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Preclinical data suggest that combined gamma-tocotrienol with pentoxifylline ameliorates radiotherapy-induced gastrointestinal damage.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.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.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.

Purpose: A molecular understanding of tissue sensitivity to radiotherapy fraction size is missing. Here, we test the hypothesis that sensitivity to fraction size is influenced by the DNA repair system activated in response to DNA double-strand breaks (DSB). Human epidermis was used as a model in which proliferation and DNA repair were correlated over 5 weeks of radiotherapy.Experimental design: Radiotherapy (25 fractions of 2 Gy) was prescribed to the breast in 30 women with early breast cancer. Breast skin biopsies were collected 2 hours after the 1st and 25th fractions. Samples of contralateral breast skin served as controls. Sections were coimmunostained for Ki67, cyclin A, p21, RAD51, 53BP1, and β1-integrin.Results: After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per millimeter of basement membrane (P = 0.002), of which the majority were in S/G2 phase, as judged by cyclin A staining (P < 0.0003). The p21 index rose from 2.8% to 87.4% (P < 0.0001) after 25 fractions, indicating cell cycle arrest. By week 5, there was a 4-fold increase (P = 0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, suggesting increasing activation of homologous recombination.Conclusions: Cell cycle arrest in S/G2 phase in the basal epidermis after a 5-week course of radiotherapy is associated with greater use of homologous recombination for repairing DSB. The high fidelity of homologous recombination, which is independent of DNA damage levels, may explain the low-fractionation sensitivity of tissues with high-proliferative indices, including self-renewing normal tissues and many cancers. Clin Cancer Res; 18(19); 5479–88. ©2012 AACR.

PDF file, 105K, Serial 3�m sections of epidermis stained with Cyclin A and p21 (brown) and counterstained with haematoxylin (blue). Overlay of serial sections using MATLAB 7.9 R2009b software shows strong uptake of p21 in Cyclin A positive cells.

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Purpose Hydrogen peroxide (H2O2) 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 H2O2 + 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 H2O2 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. H2O2 was mixed in 1% sodium hyaluronate gel (final H2O2 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 H2O2 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 H2O2 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.

Background This communication reports the identification of a new panel of transcriptional changes in inflammation-associated genes observed in response to ionising radiation received by radiotherapy patients. Methods Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 h post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Results Statistical analysis with BRB-ArrayTools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Conclusion Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specific responses in support of personalised approaches to radiation medicine.

This communication reports the identification of a new panel of transcriptional changes in inflammation-associated genes observed in response to ionising radiation received by radiotherapy patients. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 hours post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Statistical analysis with BRB Array Tools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specific responses in support of personalised approaches to radiation medicine.

Summary 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.

The RTGene study was focused on the development and validation of new transcriptional biomarkers for prediction of individual radiotherapy patient responses to ionizing radiation. In parallel, for validation purposes, this study incorporated conventional biomarkers of radiation exposure, including the dicentric assay. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumors. For the dicentric assay, two samples were taken from each patient: prior to radiotherapy and before the final fraction. Blood samples were set up using standard methods for the dicentric assay. All the baseline samples had dicentric frequencies consistent with the expected background for the normal population. For blood taken before the final fraction, all the samples displayed distributions of aberrations, which are indicative of partial-body exposures. Whole-body and partial-body cytogenetic doses were calculated with reference to a 250-kVp X-ray calibration curve and then compared to the dose to blood derived using two newly developed blood dosimetric models. Initial comparisons indicated that the relationship between these measures of dose appear very promising, with a correlation of 0.88 (P = 0.001). A new Bayesian zero-inflated Poisson finite mixture method was applied to the dicentric data, and partial-body dose estimates showed no significant difference (P > 0.999) from those calculated by the contaminated Poisson technique. The next step will be further development and validation in a larger patient group.

International audience; For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.

Voluntary inspiration breath hold (VIBH) for left breast cancer patients has been shown to be a safe and effective method of reducing radiation dose to the heart. Currently, VIBH protocol compliance is monitored visually. In this work, we establish whether it is possible to gate the delivery of radiation from an Elekta linac using the Microsoft Kinect version 2 (Kinect v2) depth sensor to measure a patient breathing signal. This would allow contactless monitoring during VMAT treatment, as an alternative to equipment–assisted methods such as active breathing control (ABC). Breathing traces were acquired from six left breast radiotherapy patients during VIBH. We developed a gating interface to an Elekta linac, using the depth signal from a Kinect v2 to control radiation delivery to a programmable motion platform following patient breathing patterns. Radiation dose to a moving phantom with gating was verified using point dose measurements and a Delta4 verification phantom. 60 breathing traces were obtained with an acquisition success rate of 100%. Point dose measurements for gated deliveries to a moving phantom agreed to within 0.5% of ungated delivery to a static phantom using both a conventional and VMAT treatment plan. Dose measurements with the verification phantom showed that there was a median dose difference of better than 0.5% and a mean (3% 3 mm) gamma index of 92.6% for gated deliveries when using static phantom data as a reference. It is possible to use a Kinect v2 device to monitor voluntary breath hold protocol compliance in a cohort of left breast radiotherapy patients. Furthermore, it is possible to use the signal from a Kinect v2 to gate an Elekta linac to deliver radiation only during the peak inhale VIBH phase.

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.

This study aimed to test whether induction of apoptosis following ex vivo X-irradiation of unstimulated blood lymphocytes correlated with clinical radiosensitivity and DNA double-strand break (DSB) repair in breast radiotherapy patients and healthy volunteers. Using small molecule inhibitors, the relationship between DSB repair and radiation-induced apoptosis was examined. Sixteen breast cancer patients with minimal (controls, n = 8) or extremely marked late radiation-induced change (cases, n = 8) and eight healthy volunteers were selected. DSBs were quantified by γH2AX/53BP1 immunofluorescence, and apoptosis was measured using a fluorogenic inhibitor of caspases assay. Mean γH2AX/53BP1 focus levels 24 h after exposure to 4 Gy were higher in cases (12.7 foci per cell) than in controls (10.3 foci per cell, p = 0.002). In contrast, the mean apoptotic fraction 48 h after 8 Gy was comparable, 37.2 % in cases and 34.7 % in controls (p = 0.442). Residual focus and apoptosis levels were not correlated within individuals (Spearman's R = -0.0059, p = 0.785). However, cells treated with DNA-PK inhibitor Nu7441 had higher focus and apoptosis levels 48 h after 1 Gy compared to mock-treated cells, suggesting that apoptosis induction following irradiation is modulated by DSB repair. This effect required functional ATM since cells treated simultaneously with Nu7441 and the ATM inhibitor Ku55933 were resistant to apoptosis despite high levels of residual foci. One clinical case displayed an impaired DNA-PK-dependent end-joining cellular phenotype. In summary, clinical radiosensitivity may be associated with impaired DSB repair in some patients. Although pharmaceutical inhibition of ATM and DNA-PK affected apoptosis induction and DSB repair, no association was observed between apoptosis and residual focus levels in patients and volunteers.

Many women diagnosed with early breast cancer will be cured and survive for many years after their initial diagnosis. Therefore it is important to take into account the risk of the development of new primary malignancies in such patients. Patients may be predisposed to develop new primary cancers as a result of genetic predisposition, environmental factors, or as a result of the adjuvant therapies used to treat early breast cancer. Treatment-related second primary cancers include an increased risk of breast and lung cancers following adjuvant radiotherapy, hematological malignancies following adjuvant chemotherapy, and endometrial cancer following tamoxifen. As a result of confounding factors the increased risk of these events differs between analyses, but the overall absolute risk of treatment-related second primary cancers remains low. Nonetheless it is important that patients are informed of these risks, modifiable risk factors are addressed and measures to minimize risks are undertaken, particularly when considering adjuvant therapies in women at low risks of recurrence.

Purpose: A molecular understanding of tissue sensitivity to radiotherapy fraction size is missing. Here, we test the hypothesis that sensitivity to fraction size is influenced by the DNA repair system activated in response to DNA double-strand breaks (DSB). Human epidermis was used as a model in which proliferation and DNA repair were correlated over 5 weeks of radiotherapy. Experimental design: Radiotherapy (25 fractions of 2 Gy) was prescribed to the breast in 30 women with early breast cancer. Breast skin biopsies were collected 2 hours after the 1st and 25th fractions. Samples of contralateral breast skin served as controls. Sections were coimmunostained for Ki67, cyclin A, p21, RAD51, 53BP1, and β1-integrin. Results: After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per millimeter of basement membrane (P = 0.002), of which the majority were in S/G2 phase, as judged by cyclin A staining (P < 0.0003). The p21 index rose from 2.8% to 87.4% (P < 0.0001) after 25 fractions, indicating cell cycle arrest. By week 5, there was a 4-fold increase (P = 0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, suggesting increasing activation of homologous recombination. Conclusions: Cell cycle arrest in S/G2 phase in the basal epidermis after a 5-week course of radiotherapy is associated with greater use of homologous recombination for repairing DSB. The high fidelity of homologous recombination, which is independent of DNA damage levels, may explain the low-fractionation sensitivity of tissues with high-proliferative indices, including self-renewing normal tissues and many cancers. Clin Cancer Res; 18(19); 5479–88. ©2012 AACR.

Purpose To test the association of DNA double-strand break (DSB) repair and chromosomal radiosensitivity in ex vivo irradiated blood lymphocytes with late-onset normal tissue responses following breast radiotherapy. Methods Breast cancer patients with minimal (controls) or marked late radiotherapy changes (cases) were retrospectively selected. DSB were quantified by γH2AX/53BP1 immunofluorescence microscopy 0.5 and 24h after exposure of unstimulated blood lymphocytes to 0.5 and 4Gy X-rays, respectively. Chromosomal aberrations were scored in blood lymphocyte metaphases after 6Gy X-rays. Results Despite similar foci levels at 0.5h in cases ( n =7) and controls ( n =7), foci levels 24h after 4Gy irradiation differed significantly between them (foci per cell were 12.8 in cases versus 10.2 in controls, p =0.004). Increased chromosomal radiosensitivity was also observed in cases (aberrations per cell were 5.84 in cases versus 3.79 in controls, p =0.001) with exchange and deletion type aberrations contributing equally to the difference between cases and controls. Residual foci correlated with formation of deletions (Spearman's R= 0.589, p =0.027) but not exchanges ( R =0.367, p =0.197) in blood lymphocytes from the same patients. Conclusions Higher levels of exchange type aberrations observed among radiosensitive breast cancer patients suggest a role for DSB misrepair, in addition to residual damage, as determinants of late normal tissue damage. Correlation of residual foci levels with deletion type aberration yields in the same cohort confirms their mechanistic linkage.

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.

Radiation-induced tissue fibrosis is a common adverse effect of curative treatment for pelvic cancer. Pilot studies testing alpha-tocopherol and pentoxifylline provide evidence of clinical regression of superficial radiation fibrosis after radiotherapy.Twenty-seven eligible research volunteers with a minimum of one grade 3 or 4 disability (LENT SOMA) due to previous radiotherapy were entered into the study. Volunteers were given dl-alpha tocopheryl acetate 500 mg twice a day orally plus pentoxifylline 400mg twice a day orally over a period of 6 months. Clinical assessment of late side effects recorded using LENT SOMA scales was selected as the primary endpoint, taken at baseline and at 6 and 12 months post-registration. Patient self-assessment of function and quality of life was assessed as a secondary endpoint using the EORTC QLQ-C30 core questionnaire and the EORTC QLQ-CR38 pelvic module. Magnetic resonance imaging was undertaken in 13/23 evaluable volunteers before and after 6 months of therapy.At 12 months post-registration there were 4 out of 23 responders. At 6 months post-registration there was a statistically significant improvement (i.e. reduction) in the median of the LENT SOMA summed scores in all areas assessed apart from 'male sexual dysfunction', 'vulva' and 'vagina' which were unchanged at 6 months. The median total LENT SOMA score at baseline and 6 months was 49 and 34, respectively, with a median change in total LENT SOMA score between baseline and 6 months of 9 (IQR 7-18) (P0.001). The maximum LENT SOMA scores improved over the study period, with a total number of 82 maximum grade 3 or 4 normal tissue scores at baseline (median of four complications per person) reduced to a total number of 67 maximum grade 3 or 4 scores at 6 months post-registration (median of 3 complications per person), i.e. a median reduction in severe complications of one per person. LENT SOMA scores at 12 months were similar to those observed at 6 month suggesting no further improvement nor deterioration in late side effects. These findings were, however, not reflected in the patient self-assessment of function and quality of life, raising question about the possibility of observer bias in recording LENT SOMA scores. No significant changes were reported on magnetic resonance images at 6 months from baseline.Despite only seeing four a priori defined responders in this pilot study testing dl-alpha tocopheryl acetate plus pentoxifylline in patients suffering complications of pelvic radiotherapy, changes in LENT SOMA scores suggest beneficial effects. However, we are not convinced that these effects are real, since no significant changes in symptoms and functional status were recorded by detailed prospective patient self-assessments.

Background and purpose Treatment-induced arm lymphoedema is a common and distressing complication of curative surgery and radiotherapy for early breast cancer. A number of studies testing alpha-tocopherol (vitamin E) and pentoxifylline suggest evidence of clinical regression of superficial radiation-induced fibrosis but there is only very limited evidence from randomised trials. Arm lymphoedema after lymphatic radiotherapy and surgery has been used in the present study as a clinical system for testing these drugs in a double-blind placebo-controlled randomised phase II trial. Patients and methods Sixty-eight eligible research volunteers with a minimum 20% increase in arm volume at a median 15.5 years (range 2–41) after axillary/supraclavicular radiotherapy (plus axillary surgery in 51/68 (75%) cases) were randomised to active drugs or placebo. All volunteers were given dl-alpha tocopheryl acetate 500mg twice a day orally plus pentoxifylline 400mg twice a day orally, or corresponding placebos, for 6 months. The primary endpoint was volume of the ipsilateral limb measured opto-electronically using a perometer and expressed as a percentage of the contralateral limb volume. Results At 12 months post-randomisation, there was no significant difference between treatment and control groups in terms of arm volume. Absolute change in arm volume at 12 months was 2.5% (95% CI −0.40 to 5.3) in the treatment group compared to 1.2% (95% CI −2.8 to 5.1) in the placebo group. The difference in mean volume change between randomisation groups at 12 months was not statistically significant ( P =0.6), −1.3% (95% CI −6.1 to 3.5), nor was there a significant difference in response at 6 months ( P =0.7), where mean change in arm volume from baseline in the treatment and placebo groups was −2.3% (95% CI −7.9 to 3.4) and −1.1% (95% CI −3.9 to 1.7), respectively. There were no significant differences between randomised groups in terms of secondary endpoints, including tissue induration (fibrosis) in the irradiated breast or chest wall, pectoral fold or supraclavicular fossa, change in photographic breast/chest wall appearance or patient self-assessment of function and Quality of Life at either 6 or 12 months. Conclusions The study fails to demonstrate efficacy of dl-alpha tocopheryl acetate plus pentoxifylline in patients with arm lymphoedema following axillary surgery and lymphatic radiotherapy, nor does it suggest any benefits of these drugs in radiation-induced induration (fibrosis) in the breast, chest wall, pectoral fold, axilla or supraclavicular fossa.

Background and purpose : To evaluate radiation-induced defects in myocardial perfusion imaging in early breast cancer patients treated with modern technique radiotherapy. Patients and methods : Twenty-four patients with left-breast tumours and 12 control patients with right-breast tumours, relapse-free since treatment for primary disease, who had undergone radiotherapy at least 5 years previously and with no history of ischaemic heart disease prior to radiotherapy underwent study. In left-breast patients, at least 1 cm of heart was required to have been in the treatment field. Patients underwent cardiac assessment and single photon emission computerized tomography myocardial perfusion imaging. Results : Myocardial perfusion tracer uptake was abnormal in 17 (70.8%) left-breast and two (16.7%) right-breast patients ( P =0.002). Of the 17 abnormal scans in left-breast patients, abnormalities were confined to the cardiac apex in 16 patients, and perfusion defects were reversible ( n =7), fixed ( n =7) or mixed ( n =3). Reversible perfusion defects that were not confined to the cardiac apex were observed in two right-breast patients. Left ventricular ejection fraction was normal in all 33 patients in whom it was measured, and no myocardial perfusion abnormalities were judged to require treatment or follow-up. Conclusions : In this selected study population modern technique radiotherapy to the left breast was associated with a significantly greater number of myocardial perfusion abnormalities than radiotherapy to the right breast. These abnormalities were both reversible and irreversible, suggesting that radiotherapy can lead to both myocardial damage and to epicardial coronary disease. With a minimum of 5 years follow-up since treatment, no abnormalities were considered to be clinically significant.

Until recently, elective treatment of the lymphatic pathways in women with early invasive breast cancer was assumed to impact on quality of life rather than on overall survival. In a multidisciplinary breast clinic these considerations underpinned a policy of observation of the lymphatic pathways if axillary lymph nodes were not palpably enlarged and if recommendations for adjuvant systemic therapy did not depend on knowledge of pathological node status. This paper evaluates the long-term outcome of the observation policy in terms of lymphatic morbidity due to cancer recurrence.Seven hundred and fifty-nine patients with operable breast cancer and suitable for breast conserving surgery were seen between January 1984 and December 1994. Of these, 291 (38.3%) were recommended a policy of observation to the lymphatic pathways. The case records of these patients were reviewed to record regional relapse patterns and morbidity.At a median follow up of 60 months, 32/291 (11%) patients suffered ipsilateral lymphatic relapse at some stage prior to death or last follow up, representing a 22% actuarial 10-year risk of lymphatic relapse. Metastases coincided with, or preceded, lymphatic relapse in 8/32 (25%) patients. Eighteen out of 32 (56%) patients suffered symptoms of lymphatic relapse prior to death or last follow up, despite subsequent surgery, radiotherapy and/or systemic therapies. The absolute risk of symptomatic ipsilateral lymphatic relapse in the observation group was 18/291 (6.2%), representing an actuarial 10-year risk of 17%.A policy of observation on the axilla with deferred treatment of lymphatic relapse has benefited 273/291 (94%) patients, but at the expense of cancer-related regional morbidity in 18 (6%) patients. We conclude that the cancer-related morbidity suffered by a minority of patients and the strengthening evidence of an overall survival benefit conferred by elective local-regional therapy favours a policy of elective treatment of the lymphatic pathways in the routine setting.

The aim of this study was to review the ability to control symptoms of regional lymphatic relapse in women with early breast cancer. A retrospective study was made of 759 consecutive women presenting with stage 1 or 2 breast cancer treated by breast conserving surgery and radiotherapy between June 1984 and December 1994, 291 (38.3%) of whom were managed by a policy of observation on the lymphatic pathways. Patterns of lymphatic relapse, relapse management and morbidity caused by recurrent malignancy were reviewed from the case notes. The overall rate of relapse in the ipsilateral axilla and/or supraclavicular fossa was 76/759 (10%) at any time prior to death or last follow-up. 34 of 65 patients who relapsed in the axilla did so despite prior axillary surgery and/or radiotherapy. 41 of 76 patients with regional recurrence presented with symptoms, including lymphoedema, arm pain or sensory motor changes. These symptoms were poorly controlled by palliative surgery, radiotherapy or systemic therapy in 23 cases, including 12 who progressed to arm paralysis. Symptomatic control of patients with regional lymphatic relapse can be very difficult, even in women under regular surveillance in a multidisciplinary breast cancer clinic.

Objective: The purpose of this UK study was to evaluate interfraction reproducibility and body image score when using ultraviolet (UV) tattoos (not visible in ambient lighting) for external references during breast/chest wall radiotherapy and compare with conventional dark ink. Methods: In this non-blinded, single-centre, parallel group, randomized control trial, patients were allocated to receive either conventional dark ink or UV ink tattoos using computer-generated random blocks. Participant assignment was not masked. Systematic (∑) and random (σ) setup errors were determined using electronic portal images. Body image questionnaires were completed at pre-treatment, 1 month and 6 months to determine the impact of tattoo type on body image. The primary end point was to determine that UV tattoo random error (σsetup) was no less accurate than with conventional dark ink tattoos, i.e.

Background: a non-randomised phase II study suggested a therapeutic effect of hyperbaric oxygen (HBO) therapy on arm lymphoedema following adjuvant radiotherapy for early breast cancer, justifying further investigation in a randomised trial.Methods: fifty-eight patients with ?15% increase in arm volume after supraclavicular ± axillary radiotherapy (axillary surgery in 52/58 patients) were randomised in a 2:1 ratio to HBO (n = 38) or to best standard care (n = 20). The HBO group breathed 100% oxygen at 2.4 atmospheres absolute for 100 min on 30 occasions over 6 weeks. Primary endpoint was ipsilateral limb volume expressed as a percentage of contralateral limb volume. Secondary endpoints included fractional removal rate of radioisotopic tracer from the arm, extracellular water content, patient self-assessments and UK SF-36 Health Survey Questionnaire.Findings: of 53/58 (91.4%) patients with baseline assessments, 46 had 12-month assessments (86.8%). Median volume of ipsilateral limb (relative to contralateral) at baseline was 133.5% (IQR 126.0–152.3%) in the control group, and 135.5% (IQR 126.5–146.0%) in the treatment group. Twelve months after baseline the median (IQR) volume of the ipsilateral limb was 131.2% (IQR 122.7–151.5%) in the control group and 133.5% (IQR 122.3–144.9%) in the treatment group. Results for the secondary endpoints were similar between randomised groups.Interpretation: no evidence has been found of a beneficial effect of HBO in the treatment of arm lymphoedema following primary surgery and adjuvant radiotherapy for early breast cancer

AimsSerial photographs have been collected prospectively to evaluate the effect of radiotherapy on normal tissues in the breast. The aim of this study was to compare two methods of scoring radiation-induced changes.Materials and methodsFive-year photographs of 400 patients randomised to receive either 42.9 or 39 Gy in 13 fractions to the whole breast after tumour excision of early breast cancer were compared with a post-surgery baseline and scored for change in breast appearance on a three-point graded scale. Two alternative methods of scoring using three observers were compared: (a) scores allocated independently, with independent resolution of discrepancies, and (b) scores allocated by consensus.ResultsTreatment effects estimated from the consensus and independent scores were very similar (odds ratio 1.89, 95% confidence interval 1.21–2.96 vs 2.28, 95% confidence interval 1.50–3.47, respectively). Agreement between the scores obtained from each method was reasonable, and the repeatability of the consensus method was good.ConclusionsThe consensus method of scoring photographic change in breast appearance seems to be no less sensitive to randomised dose as the independent method of assessment, but is much quicker to administer. The consensus method has been used to score over 3000 sets of photographs in the National Cancer Research Institute Standardisation of Breast Radiotherapy trial.

Background Radiation dose distributions created by two dimensional (2D) treatment planning are responsible for partial volumes receiving >107% of the prescribed dose in a proportion of patients prescribed whole breast radiotherapy after tumour excision of early breast cancer. These may contribute to clinically significant late radiation adverse effects. Aim To test three dimensional (3D) intensity modulated radiotherapy (IMRT) against 2D dosimetry using standard wedge compensators in terms of late adverse effects after whole breast radiotherapy. Methods Three hundred and six women prescribed whole breast radiotherapy after tumour excision for early stage cancer were randomised to 3D IMRT (test arm) or 2D radiotherapy delivered using standard wedge compensators (control arm). All patients were treated with 6 or 10MV photons to a dose of 50Gy in 25 fractions to 100% in 5 weeks followed by an electron boost to the tumour bed of 11.1Gy in 5 fractions to 100%. The primary endpoint was change in breast appearance scored from serial photographs taken before radiotherapy and at 1, 2 and 5 years follow up. Secondary endpoints included patient self-assessments of breast discomfort, breast hardness, quality of life and physician assessments of breast induration. Analysis was by intention to treat. Results 240 (79%) patients with 5-year photographs were available for analysis. Change in breast appearance was identified in 71/122 (58%) allocated standard 2D treatment compared to only 47/118 (40%) patients allocated 3D IMRT. The control arm patients were 1.7 times more likely to have a change in breast appearance than the IMRT arm patients after adjustment for year of photographic assessment (95% confidence interval 1.2–2.5, p =0.008). Significantly fewer patients in the 3D IMRT group developed palpable induration assessed clinically in the centre of the breast, pectoral fold, infra-mammary fold and at the boost site. No significant differences between treatment groups were found in patient reported breast discomfort, breast hardness or quality of life. Conclusion This analysis suggests that minimisation of unwanted radiation dose inhomogeneity in the breast reduces late adverse effects. Incidence of change in breast appearance was statistically significantly higher in patients in the standard 2D treatment arm compared with the IMRT arm. A beneficial effect on quality of life remains to be demonstrated.

Background and purpose Tissue hardness (induration), pain and tenderness are common late adverse effects of curative radiotherapy for early breast cancer. The purpose of this study was to test the efficacy of IH636 grape seed proanthocyanidin extract (GSPE) in patients with tissue induration after high-dose radiotherapy for early breast cancer in a double-blind placebo-controlled randomised phase II trial. Patients and methods Sixty-six eligible research volunteers with moderate or marked breast induration at a mean 10.8 years since radiotherapy for early breast cancer were randomised to active drug ( n =44) or placebo ( n =22). All patients were given grape seed proanthocyanidin extract (GSPE) 100mg three times a day orally, or corresponding placebo capsules, for 6 months. The primary endpoint was percentage change in surface area (cm 2 ) of palpable breast induration measured at the skin surface 12 months after randomisation. Secondary endpoints included change in photographic breast appearance and patient self-assessment of breast hardness, pain and tenderness. Results At 12 months post-randomisation, ≥50% reduction in surface area (cm 2 ) of breast induration was recorded in13/44 (29.5%) GSPE and 6/22 (27%) placebo group patients (NS). At 12 months post-randomisation, there was no significant difference between treatment and control groups in terms of external assessments of tissue hardness, breast appearance or patient self-assessments of breast hardness, pain or tenderness. Conclusions The study failed to show efficacy of orally-adminstered GSPE in patients with breast induration following radiotherapy for breast cancer.

Background : Radiation-induced arm lymphoedema is a common and distressing complication of curative treatment for early breast cancer. Hyperbaric oxygen (HBO 2 ) therapy promotes healing in bone rendered ischaemic by radiotherapy, and may help some soft-tissue injuries too, but is untested in arm lymphoedema. Methods: Twenty-one eligible research volunteers with a minimum 30% increase in arm volume in the years after axillary/supraclavicular radiotherapy (axillary surgery in 18/21 cases) were treated with HBO 2 . The volunteers breathed 100% oxygen at 2.4 ATA for 100 min in a multiplace hyperbaric chamber on 30 occasions over a period of 6 weeks. The volume of the ipsilateral limb, measured opto-electronically by a perometer and expressed as a percentage of contralateral limb volume, was selected as the primary endpoint. A secondary endpoint was local lymph drainage expressed as fractional removal rate of radioisotopic tracer, measured using lymphoscintigraphy. Results : Three out of 19 evaluable patients experienced >20% reduction in arm volume at 12 months. Six out of 13 evaluable patients experienced a >25% improvement in 99 Tc-nanocolloid clearance rate from the ipsilateral forearm measured by quantitative lymphoscintigraphy at 12 months. Overall, there was a statistically significant, but clinically modest, reduction in ipsilateral arm volume at 12 months follow-up compared with baseline ( P =0.005). The mean percentage reduction in arm volume from baseline at 12 months was 7.51. Moderate or marked lessening of induration in the irradiated breast, pectoral fold and/or supraclavicular fossa was recorded clinically in 8/15 evaluable patients. Twelve out of 19 evaluable patients volunteered that their arms felt softer, and six reported improvements in shoulder mobility at 12 months. No significant improvements were noted in patient self-assessments of quality of life. Conclusion : Interpretation is limited by the absence of a control group. However, measurement of limb volume by perometry is reportedly reliable, and lymphoscintigraphy is assumed to be operator-independent. Taking all data into account, there is sufficient evidence to justify a double-blind randomised controlled trial of hyperbaric oxygen in this group of patients.

Background: radiation fibrosis is not easily measurable although clinical scores have been developed for this purpose. Biomarkers present an alternative more objective approach to quantification, and estimation in blood provides accessible samples. We investigated if blood cytokines could be used to measure established fibrosis in patients who have undergone radiotherapy for breast cancer.Methods: we studied two cohorts treated by breast-conserving surgery and radiotherapy in the UK START Trial A, one with breast fibrosis (cases) and one with no or minimal fibrosis (controls). Two candidate cytokines, plasma connective tissue growth factor (CTGF) and serum interleukin-6 (IL6) were estimated by ELISA. Comparisons between cases and controls used the t-test or Mann–Whitney test and associations between blood concentration and clinical factors were assessed using the Spearman rank correlation coefficient.Results: seventy patients were included (26 cases, 44 controls). Mean time since radiotherapy was 9.9 years (range 8.3-12.0). No statistically significant differences between cases and controls in serum IL6 (median (IQR) 0.84 pg/ml (0.57-1.14), 0.75 pg/ml (0.41-1.43) respectively) or plasma CTGF (331.4 pg/ml (234.8-602.9), 334.5 pg/ml (270.0-452.8) were identified. There were no significant associations between blood cytokine concentration and age, fibrosis severity, breast size or time since radiotherapy.Conclusions: no significant difference in IL6 or CTGF concentrations was detected between patients with breast fibrosis and controls with minimal or no fibrosis

Background : Radiation-induced brachial plexopathy (RIBP) is an untreatable complication of curative radiotherapy for early breast cancer, characterized by chronic neuropathic pain and limb paralysis. Hyperbaric oxygen (HBO 2 ) therapy is known to promote healing of tissue rendered ischaemic by radiotherapy, but is untested in RIBP. Methods : Thirty four eligible research volunteers suffering from RIBP were randomized to HBO 2 or control group. The HBO 2 group breathed 100% oxygen for 100 min in a multiplace hyperbaric chamber on 30 occasions over a period of 6 weeks. The control group accompanied the HBO 2 group and breathed a gas mixture equivalent to breathing 100% oxygen at surface pressure. All volunteers and investigators, except the operators of the hyperbaric chamber and the trial statistician, were blind to treatment assignments. The warm sensory threshold, which measures the function of small sensory fibres, was selected as the primary endpoint. Findings : Pre-treatment neurophysiological tests were grossly abnormal in the affected hand compared to the unaffected hand in both HBO 2 and control groups, as expected, but no statistically significant differences were noted in either group at any time up to 12 months post-treatment. However, normalization of the warm sensory threshold in two of the HBO 2 group was reliably recorded. Two cases with marked chronic arm lymphoedema reported major and persistent improvements in arm volume for at least 12 months after treatment with HBO 2 . Interpretation : There is no reliable evidence to support the hypothesis that HBO 2 therapy slows or reverses RIBP in a substantial proportion of affected individuals, although improvements in warm sensory threshold offer some suggestion of therapeutic effect. Improvement in long-standing arm lymphoedema was not anticipated, and justifies further investigation.

11116 Background: Critical opinions for the lack of success of DNA double-strand break (DSB) repair as a predictive marker of normal tissue radiosensitivity include the argument that in vitro cellular responses correlate poorly with in vivo responses due to the modifying influence of tissue environment. In this study, we test the hypothesis that a DNA damage assay based on in vivo irradiated skin tissues better predicts clinical responses in human skin, as opposed to the same assay performed in ex vivo irradiated lymphocytes. Methods: DSB levels (24 h post-4 Gy) were quantified using γH2AX/53BP1 immunostaining in irradiated skin tissues and G0 lymphocytes of 35 breast RT patients. Patients were selected on the basis of late RT effects in their breast and individuals with marked or minimal effects were classified as cases and controls, respectively. Risk factors of late effects established from multivariate analyses of outcomes of two breast RT trials were also considered in patient selection. They were 1) total RT dose, 2) RT dosimetry, 3) tumour bed boost, 4) breast size, 5) surgical cavity, and 6) axillary treatment. Results: Clinical parameters were balanced in both patient groups. Residual foci levels in skin epidermis and dermis were comparable between cases (n = 20) and controls (n = 15). Mean foci per cell were 3.29 in cases, 2.80 in controls for dermal fibroblasts (p = 0.07); 3.28 in cases, 2.60 in controls for endothelial cells (p = 0.08); 2.87 in cases, 2.41 in controls for superficial keratinocytes (p = 0.45); 2.32 in cases, 2.35 in controls for basal keratinocytes (p = 0.27). Residual foci levels in lymphocytes were however significantly higher among cases (foci per cell = 12.1) compared to controls (foci per cell = 10.3, p = 0.01). Of the different cell types, only residual foci levels of dermal fibroblasts and lymphocytes correlated with clinical severity (R = 0.722, p < 0.001; 0.593, p = 0.01, respectively). Interestingly, foci levels were not correlated between skin cells and lymphocytes of the same patients. Conclusions: DSB repair of ex vivo irradiated lymphocytes appears to be a better predictive marker of late effects to breast RT than DSB repair of in vivo irradiated skin.

Department of Radiotherapy, The Royal Marsden NHS Foundation Trust, Sutton, UK; b The Institute of Cancer Research Clinical Trials and Statistics Unit (ICR-CTSU), Sutton, UK; Diving Diseases Research Centre, The Hyperbaric Medical Centre, Plymouth, UK; Hyperbaric Unit, BUPA Hospital Hull & East Riding, Kingston-Upon-Hull, UK; Hyperbaric Medicine Unit, Royal Hospital Haslar, Gosport, UK; f London Hyperbaric, Whipps Cross University Hospital, Leytonstone, UK; g Lymphoedema Services, The Royal Marsden NHS Foundation Trust, Sutton, UK; Department of Nuclear Medicine, The Royal Marsden NHS Foundation Trust, Sutton, UK; Division of Physiological Medicine (Dermatology), St. George’s Hospital Medical School, London, UK

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.