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579: Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 decreased b-catenin protein level both in nuclear extracts and in cytoplasmic fractions. Conclusion: In conclusion, 1,25-D3 induces differentiation, reduces IGFinduced proliferation and inhibits Wnt signaling in the adenoma cell line LT97, which suggests a protective role against tumor progression. This project is funded by Herzfelder’sche Familienstiftung and WWTF. No conflict of interest. 577 Lats1 knockout mouse model recapitulating human dedifferentiated liposarcoma S. Kim1 , D. Lim2 , N. Cho1 , W. Yang1 . 1 Yonsei University Medical Center, Department of Pathology, Seoul, South Korea, 2 KAIST, Department of Biological Sciences, Daejeon, South Korea Introduction: Human dedifferentiated liposarcoma (DDLS) is a malignant adipocytic tumor defined as an atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WLS), which progress to sarcoma of variable histologic grades. The pathogenesis of this tumor remains poorly understood, but it has been reported that murine double minute 2 (MDM2) gene amplification/ overexpression is associated with the development of ALT/WLS and DDSL. The purpose of this study was to establish a spontaneous de novo DDLS tumor model which could serve as an experimental platform of this disease entity. Material and Method: The Lats1 deficient mice developed soft tissue tumors by the age of approximately 15 moths. We examined the histologic features of these tumors and performed fluorescent in situ hybridization (FISH) assay and PT-PCR to measure MDM2 expression levels. In parallel, we analyzed 22 human liposarcoma surgical specimens obtained from patients who were diagnosed and operated in Yonsei University medical center from 1995 to 2013. These included different subtypes as follows: ALT/WLSs (n = 3), DDLSs (n = 12), pleomorphic liposarcomas (PLSs, n = 3), and myxoid liposarcomas (MLSs, n = 4). Immunohistochemical analysis, FISH assay, and RT-PCR were performed to assess the occurrence of MDM2 gene amplification/ overexpression and Lats1 status. Results and Discussion: Histologic examination of the tumors produced by Lats1 deficient mice revealed low-grade spindle cells consisting of solid sheets or fascicles and showed abrupt transition to ALT/WLS component. These are typical histologic features of human DDLS. Immunohistochemical analysis, FISH assay, and RT-PCR of these tumors revealed over-expressed levels of MDM2 in comparison with those of adipose tissues obtained from wild type mice. Next, we examined the Lats1 expression levels in different types of human liposarcomas by RT-PCR. Human DDLSs demonstrated Lats1 down-regulation in 10 out of 12 cases (83.33%), but the other types of liposarcomas such as ALT/WLS, MLS and PLS did not show significant difference. Conclusion: We propose a spontaneous tumor model based on genetically engineered mice (Lats1−/− ) that recapitulates the features of a subtype of human liposarcoma (DDLS). This model not only carries the precise histologic features of human DDLS, but also certain genetic traits. We hope that our tumor model could serve as a study platform to elucidate the pathogenesis and develop targeted therapies of this disease entity. No conflict of interest. 578 Critical age windows of radiation exposure for cancer risk in experimental animal models Y. Shimada1 , M. Nishimura1 , T. Imaoka1 , K. Daino1 , Y. Yamada1 , K. Ariyoshi1 , C. Tsuruoka1 , S. Kakinuma1 . 1 National Institute of Radiological Sciences, Radiobiology for Children’s Health Program, Chiba, Japan Background: Children are susceptible to environmental carcinogens compared to adults. Several animal cancer models have shown the existence of critical age window of radiation carcinogenesis. However, only fragmental information is available for the underlying mechanism. In this study, we introduce the panel of critical age windows of radiation exposure for cancer risk of each organ in experimental animal models. Material and Methods: The animal models we used are B6C3F1 mouse, C3H mouse, Apc Min/+ mouse, Ptch1 mouse, and Eker rat, SD rat and Wistar rat. They were exposed to gamma or X rays at the doses of 0.05 to 4 Gy at 1 week to 15 weeks after birth. Results: Both medulloblastoma in Ptch1 mouse and renal cell carcinoma in Eker rat showed the highest sensitivity to ionizing radiation at perinatal stage. The highest incidence of liver tumors and thymic lymphomas in B6C3F1 mice and intestinal tumors in Apc Min/+ mouse was observedd after irradiation at infant. In contrast, myeloid leukemia in C3H mouse was induced by adulthood. Age dependence was relatively small for radiation-induced lung carcinogenesis in Wistar rat. The number of cell at risk and the mode of cell division (symmetric or asymmetric) change with age, which may affect the cancer risk of radiation. Intestinal crypts, where stem cells are abundant, grow rapidly by symmetric division during infant stage. Bone marrow stem cells are also increasing in
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number during juvenile stage. Interestingly, infant crypt cells are resistant to radiation-induced apoptosis, whereas adult intestinal cryptic cells display frequent apoptosis after irradiation. In contrast, infant myeloid stem cells as determined by CFU-S12 were highly sensitive to radiation-induced cell killing, while the cells were resistant at adult. Considered that intestinal tumors and myeloid leukemia are induced efficiently by infant and adulthood exposures, respectively, the cells at tumor susceptible period are rather resistance to radiation-induced cell killing, thereby, possibly, surviving with DNA damages (mutations). Granule cell precursors migrate over cerebellar anlage in late fetal stage to form external granule layer (EGL), where they undergo rapid clonal expansion. Ten days after birth, they migrate inside to form IGL generating post-mitotic neuron. The susceptible window ends after migration into IGL. Conclusion: There is a critical age window for radiation-induced tumors in a tissue dependent manner. The age window appears to be related to proliferative status and cellular response to radiation, including apoptosis. No conflict of interest. 579 Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer M. Mancino1 , P. Fernandez-Nogueira2 , E. Enreig2 , E. Ametller2 , P. Gascon1 , V. Almendro1 . 1 Hospital Clinic − Institut d’Investigacions Biomediques August Pi i Sunyer, Oncologia, Barcelona, Spain, 2 Hospital Clinic, Oncologia, Barcelona, Spain Background: Histamine is a monoamine produced by a-decarboxylation of histidine by the enzyme histidine decarboxylase (HDC). Histamine acts as an autocrine regulator of cell proliferation through its binding to HRH1−4 receptors. In breast cancer (BC), there are controversial results about the therapeutic efficacy of HRH1 or HRH2 inhibitors and the role of histamine receptors in tumor progression. Objective: Given the phenotypic heterogeneity of BC and the presence of different molecular subtypes with different clinical outcome, we aimed to investigate the contribution of histamine receptors in the proliferation, migration and cell survival of BC cells of different molecular subtypes. Experimental procedure: The expression of histamine receptors HRH1−4 and HDC in human breast tumors of different subtypes, and their correlation with clinical variables were gathered through informatics analysis using the ‘Gene expression based Outcome for Breast cancer Online’ (GOBO) webbased tool. The measurement of the mRNA levels of HRH1−4 and HDC in a panel of breast cancer cell lines was used to confirm the subtype specific expression of each protein. Using specific HRH inhibitors in luminal and basallike breast cancer cell lines we assessed the functional role of histamine receptors in cell proliferation, migration and survival. No conflict of interest. 580 Sensitive detection of NRAS mutations using mutant-enriched PCR and reverse-hybridization teststrips M. Novy1 , B. Rauscher1 , N. Fakhreddin2 , R. Mahfouz2 , C. Oberkanins1 . 1 ViennaLab Diagnostics GmbH, R&D, Vienna, Austria, 2 American University of Beirut Medical Center, Department of Pathology and Laboratory Medicine, Beirut, Lebanon Introduction: RAS proteins are key players in growth factor receptor induced signaling pathways. The clinically most important RAS family members are the proto-oncogenes KRAS and NRAS. Somatic mutations are frequently found in these genes. According to the COSMIC database KRAS is mutated in 35% of all large intestine tumors. In contrast, somatic NRAS mutations are much less frequent in colorectal cancer (~4%) but prominent in melanomas (~15%). Clinical studies showed that KRAS and NRAS mutations restrict the therapeutic use of anti-EGFR monoclonal antibodies in colorectal cancer therapies. Materials and Methods: We have developed a reverse-hybridization StripAssay for the detection of five mutations in codon 12, three mutations in codon 13 and four mutations in codon 61 of the NRAS gene. The test is based on mutant-enriched PCR in the presence of NRAS wild-type suppressors, followed by hybridization of biotinylated PCR products to teststrips presenting a parallel array of allele-specific oligonucleotide probes. The hybridization and detection steps can be carried out fully automated using commercially available instrumentation. Results and Discussion: StripAssay performance was evaluated using genomic DNA obtained from cultured cell lines and formalin-fixed paraffineembedded (FFPE) tumor tissue. Plasmid clones harbouring the respective NRAS mutations served as reference templates to control for test specificity. By using normal DNA spiked with serial dilutions of DNA from NRAS-mutant cell lines all mutations were shown to be detectable at a level of 1%. In an ongoing study we are testing 270 thyroid FFPE tissue samples for the presence of NRAS mutations.
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number during juvenile stage. Interestingly, infant crypt cells are resistant to radiation-induced apoptosis, whereas adult intestinal cryptic cells display frequent apoptosis after irradiation. In contrast, infant myeloid stem cells as determined by CFU-S12 were highly sensitive to radiation-induced cell killing, while the cells were resistant at adult. Considered that intestinal tumors and myeloid leukemia are induced efficiently by infant and adulthood exposures, respectively, the cells at tumor susceptible period are rather resistance to radiation-induced cell killing, thereby, possibly, surviving with DNA damages (mutations). Granule cell precursors migrate over cerebellar anlage in late fetal stage to form external granule layer (EGL), where they undergo rapid clonal expansion. Ten days after birth, they migrate inside to form IGL generating post-mitotic neuron. The susceptible window ends after migration into IGL. Conclusion: There is a critical age window for radiation-induced tumors in a tissue dependent manner. The age window appears to be related to proliferative status and cellular response to radiation, including apoptosis. No conflict of interest. 579 Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer M. Mancino1 , P. Fernandez-Nogueira2 , E. Enreig2 , E. Ametller2 , P. Gascon1 , V. Almendro1 . 1 Hospital Clinic − Institut d’Investigacions Biomediques August Pi i Sunyer, Oncologia, Barcelona, Spain, 2 Hospital Clinic, Oncologia, Barcelona, Spain Background: Histamine is a monoamine produced by a-decarboxylation of histidine by the enzyme histidine decarboxylase (HDC). Histamine acts as an autocrine regulator of cell proliferation through its binding to HRH1−4 receptors. In breast cancer (BC), there are controversial results about the therapeutic efficacy of HRH1 or HRH2 inhibitors and the role of histamine receptors in tumor progression. Objective: Given the phenotypic heterogeneity of BC and the presence of different molecular subtypes with different clinical outcome, we aimed to investigate the contribution of histamine receptors in the proliferation, migration and cell survival of BC cells of different molecular subtypes. Experimental procedure: The expression of histamine receptors HRH1−4 and HDC in human breast tumors of different subtypes, and their correlation with clinical variables were gathered through informatics analysis using the ‘Gene expression based Outcome for Breast cancer Online’ (GOBO) webbased tool. The measurement of the mRNA levels of HRH1−4 and HDC in a panel of breast cancer cell lines was used to confirm the subtype specific expression of each protein. Using specific HRH inhibitors in luminal and basallike breast cancer cell lines we assessed the functional role of histamine receptors in cell proliferation, migration and survival. No conflict of interest. 580 Sensitive detection of NRAS mutations using mutant-enriched PCR and reverse-hybridization teststrips M. Novy1 , B. Rauscher1 , N. Fakhreddin2 , R. Mahfouz2 , C. Oberkanins1 . 1 ViennaLab Diagnostics GmbH, R&D, Vienna, Austria, 2 American University of Beirut Medical Center, Department of Pathology and Laboratory Medicine, Beirut, Lebanon Introduction: RAS proteins are key players in growth factor receptor induced signaling pathways. The clinically most important RAS family members are the proto-oncogenes KRAS and NRAS. Somatic mutations are frequently found in these genes. According to the COSMIC database KRAS is mutated in 35% of all large intestine tumors. In contrast, somatic NRAS mutations are much less frequent in colorectal cancer (~4%) but prominent in melanomas (~15%). Clinical studies showed that KRAS and NRAS mutations restrict the therapeutic use of anti-EGFR monoclonal antibodies in colorectal cancer therapies. Materials and Methods: We have developed a reverse-hybridization StripAssay for the detection of five mutations in codon 12, three mutations in codon 13 and four mutations in codon 61 of the NRAS gene. The test is based on mutant-enriched PCR in the presence of NRAS wild-type suppressors, followed by hybridization of biotinylated PCR products to teststrips presenting a parallel array of allele-specific oligonucleotide probes. The hybridization and detection steps can be carried out fully automated using commercially available instrumentation. Results and Discussion: StripAssay performance was evaluated using genomic DNA obtained from cultured cell lines and formalin-fixed paraffineembedded (FFPE) tumor tissue. Plasmid clones harbouring the respective NRAS mutations served as reference templates to control for test specificity. By using normal DNA spiked with serial dilutions of DNA from NRAS-mutant cell lines all mutations were shown to be detectable at a level of 1%. In an ongoing study we are testing 270 thyroid FFPE tissue samples for the presence of NRAS mutations.