To investigate the relationship of HIF1 alpha signaling to oxidative stress, tissue hypoxia, angiogenesis and inflammation, female Fischer 344 rats were irradiated to the right hemithorax with a fractionated dose of 40 Gy (8 Gy X 5 days). The lung tissues were harvested before and at 4, 6, 10, 14, 18, 22 and 26 weeks after irradiation for serial studies of biological markers, including markers for hypoxia (HIF1 alpha, pimonidazole and CA IX), oxidative stress (8-OHdG), and angiogenesis/capillary proliferation (VEGF/CD 105), as well as macrophage activation (ED-1) and cell signaling/fibrosis (NF kappa B, TGF beta 1), using immunohistochemistry and Western blot analysis. HIF1 alpha staining could be observed as early as 4 weeks postirradiation and was significantly increased with time after irradiation. Importantly, HIF1 alpha levels paralleled oxidative stress (8-OHdG), tissue hypoxia (pimonidazole and CA IX), and macrophage accumulation consistent with inflammatory response. Moreover, changes in HIF1 alpha expression identified by immunohistochemistry assay parallel the changes in TGF beta 1, VEGF, NF kappa B and CD 105 levels in irradiated lungs. These results support the notion that oxidative stress and tissue hypoxia might serve as triggering signals for HIF1 alpha activity in irradiated lungs, relating to radiation-induced inflammation, angiogenesis and fibrosis. (C) 2010 by Radiation Research Society

Chronic production of reactive oxygen and nitrogen species is an underlying mechanism of irradiation (IR)-induced lung injury The purpose of this study was to determine the optimum time of delivery of an antioxidant and redox-modulating Mn porphyrin, MnTE-2-PyP5+, to mitigate and/or treat IR-induced lung damage Female Fischer-344 rats were irradiated to their right hemithorax (28 Gy) Irradiated animals were treated with PBS or MnTE-2-PyP5+ (6 mg /kg/24 h) delivered tot 2 weeks by sc-implanted osmotic pumps (beginning after 2, 6, 12, 24, or 72 h or 8 weeks) Animals were sacrificed 10 weeks post-IR Endpoints were body weight, breathing frequency. histopathology, and immunohistochemistry (8-OHdG, ED-1, TGE-beta, HIF-1 alpha,VEGF A) A significant radioprotective effect on functional injury, measured by breathing frequency, was observed for all animals treated with MnTE-2-PyP5+ Treatment with MnTE-2-PyP5+ starting 2, 6, and 12 h but not after 24 or 72 h resulted in a significant decrease in immunostaining for 8-OHdG, HIF-1 alpha, TGF-beta and VEGF A A significant decrease in HIF-1 alpha, TGF-beta, and VEGF A, as well as an over all reduction in lung damage (histopathology), was observed in animals beginning treatment at the time of fully developed lung injury (8 weeks post-IR) The catalytic manganese porphyrin antioxidant and modulator of redox-based signaling pathways MnTE-2-PyP5+ mitigates radiation-induced lung injury when given within the first 12 h after IR More importantly. this is the first study to demonstrate that MnTE-2-PyP5+ can reverse overall lung damage when started at the time of established lung injury 8 weeks post-IR The radioprotective effects are presumably mediated through its ability both to suppress oxidative stress and to decrease activation of key transcription factors and proangiogenic and profibrogenic cytokines (C) 2010 Elsevier Inc All rights reserved

To understand the molecular events involved in radiation-induced pulmonary injury, we analyzed expression and post-translational changes in regulatory proteins of the lung after irradiation. Lung lysates from Fisher 344 rats were prepared 1,3 and 7 days, 2, 4, 6, 8, and 14 weeks after hemithoracic radiation (28 Gy) and probed by Western blot for nitric oxide synthase-2 (NOS-2), heme oxygenase-1 (HO-1), β-catenin, IκBα, NF-κB p65, Akt-1, hypoxia-inducible factor-1α (HIF-1α), and factor inhibiting HIF-1 (FIH-1). Loading controls were actin and Mn-superoxide dismutase. Radiation stimulates parallel biphasic expression changes in iNOS, HO-1 and p65 with significant increases days 1-7 that partially decrease by 2 weeks followed by a secondary phase of increased expression at weeks 6-8. At 14 weeks NOS-2 protein levels remain elevated compared to non-irradiated controls whereas changes in the levels of the other proteins are negligible. Changes in NOS-2 protein were confirmed by immunohistochemistry showing parallel changes in protein tyrosine nitration. Radiation stimulates HIF-1 expression from day 1 through 8 weeks but does not alter FIH-1 protein levels. IκBα, β-catenin, and Akt-1 levels are unchanged through week 1 but by 2 weeks drop significantly. These molecular changes correlate temporally with physiological changes previously measured including transient hypoxia during the first week post-irradiation and early acute inflammatory, intermediate acute, and chronic inflammatory responses to radiation (Fleckenstein et al., 2007, IJROBP 68:196-204). In vitro radiation stimulates the tyrosine nitration of IκBα, activating the pro-inflammatory transcription factor NF-κB (Yakovlev et al., 2007 Biochemistry 46:11671-83). In the lung, nitration of IκBα was also observed with similar oscillating changes in nitration as observed in vitro. Since protein tyrosine nitration is a marker of pro-inflammatory conditions, we tested whether a NOS inhibitor in the drinking water (L-N-nitroarginine, 0.5 gm/l) of rats 2 days prior to irradiation blocked protein nitration in the rat lung measured immunohistochemically 2 days post-radiation. The anti-inflammatory activity of HO-1 may be a consequence of its enzymatic product, carbon monoxide. We therefore tested whether ip injection once daily (150 μg) of the CO donor, CORM-3, also blocked the initial inflammatory response to radiation. Both the NOS inhibitor and CO donor blocked the initial inflammatory response to radiation. These results suggest possible approaches to prevent late pulmonary injury as a consequence of radiation.

Zielsetzung: Die Rezidivrate nach brusterhaltender Chirurgie des Mammakarzinoms wird durch adjuvante Ganzbrustbestrahlung mit zusätzlichem Boost deutlich gesenkt. Ein Boost als Intraoperative Strahlentherapie (IORT) könnte theoretisch das Risiko verringern, verbleibende Tumorzellen im Tumorbett mit einem externen Boost zu verfehlen. Ziel war es das Normalgewebsrisiko nach der Kombination eines intraoperativen Boost mit 50kV Röntgenstrahlung und externer, konventioneller Ganzbrustbestrahlung zu modellieren.

Accelerated partial breast radiotherapy with low-energy photons from a miniature X-ray machine is undergoing a randomized clinical trial (Targeted Intra-operative Radiation Therapy [TARGIT]) in a selected subgroup of patients treated with breast-conserving surgery. The steep radial dose gradient implies reduced tumor cell control with increasing depth in the tumor bed. The purpose was to compare the expected risk of local recurrence in this nonuniform radiation field with that after conventional external beam radiotherapy.The relative biologic effectiveness of low-energy photons was modeled using the linear-quadratic formalism including repair of sublethal lesions during protracted irradiation. Doses of 50-kV X-rays (Intrabeam) were converted to equivalent fractionated doses, EQD2, as function of depth in the tumor bed. The probability of local control was estimated using a logistic dose–response relationship fitted to clinical data from fractionated radiotherapy.The model calculations show that, for a cohort of patients, the increase in local control in the high-dose region near the applicator partly compensates the reduction of local control at greater distances. Thus a “sphere of equivalence” exists within which the risk of recurrence is equal to that after external fractionated radiotherapy. The spatial distribution of recurrences inside this sphere will be different from that after conventional radiotherapy.A novel target volume concept is presented here. The incidence of recurrences arising in the tumor bed around the excised tumor will test the validity of this concept and the efficacy of the treatment. Recurrences elsewhere will have implications for the rationale of TARGIT.

Purpose: Intraoperative radiotherapy (IORT) as a boost for breast cancer delivers a high single dose of radiation to a late-reacting tissue; therefore late toxicity is of particular interest, and long-term follow-up is warranted. To date there are only limited data available on breast cancer patients treated with IORT using low energy X-rays. We analyzed toxicity and cosmesis after IORT as a boost with a minimum follow-up of 18 months.Methods and Materials: A total of 73 patients treated with IORT (20 Gy/50 kV X-rays; INTRABEAM [Carl Zeiss Surgical, Oberkochen, Germany]) to the tumor bed during breast-conserving surgery as a boost followed by whole-breast radiotherapy (WBRT, 46 Gy) underwent a prospective, predefined follow-up (median, 25 months; range 18 44 months), including clinical examination and breast ultrasound at 6-months and mammographies at 1-year intervals. Toxicities were documented using the common toxicity criteria (CTC)/European Organization for Research and Treatment of Cancer and the LENT-SOMA score. Cosmesis was evaluated with a score from I to 4.Results: The IORT in combination with WBRT was well tolerated, with no Grade 3 or 4 skin toxicities and no telangiectasias. Fibrosis of the entire breast was observed in 5% of the patients. A circumscribed fibrosis around the tumor bed was palpable in up to 27% with a peak around 18 months after therapy and a decline thereafter. The observed toxicitiy rates were not influenced by age, tumor stage, or systemic therapy. The cosmetic outcome was good to excellent in >= 90% of cases.Conclusions: After IORT of the breast using low-energy X-rays, no unexpected toxicity rates were observed during long-term-follow-up. (c) 2006 Elsevier Inc.

An important strategy for improving cancer therapy is the protection of healthy cells against therapy-induced damage. In this context gene therapy can complement radio- and chemotherapy in the treatment of tumours. Retroviral overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, can deliver such a protective effect. P-gp possesses two relevant functions: its detoxifying effect acts as an efflux pump and could be important in high-dose chemotherapy, the anti-apoptotic effect of overexpressed P-gp could protect normal cells in tumour radio- or chemotherapy. The protection of haematopoietic stem cells in lymphoma, myeloma or solid tumor therapy is a promising candidate for this approach. However, little is known which other gene expressions might be influenced by MDR1-overexpression. Therefore we analyzed differentially expressed genes in the human lymphoblastoid cell line TK6 transduced with onco-retrovirus SF91m3 encoding MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). 61 annotated genes showed a significant change in expression (p<10−4) in MDR1 overexpressing compared to control transduced (overexpression the neomycin resistance gene) and untransduced cells. We found that genes coding for detoxifying and exocytotis proteins (e.g. ABCB4, ALDH1A, unc13) were up-regulated. Furthermore several proapoptotic genes were down-regulated (e.g. Casp1, Casp4) with concomitant increased expression of antiapoptotic genes (e.g. Akt3). The differential expressions for several genes were verified with real-time PCR. The results confirmed the dependence of these gene regulations on MDR1 overexpression. The influence of MDR1/P-gp overexpression on apoptotic signalling and cell survival was further corroborated by reduced apoptosis rates in response to irradiation in SF91m3 transduced cells compared to the control cell lines. Our genomics results support the findings from other studies that overexpression of MDR1 confers protection against apoptotic stimuli. The multidrug resistance phenotype does not seem to be only due to high P-gp expression but also to other metabolic proteins. Our results could have important implications for MDR1-gene therapy in patients receiving chemotherapy regimens in combination with radiotherapy.

Purpose/Objective: To prospectively measure and characterize neurocognitive functioning early after the beginning of radiation therapy (RT) depending on radiation dose and volume in patients with and without brain tumors.Materials/Methods: Eighty-six patients (median age 58.0 years, age range, 26–76 yr) were studied before and 1–4 fractions after the beginning of RT. Most of the patients had breast carcinoma, small-cell lung cancer (SCLC), non-SCLC, or meningioma. The neuropsychological evaluation consisted of the Auditory Verbal Learning Test (AVLT), the Medical College of Georgia (MCG) Complex Figures, and a highly sensitive, computerized test battery developed for the assessment of attentional deficits in patients with cerebral lesions in the German-speaking part (Test for Attentional Performance, TAP). Only standardised neuropsychological instruments (all with published normative data) were used. Each session lasted about 1 hour. Premorbid intellectual functioning, emotional distress, KPS, cancer diagnoses, and brain lesion location were controlled.Results: Analyses of covariance with baseline score and proactive interference score as covariates found a significant effect for supra-span working memory, divided attention (error rate), and treatment group (p<.02), favoring control (non-CNS RT) patients and patients without brain tumors in contrast to patients with brain tumors. Furthermore, the patients after 3 Gy whole brain radiation therapy had worse neurocognitive functioning compared to patients after 1.8 Gy partial brain RT. There was no significant difference between treatment groups based on verbal learning, immediate and delayed recall, recognition, visuoconstruction, focused attention, alertness, and visual memory previously shown to be sensitive in detecting radiotherapy-associated impairment. Figure 1 presents the group mean pre-post percentile scores on the supra-span immediate memory task relative to the median (50th percentile) score of the standardization sample (CG: control group, PB: partial brain irradiation, PCI: prophylactic cranial irradiation, TCI: therapeutic cranial irradiation, RS: radiosurgery). Purpose/Objective: To prospectively measure and characterize neurocognitive functioning early after the beginning of radiation therapy (RT) depending on radiation dose and volume in patients with and without brain tumors. Materials/Methods: Eighty-six patients (median age 58.0 years, age range, 26–76 yr) were studied before and 1–4 fractions after the beginning of RT. Most of the patients had breast carcinoma, small-cell lung cancer (SCLC), non-SCLC, or meningioma. The neuropsychological evaluation consisted of the Auditory Verbal Learning Test (AVLT), the Medical College of Georgia (MCG) Complex Figures, and a highly sensitive, computerized test battery developed for the assessment of attentional deficits in patients with cerebral lesions in the German-speaking part (Test for Attentional Performance, TAP). Only standardised neuropsychological instruments (all with published normative data) were used. Each session lasted about 1 hour. Premorbid intellectual functioning, emotional distress, KPS, cancer diagnoses, and brain lesion location were controlled. Results: Analyses of covariance with baseline score and proactive interference score as covariates found a significant effect for supra-span working memory, divided attention (error rate), and treatment group (p<.02), favoring control (non-CNS RT) patients and patients without brain tumors in contrast to patients with brain tumors. Furthermore, the patients after 3 Gy whole brain radiation therapy had worse neurocognitive functioning compared to patients after 1.8 Gy partial brain RT. There was no significant difference between treatment groups based on verbal learning, immediate and delayed recall, recognition, visuoconstruction, focused attention, alertness, and visual memory previously shown to be sensitive in detecting radiotherapy-associated impairment. Figure 1 presents the group mean pre-post percentile scores on the supra-span immediate memory task relative to the median (50th percentile) score of the standardization sample (CG: control group, PB: partial brain irradiation, PCI: prophylactic cranial irradiation, TCI: therapeutic cranial irradiation, RS: radiosurgery).