Immunoisolation of Pancreatic Epithelial Cells from EUS-FNA with Magnetic Beads for Downstream Molecular Application
Alaa Afify, Shiro Urayama. UC Davis, Sacramento, CA
Background: Evaluation of pancreatic masses is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells in FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique for isolating pancreatic epithelial cells from needle aspirates and to demonstrate that the isolated cells could be utilized for molecular analysis.
Design: All samples included in the study were obtained by EUS-FNA. After adequate sampling for cytopathological evaluation, three additional needle passes were collected individually into a microfuge tube with RNAlater and stored at –80oC. Based on the final cytopathological diagnosis, 17 adenocarcinoma, 2 lymphoma, and 2 benign cases were retrieved from the collection for further analyses. After thawing the frozen specimen, a cytospun slide was prepared and stained with Pap stain for cytology evaluation to confirm the diagnosis and determine the types of cells present. Epithelial cells were isolated using anti-human epithelial cell specific antibody-bound magnetic beads (Invitrogen, Carlsbad, CA). The isolated cellular component was examined cytologically and Genomic DNA was extracted, quantitated and evaluated with methylation-specific PCR for methylation of the promoter region of the cyclin D2 gene.
Results: Evaluation of cytospun slides prepared directly after thawing the frozen specimen confirmed the original diagnosis and the presence of the neoplastic cells admixed with histiocytes, lymphocytes, fibro-connective tissue, and RBCs. After optimization of the technique, we isolated malignant epithelial cells successfully from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from the 2 benign cases. No cells could be isolated from the cases of lymphoma. The mean DNA yields was 357 ng. Methylation-specific PCR for cyclin D2 showed a methylated state at the promoter region, demonstrating feasible evaluation of the methylation status.
Conclusions: Magnetic immunoconcentration of pancreatic EUS-FNA specimens, as described in this study, is a practical method of isolating pancreatic epithelial cells from needle aspirates. This methodology is a relatively easy allowing molecular analysis of pancreatic neoplasms, which may be adaptable for future molecular signal or biomarker assays.
Wednesday, March 5, 2014 1:00 PM
Poster Session VI # 127, Wednesday Afternoon