ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 385 Introduction: Palladin is a cytoskeletal protein involved in actin organization and cell motility. Recent research showed that a mutation in palladin is linked to a form of familial pancreatic cancer, and that palladin may be overexpressed in sporadic pancreatic cancers. The role of palladin in breast cancer has not been well elucidated. Previous work suggests that palladin expression is increased in phenotypically invasive breast cancer cells, while others report a decrease in palladin mRNA levels in metastatic lesions. Research in palladin is complicated in that multiple isoforms are generated from a single gene with nested promoters, so that different probes may detect different isoforms. Our purpose was to determine the role of palladin in human breast cancer by examining protein levels in both in vitro models and human breast cancer specimens using multiple well-characterized palladin antibodies. Materials and Methods: Three breast cancer cell lines (MCF-7, MDA-MB-231, and SUM159) were selected based on their different metastatic potentials. The presence of palladin was assessed in whole cell extracts using western blot analysis with a commercial antibody (Proteintech Group, Inc., Chicago, IL). Three primary human breast cancer specimens, three metastatic breast cancer specimens and three samples of benign breast tissue were obtained from the UNC Tissue Procurement Facility, and extracts were prepared and analyzed using western blots with two additional palladin polyclonal antibodies (4IG and 622). Results: Examination of palladin protein levels in the in vitro models demonstrated increases correlating directly with increasing metastatic potential. Although palladin was expressed in all cell lines, the highest expression was noted in the cells characterized to have the highest metastatic potential (SUM159) and the lowest expression was in the ER(⫹) MCF-7 cells. In the human breast cancer tissue specimens, the commercial antibody illustrated an increase in palladin levels in 2/3 primary tumors and 2/3 metastatic lesions when compared to normals. Evaluation with the 622 antibody demonstrated a similar pattern of palladin expression (4/6 tumors) but different isoforms were noted. Finally, the 4IG antibody revealed that all (6/6) primary and metastatic specimens had more palladin than normal breast tissue (see Figure). Conclusion: Palladin protein levels in cultured cells are directly correlated to their metastatic potential. Importantly, the expression of palladin is increased in human breast cancer specimens when compared to benign breast tissue. Further investigations are ongoing to determine the role of palladin and its isoforms in breast cancer, and whether palladin expression can be linked to metastatic potential, severity of disease, tumor type, or clinical outcome.
QS297. DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE DETECTION OF GERMLINE MUTATIONS IN PHAEOCHROMOCYTOMA. Goswin Y. Meyer-Rochow1, Janine M. Smith2, Debbie J. Marsh3, Bruce G. Robinson1, Stanley B. Sidhu1, Diana E. Benn1; 1Cancer Genetics Laboratory, Hormone and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia; 2Department of Clinical Genetics, The Children’s Hospital at Westmead, Westmead, Australia; 3Functional
Genomics, Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, Sydney, Australia Introduction: Pheochromocytomas are neuroendocrine tumours of chromaffin cell origin, arising from the adrenal medulla and less commonly from extra-adrenal sympathetic paraganglia within the abdomen, thorax and neck. For some time it has been recognised that pheochromocytomas are component tumours in the following familial syndromes: Multiple Endocrine Neoplasia type 2, von Hippel Lindau disease and Neurofibromatosis type 1 caused by mutations in RET, VHL and NF1 respectively. More recently germline mutations in the genes encoding succinate dehydrogenase subunit B (SDHB) and subunit D (SDHD) have been identified as high risk factors for the development of Pheochromocytoma/Paraganglioma syndromes. Approximately 11-24% of pheochromocytomas with an apparently sporadic presentation have been shown to carry a germline mutation indicating familial disease. Finding a germline mutation in a patient is an indication for the screening of tumours associated with the identified syndrome, thereby allowing earlier detection of associated disease. The aim of this study was to perform mutation analysis for the VHL, SDHB, and SDHD genes in a cohort of 82 patients diagnosed with a pheochromocytoma, using denaturing high performance liquid chromatography (dHPLC). Methods: A polymerase chain reaction (PCR) of all exons of the three genes was performed on leukocyte DNA extracted from stored blood samples of 82 patients treated for pheochromocytoma. Institutional ethics was obtained to use samples stored in the Kolling Institue of Medical Research Neuroendocrine tumour bank. There were 40 males with a median age of 51 (11-89) years and 42 females with a median age of 48 (11-77) years in the sample population. dHPLC was undertaken on a WAVE TM DNA fragment analysis system (Transgenomic, Omaha, NE, USA) with analysis performed using the WAVEMaker TM software. Samples demonstrating variance when compared to normal controls were selected for sequencing. Results: Using this method a total of five mutations and 13 polymorphisms were detected in SDHB and seven mutations and one polymorphism were identified in VHL. All mutations were confirmed by re-extraction of DNA from the original blood sample, repeat PCR and dHPLC, followed by sequencing. Of 52 apparently sporadic pheochromocytoma, three SDHB (6%) and one VHL (2%) mutations were identified which is consistent with previously published literature. SDHD mutation analysis is in progress. With PCR and dHPLC (followed by sequencing of variants only), the total amount of DNA sequencing required was able to be reduced by approximately 90% when compared to screening by sequence analysis alone. Conclusions: dHPLC appears to be a cost effective screening tool for the detection of germ-line mutations in SDHB and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients. The detection of germline mutations in a patient with pheochromocytoma identifies familial disease which demonstrates the clinical importance of mutation analysis in the management of pheochromocytoma. QS298. ROLE OF CPG ISLAND METHYLATOR PHENOTYPE (CIMP) IN ULCERATIVE COLITIS ONCOGENESIS. Julian A. Sanchez, Kathryn L. DeJulius, Craig A. Messick, Mary P. Bronner, Graham Casey, James M. Church, Matthew F. Kalady; Cleveland Clinic Foundation, Cleveland, OH Introduction: Patients with ulcerative colitis (UC) harbor increased colorectal cancer risk which is believed to be secondary to repetitive colonic mucosal injury. However, underlying causes for malignant transformation remain incompletely defined. Epigenetic methylation of DNA has been implicated as a mechanism for oncogenesis in a variety of inflammation-associated cancers including gastric MALT lymphoma, Barrett’s-associated esophageal cancer, and hepatocellular carcinoma. This study evaluates the relative contribution of epigenetic methylation as well as the more classical