic gastrointestinal symptoms for 12 months or more after radiotherapy and which persisted despite at least 3 months of optimal medical therapy and no evidence of cancer recurrence. Participants were stratified by participating hyperbaric centre and randomly assigned (2:1) by a computer-generated list (block size nine or 12) to receive treatment with hyperbaric oxygen therapy or sham. Participants in the active treatment group breathed 100% oxygen at 2·4 atmospheres of absolute pressure (ATA) and the control group breathed 21% oxygen at 1·3 ATA; both treatment groups received 90-min air pressure exposures once daily for 5 days per week for a total of 8 weeks (total of 40 exposures). Staff at the participating hyperbaric medicine facilities knew the allocated treatment, but patients, clinicians, nurse practitioners, and other health-care professionals associated with patients' care were masked to treatment allocation. Primary endpoints were changes in the bowel component of the modified Inflammatory Bowel Disease Questionnaire (IBDQ) score and the IBDQ rectal bleeding score 12 months after start of treatment relative to baseline. The primary outcome was analysed in a modified intention-to-treat population, excluding patients who did not provide IBDQ scores within a predetermined time-frame. All patients have completed 12 months of follow-up and the final analysis is complete. The trial is registered with the ISRCTN registry, number ISRCTN86894066., Findings: Between Aug 14, 2009, and Oct 23, 2012, 84 participants were randomly assigned: 55 to hyperbaric oxygen and 29 to sham control. 75 (89%) participants received 40 pressure exposures, all participants returned the IBDQ at baseline, 75 (89%) participants returned the IBDQ at 2 weeks post-treatment, and 79 (94%) participants returned the IBDQ at 12 months post-start of treatment. Patients were excluded from analyses of co-primary endpoints if they had missing IBDQ scores for intestinal function or rectal bleeding at baseline or at 12 months. In an analysis of 46 participants in the active treatment group and 23 participants in the control group, we found no significant differences in the change of IBDQ bowel component score (median change from baseline to 12 months of 4 (IQR -3 to 11) in the treatment group vs 4 (-6 to 9) in the sham group; Mann-Whitney U score 0·67, p=0·50). In an analysis of 29 participants in the active treatment group and 11 participants in the sham group with rectal bleeding at baseline, we also found no significant differences in the change of IBDQ rectal bleeding score (median change from baseline to 12 months of 3 [1 to 3] in the treatment group vs 1 [1 to 2] in the sham group; U score 1·69, p=0·092). Common adverse events in both groups were eye refractive changes (three [11%] of 28 patients in the control group vs 16 [30%] of 53 patients in the treatment group), increased fatigue (three [11%] vs two [4%]), and ear pain (six [21%] vs 15 [28%]). Eight serious adverse events were reported in eight patients: two were reported in two patients in the control group (tonsillitis requiring surgery [grade 3]; recurrent cancer of the vulva [grade 4]) and six serious adverse events were reported in six patients in the treatment group (malignant spinal cord compression requiring surgery [grade 3]; malignant paraortic lymph node involvement requiring surgery [grade 3]; recurrence of vomiting and dehydration [grade 3]; diarrhoea and fever associated with Campylobacter infection [grade 3]; recurrence of abdominal pain, bloating, diarrhoea, and urinary tract infection [grade 3]; aneurysm [grade 4]), none of which were deemed treatment-related., Interpretation: We found no evidence that patients with radiation-induced chronic gastrointestinal symptoms, including those patients with rectal bleeding, benefit from hyperbaric oxygen therapy. These findings contrast with evidence used to justify current practices, and more level 1 evidence is urgently needed., Funding: Cancer Research UK and National Health Service (NHS) funding to the National Institute of Health Research Biomedical Research Centre at The Royal Marsden and the Institute of Cancer Research., (Copyright © 2016 Glover et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved.)

Objective: To determine if subsets of patients may benefit from smaller or larger margins when using laser setup and bony anatomy verification of breast tumour bed (TB) boost radiotherapy (RT)., Methods: Verification imaging data acquired using cone-beam CT, megavoltage CT or two-dimensional kilovoltage imaging on 218 patients were used (1574 images). TB setup errors for laser-only setup (dlaser) and for bony anatomy verification (dbone) were determined using clips implanted into the TB as a gold standard for the TB position. Cases were grouped by centre-, patient- and treatment-related factors, including breast volume, TB position, seroma visibility and surgical technique. Systematic (Σ) and random (σ) TB setup errors were compared between groups, and TB planning target volume margins (MTB) were calculated., Results: For the study population, Σlaser was between 2.8 and 3.4 mm, and Σbone was between 2.2 and 2.6 mm, respectively. Females with larger breasts (p = 0.03), easily visible seroma (p ≤ 0.02) and open surgical technique (p ≤ 0.04) had larger Σlaser. Σbone was larger for females with larger breasts (p = 0.02) and lateral tumours (p = 0.04). Females with medial tumours (p < 0.01) had smaller Σbone., Conclusion: If clips are not used, margins should be 8 and 10 mm for bony anatomy verification and laser setup, respectively. Individualization of TB margins may be considered based on breast volume, TB and seroma visibility., Advances in Knowledge: Setup accuracy using lasers and bony anatomy is influenced by patient and treatment factors. Some patients may benefit from clip-based image guidance more than others.

The response of human normal tissues to radiotherapy fraction size is often described in terms of cellular recovery, but the causal links between cellular and tissue responses to ionising radiation are not necessarily straightforward. This article reviews the evidence for a cellular basis to clinical fractionation sensitivity in normal tissues and discusses the significance of a long-established inverse association between fractionation sensitivity and proliferative indices. Molecular mechanisms of fractionation sensitivity involving DNA damage repair and cell cycle control are proposed that will probably require modification before being applicable to human cancer. The article concludes by discussing the kind of correlative research needed to test for and validate predictive biomarkers of tumour fractionation sensitivity., (Copyright © 2015 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Purpose: To identify single-nucleotide polymorphisms (SNPs) in oxidative stress-related genes associated with risk of late toxicities in breast cancer patients receiving radiation therapy., Methods and Materials: Using a 2-stage design, 305 SNPs in 59 candidate genes were investigated in the discovery phase in 753 breast cancer patients from 2 prospective cohorts from Germany. The 10 most promising SNPs in 4 genes were evaluated in the replication phase in up to 1883 breast cancer patients from 6 cohorts identified through the Radiogenomics Consortium. Outcomes of interest were late skin toxicity and fibrosis of the breast, as well as an overall toxicity score (Standardized Total Average Toxicity). Multivariable logistic and linear regression models were used to assess associations between SNPs and late toxicity. A meta-analysis approach was used to summarize evidence., Results: The association of a genetic variant in the base excision repair gene XRCC1, rs2682585, with normal tissue late radiation toxicity was replicated in all tested studies. In the combined analysis of discovery and replication cohorts, carrying the rare allele was associated with a significantly lower risk of skin toxicities (multivariate odds ratio 0.77, 95% confidence interval 0.61-0.96, P=.02) and a decrease in Standardized Total Average Toxicity scores (-0.08, 95% confidence interval -0.15 to -0.02, P=.016)., Conclusions: Using a stage design with replication, we identified a variant allele in the base excision repair gene XRCC1 that could be used in combination with additional variants for developing a test to predict late toxicities after radiation therapy in breast cancer patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Delineation of clinical target volumes (CTVs) is a weak link in radiation therapy (RT), and large inter-observer variation is seen in breast cancer patients. Several guidelines have been proposed, but most result in larger CTVs than based on conventional simulator-based RT. The aim was to develop a delineation guideline obtained by consensus between a broad European group of radiation oncologists., Material and Methods: During ESTRO teaching courses on breast cancer, teachers sought consensus on delineation of CTV through dialogue based on cases. One teacher delineated CTV on CT scans of 2 patients, followed by discussion and adaptation of the delineation. The consensus established between teachers was sent to other teams working in the same field, both locally and on a national level, for their input. This was followed by developing a broad consensus based on discussions., Results: Borders of the CTV encompassing a 5mm margin around the large veins, running through the regional lymph node levels were agreed, and for the breast/thoracic wall other vessels were pointed out to guide delineation, with comments on margins for patients with advanced breast cancer., Conclusion: The ESTRO consensus on CTV for elective RT of breast cancer, endorsed by a broad base of the radiation oncology community, is presented to improve consistency., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)