Characterization of Neuropilin-1 in Squamous Cell Carcinoma of the Tongue: A Laboratory Study

Oral Abstract Session 2 otide Polymorphism (SNP) array and GDAS software. Both RNA and DNA were extracted from each tumor. Method of Data Analysis: Software packages: GCOS, GDAS, RMA, SAM. Results: Our work demonstrates clear patterns of chromosomal imbalances and gene expression profiles associated with both UK and Sri Lankan oral cancer and that these are grossly different. Within each cohort subgroups can be defined on the basis of gene expression patterns and close correlations are drawn with clinical features of the tumors. We shall also demonstrate how chromosomal imbalances appear to have an effect on gene expression patterns and that these are unique to each cohort. Conclusion: This work clearly demonstrates unique patterns of chromosomal imbalance and gene expression associated with oral cancer arising from two distinct aetiological groups. Furthermore these patterns can be correlated to the clinical course of the disease. References Alizadeh AA, et al: Nature 403:503, 2000 Nagata M, et al: Int J Cancer 106:683, 2003 O’Donnell R, et al: Oncogene online 2004 doi:10.1038/sj.onc.208285

Gene Expression Changes in Response to Therapeutic Doses of Irradiation and Molecular Manipulation to Change Such Responses in an Oral Cancer Cell Line Victor Lopes, FRCS, FDSRCS, Department of Maxillofacial Surgery, University Hospital Birmingham NHS Trust, Edgbaston, Birmingham, West Midlands B15 2TH, UK (Offer N; Wei WB; Murray P) Statement of the Problem: Investigating the effects of ionising radiation on cells cultured in-vitro has always been difficult because of concerns regarding dosimetry. Frequently investigators have been forced to use gamma sources and have assumed 100% dose absorption. We have developed a protocol that simulates a biological system and is carefully dose controlled. This system will allow adherent oral cancer cell lines to be irradiated in a manner similar to a tumor within the body. As a result it is possible to study genome wide gene expression changes in oral cancer cell lines; about which to date little is understood. Materials and Methods: We examined the gene expression changes in the oral cancer cell line SCC4 at 3, 6, and 12 hours post-irradiation with gamma rays. Therapeutic doses of irradiation (2-4 Gy) were used in a single fraction. RNA was extracted from the cells in culture and each irradiation was repeated 3 times. We used the Affymetrix focus array to determine gene expression at each time point. Gene expression changes were validated by RT-PCR and Western blotting. 40

Method of Data Analysis: Software packages GCOS, RMA, and SAM. Results: Using therapeutic doses of radiation (2-4 Gy) we can show that the great majority of gene expression changes occur within the first 3 hours after irradiation and that by 12 hours there are few changes in gene expression compared to unirradiated cells. We also demonstrate that a large proportion of the conventional DNA damage response genes are not activated after a single fraction of radiation to the p53 mutant cell line SCC4. However we are able to demonstrate a dramatic upregulation of one of the cohesin sub-units which is responsible for homologous recombination (DNA repair). We have also shown that this same gene is not upregulated in p53 wild type cell lines in response to irradiation but is upregulated in a proportion of ex-vivo irradiated primary oral cancers. We shall also present data showing that manipulation of the expression of this gene can have effects on the cell dramatically changing the response of the cell to irradiation. Conclusion: Modifying the cellular response to irradiation may in future prove to be a useful therapeutic intervention. In particular, amplifying cellular response may allow radiation dose reduction. This would be most advantageous when radiation is used as an adjuvant therapy. Here the benefits are less clear when compared to potential complications. Dose reduction would reduce complications and improve the safety profile of adjuvant radiotherapy. References Birkenbihl RP, et al: Nucleic Acids Res 20:6605, 1992 Zhang Y, et al: Radiat Res 161:667, 2004

Characterization of Neuropilin-1 in Squamous Cell Carcinoma of the Tongue: A Laboratory Study Sanjay Sharma, BDS, MBBS, FDSRCS, MRCSI, MRCS, 5 Cadgwith Place, Port Solent, Hampshire PO6 4TD, UK (Quintera-Ortiz M; Spedding A; Anand R; Faris K; Brennan P; Cree I) Statement of the Problem: Angiogenesis is a key process in the growth of all solid tumors. Neuropilin-1 has recently been identified as a co-receptor for vascular endothelial growth factor receptor 2 (VEGFR2) and enhances the binding of VEGF-165 to VEGFR2 promoting endothelial cell proliferation. Neuropilin-1 has been described in breast, prostate, and lung cancers. We aim to characterize neuropilin-1 in squamous cell carcinoma. Materials and Methods: Immunohistochemical staining of 38 paraffin fixed sections of primary tongue squamous cell carcinoma (SCC) with antibodies to NRP-1, VEGF-165, and CD34. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blots of SCC cell lines for mRNA and protein respectively. AAOMS • 2005

Oral Abstract Session 2 Method of Data Analysis: Pearson’s coefficient of correlation and linear regression analysis to compare neuropilin-1 with VEGF and microvessel density. KaplanMeier survival curves were plotted for neuropilin-1 positive and negative tumors. Bio-Rad i-Cycler software analysis to compare adjusted crossing thresholds of neuropilin-1 with 4 housekeeping genes. SYBR – green gel analysis of RT-qPCR products together with digital Western blot analysis of neuropilin-1 proteins. Results: Neuropilin-1 expression was seen in 58% of specimens. Most of the staining pattern was cytoplasmic with granular positivity. There is a strong correlation with VEGF distribution and no correlation with microvessel density. There was no difference in overall survival between neuropilin-1 positive and negative tumors when analyzed by Kaplan-Meier statistics. RT-qPCR showed crossing thresholds comparable with those of the housekeeping genes. Gels confirmed product specificity. Western blots were strongly positive for neuropilin-1 in all cell lines tested. Conclusion: This is the first study to characterize neuropilin-1 in squamous cell carcinoma. We postulate that neuropilin-1 may act as an intracellular transducer of VEGF signalling rather than acting purely as a co-receptor for VEGFR2.

treated for 12 months were compared with the patients who received calcitonin for 15 months. Volumetric analysis of the lesions was preformed by two radiologists, who were blinded for the therapy, on CT-scans made every 3 months. Method of Data Analysis: The Chi2-test was used to compare the distribution of tumor reduction (categorical variables) between the two populations after three months and at cessation of therapy. Results: There was no difference in reduction of the lesions between the placebo group and the calcitonin group after 3 months. There was also no difference in reduction of the lesions after 12 or 15 months of therapy. After 6 months of follow-up there was a clinically and radiologically considerable reduction in the size of the lesion in 7 patients (mean: 38% vol). One patient was out of the study after 3 months and in one patient the lesion exhibited ongoing growth during the whole treatment. Conclusion: A positive effect of calcitonin could not be demonstrated during the placebo-controlled period. Also, there was no significant difference between the 12 and 15 months treatment periods. However, after 6 months of follow-up there was a considerable reduction of the lesion in half of the patients. In the other patients calcitonin was not effective.

References Fuh G, Garcia KC, de Vos AM: The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. J Biol Chem 275:26690, 2000 Miao HQ, Lee P, Lin H, et al: Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression. FASEB J 14:2532, 2000

Calcitonin Therapy in Central Giant Cell Granuloma: A Randomized Double Blind Placebo-Controlled Study Jan de Lange, DDS, MD, Jachtlaan 17, Wezep 8091BL, The Netherlands (van den Akker HP; van Zanten V; Engelshove HA) Statement of the Problem: The successful therapeutic use of calcitonin in patients with a central giant cell granuloma (CGCG) has been shown in several case reports. However, at present a randomized controlled trial to demonstrate a positive therapeutic effect of calcitonin is not available. In this presentation the results of such a trial are discussed. Materials and Methods: In a prospective, randomized, double- blinded, placebo-controlled clinical trial 14 patients with a histologically confirmed CGCG and normal calcium and parathyroid hormone serum levels were included over a period of 2 years. They were treated with intranasally administered salmon calcitonin or a placebo. The placebo-controlled period was 3 months, after which all patients were treated with calcitonin for one year. At the end of therapy the patients who were AAOMS • 2005

References Harris M. Central giant cell granulomas of the jaws regress with calcitonin therapy. Br J Oral Maxillofac Surg 31:89, 1993 de Lange J, Rosenberg AJWP, van den Akker HP, et al: Treatment of central giant cell granuloma of the jaw with calcitonin. Int J Oral Maxillofac Surg 28:372, 1999 Pogrel MA: Calcitonin therapy for central giant cell granuloma. J Oral Maxillofac Surg 61:649, 2003 Funding Source: Novartis Pharma A.G. provided the medication.

A Role for Specialist Nurses in a Maxillofacial Outpatient Clinic: Delivery of a Motivational Interview Package to Patients With Alcohol-Related Facial Injury Ian Holland, BDS, FDSRCPS, MBBS, FRCS (OMFS), Falkirk Royal Infirmary, 1 Major Loan, Falkirk, Stirlingshire FK1 5QE, UK (Oakey F; Goodall C; Crawford A; Smith I; Gilchrist G; Bowman A; Russel A; Koppel D; Ayoub A) Statement of the Problem: Alcohol abuse and alcohol related violence are growing problems. A recent study showed that 24% of facial injuries in the UK are alcohol related (Hutchison et al, 1998). Such injuries can have long lasting effects on patients both physically and psychologically (Hull et al, 2003). Often, the injury is treated surgically while the underlying causative factors such as 41