Microdissection Based Genotyping Improves the Accuracy of EUS Guided FNA of Pancreatic Tumors

Abstract

PURPOSE: Endoscopic ultrasound guided fine needle aspiration (EUS/FNA) of pancreatic cancer is confounded by inconclusive cytological diagnoses in a significant subset of cases. We propose that Microdissection based genotyping (MBG) improves the yield of EUS/FNA in pancreatic cancer. METHODS: We studied 11 patients with pancreatic masses who underwent both EUS/FNA and surgical resection. 6 patients had positive cytology for pancreatic adenocarcinoma. 5 patients had suspicious cytology. 4 additional patients who underwent resection of inflammatory masses (chronic pancreatitis) served as negative controls. At least 2 individual aggregates of 50-200 representative cells were microdissected from each EUS/FNA sample after removal of the cover slip. Cancer and adjacent non-neoplastic (internal control) tissue were microdissected from serial unstained tissue sections. Negative control specimens were also microdissected. Polymerase chain reaction (PCR) was performed on the microdissected cells to generate sufficient DNA for broad panel genotyping of microsatellite allele loss markers situated at 1p, 3p, 5q, 9p, 10q & 17p. Loss of Heterozygosity (LOH) analysis utilized fluorescent capillary electrophoresis for quantitative determination of allelic imbalance. In addition k-ras-2 point mutation analysis was performed. RESULTS: No LOH was seen in the internal control and negative control (chronic pancreatitis) samples. Surgical pathology of one patient with suspicious cytology revealed autoimmune pancreatitis. No LOH was seen in the cytologic specimen and resected specimen from this case. All patients with cancer had multiple LOH (fractional allelic loss {FAL}-0.48) at critical sites (p=0.002). A high concordance rate (matching mutations) between tissue genotyping (FAL - 0.52) and cytology genotyping (FAL-0.48) was seen. All cases with positive (FAL-0.48) and suspicious (FAL-0.48) cytology had similar mutational damage. CONCLUSIONS: 1) MB broad panel LOH analysis can be reliably performed on EUS/FNA samples and improves the diagnostic accuracy. 2) The genotyping findings presented support the use of MBG when inconclusive cytology is encountered in the context of pancreatic cancer.