A Novel Breast Carcinoma Stromal Response Defined by the Nodular Fasciitis Gene Signature

I. J. Radiation Oncology d Biology d Physics

S686

3149

Volume 72, Number 1, Supplement, 2008

Integration of Human Papillomavirus (HPV) 16 into the Host Genome Influences the Radiosensitivity via the Disruption of the Viral Transcriptional Regulator Gene E2

K. Lindel1, S. Daffinger1, K. Weber1, E. de Villiers2, P. Beard3, J. Debus1 1

Dept. of Radio-Oncology, University of Heidelberg, Heidelberg, Germany, 2Division of Tumorvirus Characterisation, DKFZ, Heidelberg, Germany, 3ISREC, Epalinges, Switzerland Purpose/Objective(s): Integration HPV into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and a loss of the transcriptional repressor function of the viral gene E2. There is clinical evidence that patients with HPV 16 positive cancer of the uterine cervix with an intact E2 gene have a better prognosis than those with a disrupted E2 gene. This might be due to a better response to radiation treatment. Purpose of this study was to investigate the role of the E2 gene for radiosensitivity of HPV 16 positive cervical keratinocytes using the W12 cell line model. Materials/Methods: W12 cell line was derived from a low grade cervical lesion by Stanley MA et al., 1989, and is unique among HPV16-containing cell lines in carrying its HPV 16 genome as a multicopy episome. We made use of a pair of isogenic cell lines, W12 and S12 to compare the difference of survival after irradiation. W12 cells contain episomal HPV 16 genomes, whereas S12 cells, which derived from the W12 line, contain HPV DNA as integrated copies. E2 gene region was screened by using PCR with three separate primers covering the whole genome. Cells were irradiated with singles doses of 0 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, and 7 Gy. Clonogenic survival was analyzed by using the 96-well in vitro test. Survival fraction and survival curves where calculated using Sigma Plot 8.0. Cell cycle analyses were performed using flow cytometry (PI staining) 24 h and 48 h after RT with 2 and 7 Gy. At least three experiments where performed for each dose point. Results: The E2 gene of the S12 cells (passage 88 - 103) was disrupted in the E2C region. The W12 cells (passage 8 -14) with an intact E2 gene showed a higher radiosensitivity with a radiation enhancement factor of 1.5 (4 Gy). W12 cell showed a G2/M block 24 h after irradiation with 2 Gy. Both cell lines showed a G2/M block 24 h after irradiation with 7 Gy. S12 cells developed polyploidy 48 h after irradiation. Conclusions: HPV 16 positive W12 cells with an intact E2 gene showed a higher radiosensitivity compared to those with a disrupted E2 gene. Differences in cell cycle regulation especially regarding the postmitotic checkpoint might cause radioresistance. About 30% of patients with cervical cancer have tumors with an intact E2 gene. Our experiments indicate a better response to radiation treatment might be factor for their better prognosis. Author Disclosure: K. Lindel, None; S. Daffinger, None; K. Weber, None; E. de Villiers, None; P. Beard, None; J. Debus, None.

3150

A Novel Breast Carcinoma Stromal Response Defined by the Nodular Fasciitis Gene Signature

N. K. Cho1,2, A. H. Beck2, I. Espinosa2, K. Montgomery2, S. Zhu2, M. van de Rijn2, R. B. West3,2 1 Department of Radiation Oncology, Stanford University Cancer Center, Stanford, CA, 2Department of Pathology, Stanford University Medical Center, Stanford, CA, 3Pathology and Laboratory Service, Palo Alto Veterans Affairs Health Care System, Palo Alto, CA

The tumor microenvironment (TME) is comprised of many cell types in addition to the carcinoma cells, including fibroblasts, smooth muscle cells, inflammatory cells, and endothelial cells. These carcinoma-associated stromal cells are felt to play a significant role in tumorigenesis. However, due to the complexity of the TME, it is difficult to study how the multitude of different stromal cells varies from one tumor to the next and whether these variances might represent distinct stromal signatures. We hypothesize that soft tissue tumors (STTs) are neoplastic representations of stromal cell types and that these tumors can function as discovery tools for the various stroma types. Previously, we identified stromal gene signatures using DNA microarray analysis of two STTs (Desmoid Type Fibromatosis and Solitary Fibrous Tumor) and found distinct stromal gene expression patterns in breast carcinomas (West et al., 2005). This led to the identification of new stromal cell markers and potentially new targets for breast cancer therapy (Beck et al., 2008). In this study, we evaluate the gene expression profile of another fibroblastic soft tissue tumor, Nodular Fasciitis (NF). An NF-specific gene signature identifies 31 genes coordinately expressed by a subset (10) of breast cancers in a previously published large breast cancer dataset comprised of 295 breast cancer patients. This core set of NF-associated genes identified a group of breast cancer patients with significantly decreased overall survival, and when evaluated on other breast cancer datasets, defined a distinct subset of breast cancers. We examined the localization of this signature in the TME by studying two NF-associated genes, Cathepsin C and Galectin-9, by immunohistochemistry in 225 breast cancers represented on tissue microarrays. Cathepsin C and Galectin-9 were co-expressed in a subset of tumor stroma and identified a distinct subset of breast tumors when compared with SPARC and CSPG-2, two stromal markers previously identified from the fibromatosis stromal signature (West et al., 2005). Our data defines a novel stromal response in breast cancer and may represent potential targets for new cancer therapies. References: Beck AH, Espinosa I, Gilks CB, et al. The Fibromatosis Signature Defines a Robust Stromal Response in Breast Carcinoma. Laboratory Investigations 2008, in press. West RB, Nuyten DS, Subramanian S, et al. Determination of Stromal Signatures in Breast Carcinoma. PLoS Biol 2005;3:e187. Author Disclosure: N.K. Cho, None; A.H. Beck, None; I. Espinosa, None; K. Montgomery, None; S. Zhu, None; M. van de Rijn, None; R.B. West, None.

3151

microRNA Expression is Altered In Vivo by Ionizing Radiation and may be a Target to Prevent Fibrosis

N. L. Simone, B. P. Soule, A. Sowers, D. Gius, J. B. Mitchell National Cancer Institute, Bethesda, MD Purpose/Objective(s): MicroRNAs (miRNA) are small, well-conserved, non-coding RNA species that have been found to regulate protein translation by interfering with mRNA. Their role in the cellular response to external stimuli, such as radiation