Detection of KRAS Mutations by Locked Nucleic Acid PCR Sequencing in Pancreatic Cyst Fluid Cells
Cristina E Aguilar, Andre L Moreira, Hans Gerdes, Marc Ladanyi, Khedoudja Nafa, Carlie S Sigel. Memorial Sloan-Kettering Cancer Center, New York
Background: Mutation of KRAS is one of the earliest alterations in pancreatic adenocarcinoma (PA). As such, in combination with clinical and cytologic findings, detection of KRAS mutations may have a role in the pre-operative management of pancreatic cysts. As of yet, detection by standard methods has shown suboptimal sensitivity possibly due to low levels of the mutant alleles in cyst fluid. We evaluated a locked nucleic acid (LNA) PCR sequencing assay to detect low levels of mutant KRAS DNA in pancreatic cysts.
Design: DNA was extracted from 32 pancreatic cyst fluid cell pellets (CFP) and 20 formalin-fixed paraffin embedded (FFPE) cell block and resection samples (CB/RS) using a DNA extraction kit. Resection samples were routinely macro-dissected to enrich for tumor. LNA-PCR was performed in the presence of an LNA oligonucleotide that suppresses the amplification of the wild type KRAS exon 2 DNA, leading to preferential amplification of any mutant alleles. The PCR products of both standard and LNA-PCRs were purified and sequenced.
Results: Standard Sanger sequencing detected 8 KRAS mutations (8/32; 25%) from CFP; LNA-PCR detected an additional 7 mutations (7/28; 56%). Thirteen cases were negative by both methods (41%). LNA-PCR was not available in 4 cases. Analysis of corresponding FFPE CB was concordant with CFP in 8/9 cases (89%) by both methods; LNA-PCR detected one additional mutation. Of 11 paired CB/RS archived samples evaluated by both methods, all CB were KRAS negative and 3/11 RS (27%) were KRAS positive. KRAS positive cases (6/20) included intraductal pancreatic mucinous neoplasms (IPMN) (2) with low or high grade dysplasia, mucinous PA, metastatic PA, minimally invasive PA arising with IPMN, and poorly differentiated carcinoma, while KRAS negative cases included an IPMN with moderate dysplasia (1), serous cystadenoma (5), solid pseudopapillary neoplasm (1), low grade well-differentiated cystic endocrine neoplasm (3), benign retention cyst (1), and para-ampullary duodenal cysts involving pancreas (1).
Conclusions: The management of patients with pancreatic cysts requires integration of clinical, radiologic, and pathologic findings. We find that high sensitivity LNA-PCR detects more KRAS mutations than standard PCR, increasing the usefulness of KRAS mutation analysis in the evaluation of low cellularity specimens, further raising diagnostic accuracy.
Tuesday, March 20, 2012 1:30 PM
Platform Session: Section E, Tuesday Afternoon