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1124 Survival Progeny Derived From Irradiated Parental Cells – Phenotype Analysis and Correlation With Tumour Progression
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males − 14 (42%). Patients ages ranged from 14 to 38 years, median age − 17. In 20 of 33 patients (60.6%) primary tumor were localized in distal femur, in 8 (24.2%) − in proximal tibia and in 5 (15.2%) − in different parts of fibula. In 30 of 33 patients lung metastasises were detected by x-ray examination and in 3 patients by computer tomography. Results: Bilateral metastatic lesions of lungs were detected in 20 (60.6%) and unilateral in 13 (in 6 (9.4%) − in upper lobe of left lung, in 4 (12.4%) − in lower lobe of right lung and in 3 (12.1%) − in upper lobe of right lung) patients. In 31of 33 patients (94%) lung metastases developed during the period from 1 to 24 months after the primary tumor development, and in 2 patients (6%) during the primary tumor development. The numbers of metastatic nodules were 1−7, middle 2 nodules. Single lung metastases were detected in 9 (27.2%) and multiple in 24 (72.8%) patients. Metastatic nodules sizes in detection time were from 5 mm up to 25 mm, median − 14.3 mm. In 4 of 33 patients after lung x-ray examination were performed computer tomography there were detected additional metastatic nodules and difference in nodules sizes. Conclusion: The target organ of osteosarcoma metastatic spread is lung and in most cases had been affected both lungs. Feature of metastatic osteosarcoma is multiple affecting of lung. 1121 BRAF Mutations in Thyroid Carcinomas Following Childhood Scalp Irradiation P. Boaventura1 , D. Pereira1 , J. Teixeira-Gomes1 , P. Soares1,2 . 1 IPATIMUP − Institute of Molecular Pathology and Immunology of the University of Porto, Cancer Biology, Porto, Portugal, 2 Medical Faculty, University of Porto, ˆ Monteiro, Portugal Alameda Prof. Hernani Introduction: Exposure to ionizing radiation at young age is known as the strongest risk factor for thyroid carcinoma development, namely papillary thyroid carcinoma (PTC). The genetic alterations associated with PTC are BRAF mutations, RAS mutations and RET/PTC rearrangement. Mutation of one of these genes can be found in up to 70% of papillary carcinomas and they rarely overlap in the same tumor. The aim of this study is to evaluate these genetic alterations in PTCs following childhood scalp irradiation. Material and Methods: We have clinically observed and followed 1367 individuals irradiated in childhood for tinea capitis scalp epilation from which 35 had thyroid cancer, 16 diagnosed by us. Twenty-one PTCs tumour samples were screened, through DNA sequencing of the PCR-amplified exon 15, for the hotspot BRAFV600E mutation at nucleotide. Screening of RAS mutations and RET/PTC rearrangement are ongoing. Results and Discussion: We detected the presence of the BRAFV600E mutation in 4 out of 21 post-tinea capitis irradiation PTCs (19.0%). The mutation was only present in the tumor tissue. The 4 cases that presented the mutation did not clinically differ from the ones without mutation. In a case other than these four we detected the rare BRAFVK600E−1E mutation, discovered by our group. Our results show that the BRAFV600E mutations found in our series are less common than what is observed in series of sporadic PTCs (36−69%), but is similar to what has been observed in other series of irradiated individuals, both with internal or external radiation (4−24%). Conclusion: As far as we are aware this is the first study of BRAF mutations in a tinea capitis scalp irradiated setting. BRAF mutations in the PTCs from tinea capitis irradiated individuals were less common than what is shown in sporadic PTCs and in accordance to what has been previously described in others settings of PTCs from subjects exposed to radiation as children. 1122 Identifying Putative MiRNAs Mediators of Radiotherapy Resistance in Breast Cancer T. Molloy1 , M. Pajic2 , S. Holzhauser2 , P. Graham3 , E.K.A. Millar4 , R.L. Sutherland4 . 1 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Breast Cancer, Sydney, Australia, 2 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Pancreatic Cancer, Sydney, Australia, 3 St George Hospital, Cancer Care Centre, Sydney, Australia, 4 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Breast Cancer, Sydney, Australia Background: Radiotherapy is an important component of primary, adjuvant, and palliative treatment for many types of solid tumours. Despite this, local control of almost one-third of patients with non-resectable tumours fails due to acquired or innate resistance to radiation, and this represents an important clinical problem. Materials and Methods: We aimed to test the hypothesis that specific noncoding micro RNA (miRNA) molecules are key regulators of the response to radiotherapy in breast cancer cells, and could therefore trigger resistance to clinically relevant doses of radiation. Results: miRNA expression microarrays were used to identify miRNAs over- or under-expressed in the primary breast tumours of patients who experienced local relapse following radiotherapy (‘radiotherapy resistant’) versus those that did not (‘radiotherapy sensitive’). Mimics of several of the miRNAs identified were subsequently transfected into cultured MCF-7 breast cancer cells and 3 were shown to induce significantly increased
Sunday 8 − Tuesday 10 July 2012
resistance or sensitivity to radiation compared to scrambled miRNA-tranfected controls. Whole-genome microarray gene expression profiling of transfected cells together with miRNA target prediction algorithms were used to identify activated gene networks. DNA repair, cell cycle control, and reactive oxygen species defence were identified as activated pathways, and functional assays, including RAD51/geminin and phospho-H2AX immunoflouresence, oxidized glutathione quantification, and cell cycle analyses confirmed this. Conclusion: Together, these data have provided evidence that several gene networks associated with multiple processes important to the cellular response to radiation can be regulated by the control of a small number of key miRNA molecules. 1123 Alpha-radioimmunotherapy − Toxicity and Therapeutic Effect of 211At-mAb in a Syngeneic Rat Colon Carcinoma Model S.E. Eriksson1 , T. Back ¨ 2 , E. Elgstrom ¨ 1 , H. Jensen3 , R. Nilsson1 , S. Lindegren2 , J. Tennvall1 . 1 Lund University, Oncology, Lund, Sweden, 2 University of Gothenburg, Radiation Physics, Gothenburg, Sweden, 3 Rigshospitalet, Copenhagen, Denmark Background: Radioimmunotherapy with alpha-emitting radionuclides such as astatine-211, 211 At, is considered as a suitable treatment of small metastases. In the present study, we used an experimental syngeneic rat colon carcinoma model which expresses the epitope for the monoclonal antibody BR96. The aim of the present study was to evaluate toxicity and therapeutic effect of 211 At-labeled BR96 on established solid tumors. The maximum tolerable activity (MTA) of 211 At to inject was estimated from previous biodistribution and dosimetry data from administration of 177 Lu-BR96 in the same animal model. Material and Methods: 211 At-labeled BR96, 2.5 or 5 MBq and 150 mg mAb/animal (corresponding to 9.1 and 18.8 MBq/kg body weight) was administered intravenously to healthy rats (n = 3/group) and rats bearing syngeneic sub-peritoneal colon carcinoma tumors (n = 6/group). Control rats were given the same amount of unlabeled mAb. Tumor volumes, body weight and blood cell counts were monitored for 100 days. Histology of tumors and metastases was evaluated. Results: Complete response was seen in 5/6 rats given either 2.5 or 5 MBq 211 At-BR96 compared to 1/6 rats in the control group. Metastases were detected in all groups after treatment. Body weight decreased after treatment with a delayed recovery in the group given the higher activity. Myelotoxicity was dose-dependent, with full recovery of both white blood cells and platelets. The higher activity was probably close to the MTA. Conclusion: 211 At-BR96 radioimmunotherapy showed promising therapeutic results against established solid tumors at activities below the MTA. The administered activities resulted in tolerable transient bone marrow toxicity. The treatment did not prevent or delay onset of metastatic disease after complete remission. 1124 Survival Progeny Derived From Irradiated Parental Cells − Phenotype Analysis and Correlation With Tumour Progression L. Gon¸calves Bastos1 , P. Marcondes Guimaraes ˜ 1 , J.A. Morgado-D´ıaz1 . 1 ˆ Instituto Nacional de Cancer, Cellular Biology − Structural Biology Laboratory, Rio de Janeiro, Brazil Introduction: Colorectal cancer (CRC) is one of the most common tumors among population and ionizing radiation (IR) is used as first line of treatment. However, the local recurrence, second malignances and metastasis remain a problem of this therapy. Irradiated-tissue-microenvironment could modulate the survivor progeny, allowing potential malignant advantages for tumor repopulation at later times. The purpose of this study was to analyze events related with the tumorigenic potential in survivors CRC cells submitted to irradiation. Material and Methods: We used Caco-2, HT-29 and HCT-116 cells as CRC model. Cells were irradiated with 5 Gy in Cs137 irradiator. After 24 and 48 hours, cells were trypsinized and maintained in culture to form colonies. Cell morphology was analyzed by phase contrast. Expression and localization of junctional complex proteins and intermediate filaments were monitored by immunofluorescence and immunoblotting. Alterations of the actin cytoskeleton was monitored by using conjugated F-actin-TRICT and confocal microscopy. Wound healing assay ensures the cell migration; radioresistance were monitored by clonogenic and Caspase-glo ® assay and the anchorage independent growth were analyzed by agarose assay. Anti apoptotic protein survivin were monitored by immunoblotting. Results and Discussion: The survival progeny derived from irradiated parental cells Caco-2, HT-29 and HCT-116 showed an aberrant morphology, with lamellipodium and philopodium projections, as compared with control cells. Confocal microscopy analysis showed heritable aberrations in actin cytoskeleton organization and disorganization of cell contacts as seen by the internalization of E-cadherin and b-catenin in all cell lines progeny. In addition, reduced E-cadherin in Caco-2, HT-29 and HCT-116 and elevated b-catenin expression in HT-29 cells was also observed by immunoblotting.
Poster Sessions
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Increased expression and reorganization of vimentin filaments was observed only in HT-29 by immunoblotting and immunofluorescence. A higher migratory potential was observed in the HT-29 progeny, as compared with control cells. Clonogenic survival and viability analyses showed that Caco-2 cells were more radiorresistant than HT-29 and HCT-116, respectively, but only HT-29 progeny showed an increase of anchorage independent growth in agarose assay. Interestingly, an increase of survivin expression was observed by immunoblotting in the HT-29 and Caco-2 progenies, but not in HCT-116 cells. Conclusion: The results suggest that IR induces heritable phenotypic alterations that are correlated with a more aggressive potential in the survival progeny derived of parental irradiated cells, which could contribute to the development of refractory tumors and metastasis. More studies are in course to elucidate this hypothesis and find radiorresistance biomarkers. 1125 Association Between Variants in the VEGF Gene and Distant Metastases in Postmenopausal Breast Cancer Patients S. Krenn-Pilko1 , E.M. Thurner1 , G. Absenger2 , W. Renner3 , A. Gerger2 , K.S. Kapp1 , U. Langsenlehner4 , T. Langsenlehner1 . 1 Medical University Graz, Department of Therapeutic Radiology and Oncology, Graz, Austria, 2 Medical University Graz, Division of Oncology Department of Internal Medicine, Graz, Austria, 3 Medical University Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria, 4 GKK Graz, Internal Outpatient Department, Graz, Austria Background: Vascular endothelial growth factor (VEGF) is essential for tumor angiogenesis and metastatic spread. The purpose of the present study was to analyze the role of VEGF polymorphisms and haplotypes for the development of metastatic progression in postmenopausal women with breast cancer. Material and Methods: A total of 584 postmenopausal breast cancer patients from the Austrian TIGER study (“tumor of breast tissue: incidence, genetics, and environmental risk factors”) were eligible for analysis. The occurrence of distant metastases was evaluated in regular follow-up examinations. Seven variants in the VEGF gene were selected and genotyping was done by a 5 -nuclease assay (TaqMan). Haplotypes and linkage disequilibrium were determined using the Haploview program. Statistical analysis was performed using SPSS 18.0 for Windows. Results: Within a median follow-up time of 77 months (range 0–121 months), 122 (21%) patients developed distant metastases. In a Kaplan–Meier analysis, carriers of the −634G>C polymorphism were at decreased risk of developing distant metastasis (p = 0.027) and in additional Cox regression analysis, the hazard ratio for distant metastases was 0.69 (95% CI 0.52 to 0.92, p = 0.012). Furthermore, the CCCCC haplotype formed by 5 polymorphisms upstream of the coding sequence including the −634G>C polymorphism showed a significant association with distant metastases (HR 0.655, 95% CI 0.487 to 0.882; p = 0.004). In a multivariate analysis including tumor stage, tumor grade, initial lymph node involvement, hormone receptor status and HER2neu status as potential confounders, the CCCCC haplotype remained a significant predictor of distant metastases (HR 0.614, 95% CI 0.416 to 0.906; p = 0.014). Conclusion: We conclude that genetic variants in the gene for VEGF may influence the risk of the development of distant metastases in postmenopausal breast cancer patients. 1126 Role of Polymorphisms in the ERCC2 Gene in the Development of Severe Side Effects After Radiotherapy of Prostate Cancer Patients E.M. Thurner1 , S. Krenn-Pilko1 , W. Renner2 , J. Szkandera3 , A. Gerger3 , U. Langsenlehner4 , K.S. Kapp1 , T. Langsenlehner1 . 1 Medical University of Graz, Department of Therapeutic Radiology and Oncology, Graz, Austria, 2 Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria, 3 Medical University of Graz, Division of Oncology − Department of Internal Medicine, Graz, Austria, 4 GKK Graz, Internal Outpatient Department, Graz, Austria Background: Single nucleotide polymorphisms in genes, which are involved in DNA repair pathways, have shown associations with the development of severe late side effects after radiotherapy. ERCC2, an ATP-dependent helicase, is an essential player in the nucleotide-excision repair pathway and is also involved in transcription. It enables the unwinding of DNA helix and prepares the strands for further DNA repair processes. Aim of the present prospective study was to evaluate the association between single nucleotide polymorphisms in the ERCC2 gene and the development of side effects after radiotherapy in prostate cancer patients. Material and Methods: In the present study, a total of 603 participants from the Austrian PROCAGENE study was included. Eligible candidates were patients with histologically confirmed prostate cancer, treated with threedimensional conformal radiotherapy in a three-field technique five times a week to a total dose of 66–70.4 Gy. Late genitourinary and rectal toxicity was graded according to standard RTOG criteria. For further analysis, 2 functional polymorphisms in the ERCC2 gene (Asp312Asn and Lys751Gln) were
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selected. Genotypes were determined by a 5 exonuclease assay (TaqMan). Statistical analysis was performed using SPSS 18.0 for Windows. Results: After a median follow-up time of 35 months, 91 patients (15.7%) developed severe genitourinary and/or rectal late side effects, classified as RTOG 2. In a Kaplan–Meier analysis, no significant associations between the variants in the ERCC2 gene and the development of severe side effects RTOG 2 were found (Asp312Asn: p = 0.4 und Lys751Gln: p = 0.71). Additional multivariate Cox proportional hazard analyses, including clinical and dosimetric factors as potential confounders, did not show a significant influence on the risk of the development of severe side effects (Asp312Asn: RR = 0.743, 95% KI 0.417–1.325; p = 0.314 und Lys751Gln: RR = 1.162, 95% KI 0.608– 2.22; p = 0.650). Conclusion: We conclude that the presence of the analyzed variants in the ERCC2 gene is not associated with the risk of radiation induced severe genitourinary and/or rectal late side effects in prostate cancer patients. 1127 Increased Levels of Reactive Oxygen and Nitrogen Species in Cells Exposed to Ionizing Radiation A. Cieslar-Pobuda1 , Y. Saenko2 , M. Skonieczna1 , J. Rzeszowska-Wolny1 . 1 Silesian University of Technology, Faculty of Automatic Control Electronics and Computer Science, Gliwice, Poland, 2 Ulyanovsk State University, Research Institute of Technology, Ulyanovsk, Russian Federation Background: Free-radical products are the main cell-damaging compounds induced by ionizing radiation and include reactive oxygen (ROS) and reactive nitrogen (RNS) species. ROS are formed as a result of radiolysis of water as well as by a number of intracellular processes. Metabolically-generated secondary radicals can act as damaging agents leading to death or induction of genomic instability, and have been shown to play a role in cancer development. In this study we examine the dynamics of changes and the sources of ROS and RNS as well as strand breaks in DNA and cell death in two human cell lines irradiated with a single X-ray dose. Materials and Methods: Human leukemic K562 and HL-60 cells were exposed to 12 Gy of ionizing radiation. Intracellular ROS and nitric oxide were assayed using 2 ,7 -dichlorofluorescein (DCF) and 4-amino-5-methylamino2 ,7 -difluororescein diacetate (DAF-FM), respectively. Mitochondrial complex I was inhibited by rotenone. The kinetics of DNA strand break rejoining were measured by comet assays and cell death was quantified using Annexin-V − propidium iodide staining and flow cytometry. Results: Reactive oxygen and nitrogen species were measured at different time points after exposing cells to X-radiation. Irradiation caused an immediate increase of about 60% in the level of ROS, followed by a gradual decrease during the next 4 h after which a second increase persisting longer than 24 h was observed in both cell lines. The dynamics of the changes in ROS and RNS levels were similar in both cell lines, but K562 cells showed a higher level of ROS in the second peak. The increase in these radicals was correlated with an increased level of DNA strand breaks and of cell death in both cell lines. To establish the source of the increased ROS, we used rotenone to inhibit the mitochondrial complex I. The initial increase of ROS was not inhibited by rotenone suggesting that it was induced directly by irradiation, but the later increase was inhibited by rotenone in K562 but not in HL60 cells. Conclusions: Exposure to ionizing radiation induces long-lasting changes in the levels of reactive oxygen and nitrogen species in cells and increased cell death. The increase of these radicals observed 24 h after irradiation results from cellular metabolic processes, and the signaling pathways involved are different in different cell types. The work was financed by Polish Ministry of Science and Higher Education grant NN 518497639. 1128 Reactive Oxygen Species May Play a Role in Ionizing Radiation-induced Bystander Effects and Regulation of mRNA Levels by MicroRNAs J. Rzeszowska-Wolny1 , M. Widel1 , A. Cieslar-Pobuda1 , A. Lalik1 , M. Skonieczna1 , R. Jaksik1 . 1 Silesian University of Technology, Biosystems Group, Gliwice, Poland Background: Cellular responses to ionizing radiation depend on direct effects and also on intercellular communication manifested as bystander effects. We have shown previously that the transcriptomes of directly-irradiated and bystander cells are very similar 36 h after irradiation, and that bystander effects depend strongly on oxidative processes in the cells. This study shows an influence of mutual interactions between irradiated and unirradiated cells at the level of reactive oxygen species (ROS) and presents analyses of transcript levels and their changes in irradiated human cells. Materials and Methods: Human Me45 melanoma cells or NHDF fibroblasts were exposed to 4 Gy of ionizing radiation and co-cultured with unirradiated cells. Intracellular ROS were assayed using 2 ,7 -dichlorofluorescein and 4-amino-5-methylamino-2 ,7 -difluorescein diacetate, micronucleus and apoptosis frequencies by fluorescence and phase-contrast microscopy in DAPI-stained cells, mRNA and miRNA levels with Affymetrix HG-U133A and
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males − 14 (42%). Patients ages ranged from 14 to 38 years, median age − 17. In 20 of 33 patients (60.6%) primary tumor were localized in distal femur, in 8 (24.2%) − in proximal tibia and in 5 (15.2%) − in different parts of fibula. In 30 of 33 patients lung metastasises were detected by x-ray examination and in 3 patients by computer tomography. Results: Bilateral metastatic lesions of lungs were detected in 20 (60.6%) and unilateral in 13 (in 6 (9.4%) − in upper lobe of left lung, in 4 (12.4%) − in lower lobe of right lung and in 3 (12.1%) − in upper lobe of right lung) patients. In 31of 33 patients (94%) lung metastases developed during the period from 1 to 24 months after the primary tumor development, and in 2 patients (6%) during the primary tumor development. The numbers of metastatic nodules were 1−7, middle 2 nodules. Single lung metastases were detected in 9 (27.2%) and multiple in 24 (72.8%) patients. Metastatic nodules sizes in detection time were from 5 mm up to 25 mm, median − 14.3 mm. In 4 of 33 patients after lung x-ray examination were performed computer tomography there were detected additional metastatic nodules and difference in nodules sizes. Conclusion: The target organ of osteosarcoma metastatic spread is lung and in most cases had been affected both lungs. Feature of metastatic osteosarcoma is multiple affecting of lung. 1121 BRAF Mutations in Thyroid Carcinomas Following Childhood Scalp Irradiation P. Boaventura1 , D. Pereira1 , J. Teixeira-Gomes1 , P. Soares1,2 . 1 IPATIMUP − Institute of Molecular Pathology and Immunology of the University of Porto, Cancer Biology, Porto, Portugal, 2 Medical Faculty, University of Porto, ˆ Monteiro, Portugal Alameda Prof. Hernani Introduction: Exposure to ionizing radiation at young age is known as the strongest risk factor for thyroid carcinoma development, namely papillary thyroid carcinoma (PTC). The genetic alterations associated with PTC are BRAF mutations, RAS mutations and RET/PTC rearrangement. Mutation of one of these genes can be found in up to 70% of papillary carcinomas and they rarely overlap in the same tumor. The aim of this study is to evaluate these genetic alterations in PTCs following childhood scalp irradiation. Material and Methods: We have clinically observed and followed 1367 individuals irradiated in childhood for tinea capitis scalp epilation from which 35 had thyroid cancer, 16 diagnosed by us. Twenty-one PTCs tumour samples were screened, through DNA sequencing of the PCR-amplified exon 15, for the hotspot BRAFV600E mutation at nucleotide. Screening of RAS mutations and RET/PTC rearrangement are ongoing. Results and Discussion: We detected the presence of the BRAFV600E mutation in 4 out of 21 post-tinea capitis irradiation PTCs (19.0%). The mutation was only present in the tumor tissue. The 4 cases that presented the mutation did not clinically differ from the ones without mutation. In a case other than these four we detected the rare BRAFVK600E−1E mutation, discovered by our group. Our results show that the BRAFV600E mutations found in our series are less common than what is observed in series of sporadic PTCs (36−69%), but is similar to what has been observed in other series of irradiated individuals, both with internal or external radiation (4−24%). Conclusion: As far as we are aware this is the first study of BRAF mutations in a tinea capitis scalp irradiated setting. BRAF mutations in the PTCs from tinea capitis irradiated individuals were less common than what is shown in sporadic PTCs and in accordance to what has been previously described in others settings of PTCs from subjects exposed to radiation as children. 1122 Identifying Putative MiRNAs Mediators of Radiotherapy Resistance in Breast Cancer T. Molloy1 , M. Pajic2 , S. Holzhauser2 , P. Graham3 , E.K.A. Millar4 , R.L. Sutherland4 . 1 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Breast Cancer, Sydney, Australia, 2 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Pancreatic Cancer, Sydney, Australia, 3 St George Hospital, Cancer Care Centre, Sydney, Australia, 4 The Kinghorn Cancer Centre and Garvan Institute of Medical Research, Breast Cancer, Sydney, Australia Background: Radiotherapy is an important component of primary, adjuvant, and palliative treatment for many types of solid tumours. Despite this, local control of almost one-third of patients with non-resectable tumours fails due to acquired or innate resistance to radiation, and this represents an important clinical problem. Materials and Methods: We aimed to test the hypothesis that specific noncoding micro RNA (miRNA) molecules are key regulators of the response to radiotherapy in breast cancer cells, and could therefore trigger resistance to clinically relevant doses of radiation. Results: miRNA expression microarrays were used to identify miRNAs over- or under-expressed in the primary breast tumours of patients who experienced local relapse following radiotherapy (‘radiotherapy resistant’) versus those that did not (‘radiotherapy sensitive’). Mimics of several of the miRNAs identified were subsequently transfected into cultured MCF-7 breast cancer cells and 3 were shown to induce significantly increased
Sunday 8 − Tuesday 10 July 2012
resistance or sensitivity to radiation compared to scrambled miRNA-tranfected controls. Whole-genome microarray gene expression profiling of transfected cells together with miRNA target prediction algorithms were used to identify activated gene networks. DNA repair, cell cycle control, and reactive oxygen species defence were identified as activated pathways, and functional assays, including RAD51/geminin and phospho-H2AX immunoflouresence, oxidized glutathione quantification, and cell cycle analyses confirmed this. Conclusion: Together, these data have provided evidence that several gene networks associated with multiple processes important to the cellular response to radiation can be regulated by the control of a small number of key miRNA molecules. 1123 Alpha-radioimmunotherapy − Toxicity and Therapeutic Effect of 211At-mAb in a Syngeneic Rat Colon Carcinoma Model S.E. Eriksson1 , T. Back ¨ 2 , E. Elgstrom ¨ 1 , H. Jensen3 , R. Nilsson1 , S. Lindegren2 , J. Tennvall1 . 1 Lund University, Oncology, Lund, Sweden, 2 University of Gothenburg, Radiation Physics, Gothenburg, Sweden, 3 Rigshospitalet, Copenhagen, Denmark Background: Radioimmunotherapy with alpha-emitting radionuclides such as astatine-211, 211 At, is considered as a suitable treatment of small metastases. In the present study, we used an experimental syngeneic rat colon carcinoma model which expresses the epitope for the monoclonal antibody BR96. The aim of the present study was to evaluate toxicity and therapeutic effect of 211 At-labeled BR96 on established solid tumors. The maximum tolerable activity (MTA) of 211 At to inject was estimated from previous biodistribution and dosimetry data from administration of 177 Lu-BR96 in the same animal model. Material and Methods: 211 At-labeled BR96, 2.5 or 5 MBq and 150 mg mAb/animal (corresponding to 9.1 and 18.8 MBq/kg body weight) was administered intravenously to healthy rats (n = 3/group) and rats bearing syngeneic sub-peritoneal colon carcinoma tumors (n = 6/group). Control rats were given the same amount of unlabeled mAb. Tumor volumes, body weight and blood cell counts were monitored for 100 days. Histology of tumors and metastases was evaluated. Results: Complete response was seen in 5/6 rats given either 2.5 or 5 MBq 211 At-BR96 compared to 1/6 rats in the control group. Metastases were detected in all groups after treatment. Body weight decreased after treatment with a delayed recovery in the group given the higher activity. Myelotoxicity was dose-dependent, with full recovery of both white blood cells and platelets. The higher activity was probably close to the MTA. Conclusion: 211 At-BR96 radioimmunotherapy showed promising therapeutic results against established solid tumors at activities below the MTA. The administered activities resulted in tolerable transient bone marrow toxicity. The treatment did not prevent or delay onset of metastatic disease after complete remission. 1124 Survival Progeny Derived From Irradiated Parental Cells − Phenotype Analysis and Correlation With Tumour Progression L. Gon¸calves Bastos1 , P. Marcondes Guimaraes ˜ 1 , J.A. Morgado-D´ıaz1 . 1 ˆ Instituto Nacional de Cancer, Cellular Biology − Structural Biology Laboratory, Rio de Janeiro, Brazil Introduction: Colorectal cancer (CRC) is one of the most common tumors among population and ionizing radiation (IR) is used as first line of treatment. However, the local recurrence, second malignances and metastasis remain a problem of this therapy. Irradiated-tissue-microenvironment could modulate the survivor progeny, allowing potential malignant advantages for tumor repopulation at later times. The purpose of this study was to analyze events related with the tumorigenic potential in survivors CRC cells submitted to irradiation. Material and Methods: We used Caco-2, HT-29 and HCT-116 cells as CRC model. Cells were irradiated with 5 Gy in Cs137 irradiator. After 24 and 48 hours, cells were trypsinized and maintained in culture to form colonies. Cell morphology was analyzed by phase contrast. Expression and localization of junctional complex proteins and intermediate filaments were monitored by immunofluorescence and immunoblotting. Alterations of the actin cytoskeleton was monitored by using conjugated F-actin-TRICT and confocal microscopy. Wound healing assay ensures the cell migration; radioresistance were monitored by clonogenic and Caspase-glo ® assay and the anchorage independent growth were analyzed by agarose assay. Anti apoptotic protein survivin were monitored by immunoblotting. Results and Discussion: The survival progeny derived from irradiated parental cells Caco-2, HT-29 and HCT-116 showed an aberrant morphology, with lamellipodium and philopodium projections, as compared with control cells. Confocal microscopy analysis showed heritable aberrations in actin cytoskeleton organization and disorganization of cell contacts as seen by the internalization of E-cadherin and b-catenin in all cell lines progeny. In addition, reduced E-cadherin in Caco-2, HT-29 and HCT-116 and elevated b-catenin expression in HT-29 cells was also observed by immunoblotting.
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
Increased expression and reorganization of vimentin filaments was observed only in HT-29 by immunoblotting and immunofluorescence. A higher migratory potential was observed in the HT-29 progeny, as compared with control cells. Clonogenic survival and viability analyses showed that Caco-2 cells were more radiorresistant than HT-29 and HCT-116, respectively, but only HT-29 progeny showed an increase of anchorage independent growth in agarose assay. Interestingly, an increase of survivin expression was observed by immunoblotting in the HT-29 and Caco-2 progenies, but not in HCT-116 cells. Conclusion: The results suggest that IR induces heritable phenotypic alterations that are correlated with a more aggressive potential in the survival progeny derived of parental irradiated cells, which could contribute to the development of refractory tumors and metastasis. More studies are in course to elucidate this hypothesis and find radiorresistance biomarkers. 1125 Association Between Variants in the VEGF Gene and Distant Metastases in Postmenopausal Breast Cancer Patients S. Krenn-Pilko1 , E.M. Thurner1 , G. Absenger2 , W. Renner3 , A. Gerger2 , K.S. Kapp1 , U. Langsenlehner4 , T. Langsenlehner1 . 1 Medical University Graz, Department of Therapeutic Radiology and Oncology, Graz, Austria, 2 Medical University Graz, Division of Oncology Department of Internal Medicine, Graz, Austria, 3 Medical University Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria, 4 GKK Graz, Internal Outpatient Department, Graz, Austria Background: Vascular endothelial growth factor (VEGF) is essential for tumor angiogenesis and metastatic spread. The purpose of the present study was to analyze the role of VEGF polymorphisms and haplotypes for the development of metastatic progression in postmenopausal women with breast cancer. Material and Methods: A total of 584 postmenopausal breast cancer patients from the Austrian TIGER study (“tumor of breast tissue: incidence, genetics, and environmental risk factors”) were eligible for analysis. The occurrence of distant metastases was evaluated in regular follow-up examinations. Seven variants in the VEGF gene were selected and genotyping was done by a 5 -nuclease assay (TaqMan). Haplotypes and linkage disequilibrium were determined using the Haploview program. Statistical analysis was performed using SPSS 18.0 for Windows. Results: Within a median follow-up time of 77 months (range 0–121 months), 122 (21%) patients developed distant metastases. In a Kaplan–Meier analysis, carriers of the −634G>C polymorphism were at decreased risk of developing distant metastasis (p = 0.027) and in additional Cox regression analysis, the hazard ratio for distant metastases was 0.69 (95% CI 0.52 to 0.92, p = 0.012). Furthermore, the CCCCC haplotype formed by 5 polymorphisms upstream of the coding sequence including the −634G>C polymorphism showed a significant association with distant metastases (HR 0.655, 95% CI 0.487 to 0.882; p = 0.004). In a multivariate analysis including tumor stage, tumor grade, initial lymph node involvement, hormone receptor status and HER2neu status as potential confounders, the CCCCC haplotype remained a significant predictor of distant metastases (HR 0.614, 95% CI 0.416 to 0.906; p = 0.014). Conclusion: We conclude that genetic variants in the gene for VEGF may influence the risk of the development of distant metastases in postmenopausal breast cancer patients. 1126 Role of Polymorphisms in the ERCC2 Gene in the Development of Severe Side Effects After Radiotherapy of Prostate Cancer Patients E.M. Thurner1 , S. Krenn-Pilko1 , W. Renner2 , J. Szkandera3 , A. Gerger3 , U. Langsenlehner4 , K.S. Kapp1 , T. Langsenlehner1 . 1 Medical University of Graz, Department of Therapeutic Radiology and Oncology, Graz, Austria, 2 Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria, 3 Medical University of Graz, Division of Oncology − Department of Internal Medicine, Graz, Austria, 4 GKK Graz, Internal Outpatient Department, Graz, Austria Background: Single nucleotide polymorphisms in genes, which are involved in DNA repair pathways, have shown associations with the development of severe late side effects after radiotherapy. ERCC2, an ATP-dependent helicase, is an essential player in the nucleotide-excision repair pathway and is also involved in transcription. It enables the unwinding of DNA helix and prepares the strands for further DNA repair processes. Aim of the present prospective study was to evaluate the association between single nucleotide polymorphisms in the ERCC2 gene and the development of side effects after radiotherapy in prostate cancer patients. Material and Methods: In the present study, a total of 603 participants from the Austrian PROCAGENE study was included. Eligible candidates were patients with histologically confirmed prostate cancer, treated with threedimensional conformal radiotherapy in a three-field technique five times a week to a total dose of 66–70.4 Gy. Late genitourinary and rectal toxicity was graded according to standard RTOG criteria. For further analysis, 2 functional polymorphisms in the ERCC2 gene (Asp312Asn and Lys751Gln) were
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selected. Genotypes were determined by a 5 exonuclease assay (TaqMan). Statistical analysis was performed using SPSS 18.0 for Windows. Results: After a median follow-up time of 35 months, 91 patients (15.7%) developed severe genitourinary and/or rectal late side effects, classified as RTOG 2. In a Kaplan–Meier analysis, no significant associations between the variants in the ERCC2 gene and the development of severe side effects RTOG 2 were found (Asp312Asn: p = 0.4 und Lys751Gln: p = 0.71). Additional multivariate Cox proportional hazard analyses, including clinical and dosimetric factors as potential confounders, did not show a significant influence on the risk of the development of severe side effects (Asp312Asn: RR = 0.743, 95% KI 0.417–1.325; p = 0.314 und Lys751Gln: RR = 1.162, 95% KI 0.608– 2.22; p = 0.650). Conclusion: We conclude that the presence of the analyzed variants in the ERCC2 gene is not associated with the risk of radiation induced severe genitourinary and/or rectal late side effects in prostate cancer patients. 1127 Increased Levels of Reactive Oxygen and Nitrogen Species in Cells Exposed to Ionizing Radiation A. Cieslar-Pobuda1 , Y. Saenko2 , M. Skonieczna1 , J. Rzeszowska-Wolny1 . 1 Silesian University of Technology, Faculty of Automatic Control Electronics and Computer Science, Gliwice, Poland, 2 Ulyanovsk State University, Research Institute of Technology, Ulyanovsk, Russian Federation Background: Free-radical products are the main cell-damaging compounds induced by ionizing radiation and include reactive oxygen (ROS) and reactive nitrogen (RNS) species. ROS are formed as a result of radiolysis of water as well as by a number of intracellular processes. Metabolically-generated secondary radicals can act as damaging agents leading to death or induction of genomic instability, and have been shown to play a role in cancer development. In this study we examine the dynamics of changes and the sources of ROS and RNS as well as strand breaks in DNA and cell death in two human cell lines irradiated with a single X-ray dose. Materials and Methods: Human leukemic K562 and HL-60 cells were exposed to 12 Gy of ionizing radiation. Intracellular ROS and nitric oxide were assayed using 2 ,7 -dichlorofluorescein (DCF) and 4-amino-5-methylamino2 ,7 -difluororescein diacetate (DAF-FM), respectively. Mitochondrial complex I was inhibited by rotenone. The kinetics of DNA strand break rejoining were measured by comet assays and cell death was quantified using Annexin-V − propidium iodide staining and flow cytometry. Results: Reactive oxygen and nitrogen species were measured at different time points after exposing cells to X-radiation. Irradiation caused an immediate increase of about 60% in the level of ROS, followed by a gradual decrease during the next 4 h after which a second increase persisting longer than 24 h was observed in both cell lines. The dynamics of the changes in ROS and RNS levels were similar in both cell lines, but K562 cells showed a higher level of ROS in the second peak. The increase in these radicals was correlated with an increased level of DNA strand breaks and of cell death in both cell lines. To establish the source of the increased ROS, we used rotenone to inhibit the mitochondrial complex I. The initial increase of ROS was not inhibited by rotenone suggesting that it was induced directly by irradiation, but the later increase was inhibited by rotenone in K562 but not in HL60 cells. Conclusions: Exposure to ionizing radiation induces long-lasting changes in the levels of reactive oxygen and nitrogen species in cells and increased cell death. The increase of these radicals observed 24 h after irradiation results from cellular metabolic processes, and the signaling pathways involved are different in different cell types. The work was financed by Polish Ministry of Science and Higher Education grant NN 518497639. 1128 Reactive Oxygen Species May Play a Role in Ionizing Radiation-induced Bystander Effects and Regulation of mRNA Levels by MicroRNAs J. Rzeszowska-Wolny1 , M. Widel1 , A. Cieslar-Pobuda1 , A. Lalik1 , M. Skonieczna1 , R. Jaksik1 . 1 Silesian University of Technology, Biosystems Group, Gliwice, Poland Background: Cellular responses to ionizing radiation depend on direct effects and also on intercellular communication manifested as bystander effects. We have shown previously that the transcriptomes of directly-irradiated and bystander cells are very similar 36 h after irradiation, and that bystander effects depend strongly on oxidative processes in the cells. This study shows an influence of mutual interactions between irradiated and unirradiated cells at the level of reactive oxygen species (ROS) and presents analyses of transcript levels and their changes in irradiated human cells. Materials and Methods: Human Me45 melanoma cells or NHDF fibroblasts were exposed to 4 Gy of ionizing radiation and co-cultured with unirradiated cells. Intracellular ROS were assayed using 2 ,7 -dichlorofluorescein and 4-amino-5-methylamino-2 ,7 -difluorescein diacetate, micronucleus and apoptosis frequencies by fluorescence and phase-contrast microscopy in DAPI-stained cells, mRNA and miRNA levels with Affymetrix HG-U133A and