title: Immunoisolation of pancreatic epithelial cells from endoscopic ultrasound-guided fine needle aspirates with magnetic beads for downstream molecular application.
abstract: BACKGROUND
  Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis.
  
  
  METHODS
  Pancreatic EUS-FNA specimens were processed and stored at -80°C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases.
  
  
  RESULTS
  After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases.  DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status.
  
  
  CONCLUSIONS
  The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.
authors.affiliation: Division of Cytopathology, Department of Pathology and Laboratory Medicine, University of California, Davis, Sacramento, California, 95817.
authors.forename: Alaa
authors.initials: A
authors.lastname: Afify
authors.affiliation: Division of Cytopathology, Department of Pathology and Laboratory Medicine, University of California, Davis, Sacramento, California, 95817.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Huang
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California, Davis, California.
authors.forename: Matthew
authors.initials: M
authors.lastname: Jeong
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California, Davis, California.
authors.forename: Shiro
authors.initials: S
authors.lastname: Urayama
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014408:Biomarkers, Tumor / Q000235:genetics*; D056742:Cyclin D2 / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D003937:Diagnosis, Differential; D061765:Endoscopic Ultrasound-Guided Fine Needle Aspiration; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D008223:Lymphoma / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010179:Pancreas / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic
chemical_list: D014408:Biomarkers, Tumor; C080336:CCND2 protein, human; D056742:Cyclin D2
keywords: EUS-FNA; immunoconcentration; magnetic beads; pancreatic adenocarcinoma