Abstract B8: Proteomic analysis of cyst fluids from intraductal papillary mucinous neoplasms

Abstract

Introduction: As a result of the increasing use and improvements in imaging technologies such as computed tomography and magnetic resonance imaging, the frequency at which pancreatic cysts are detected has been growing. Diagnosing the biological nature of such pancreatic cystic lesions remains a difficult challenge to clinicians, as diagnosis by cytology is insensitive and there are currently no reliable methods to measure the degree malignant potential of cysts. Therefore, new markers capable of differentiating benign from malignant IPMNs would assist in establishing an optimal therapeutic strategy for treatment by identifying patients who would benefit from surgery or surveillance. We applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics and multiple reaction monitoring (LC-MRM-MS) analyses on a collection of IPMN cyst fluid samples to identify and verify potential biomarker which may serve as diagnostic indicators of malignant potential and assist in risk stratification. Methods: A total of 15 IPMN pancreatic cyst fluid samples (5 adenomas, 5 carcinomas in situ and 5 invasive carcinomas) were obtained from freshly resected cysts and were analyzed by shotgun proteomics. Aliquots of each sample were reduced, alkylated and digested with trypsin and the resulting peptides were fractionated by isoelectric focusing (pI 3.5-4.7) into 15 fractions. Reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using data-dependent scanning on a Thermo Orbitrap Velos instrument. Following LC-MS/MS analysis, tandem mass spectra were searched against the human IPI database (v.3.56) using the Myrimatch algorithm and the search results filtered using IDPicker at a 5% FDR. A spectral counting approach was applied to detect statistically significant differences in protein abundances with the Quasitel algorithm. MRM assays were configured for differential proteins and further verified in a new collection of unfractionated IPMN cyst fluids with varying degrees of dysplasia (mild, moderate, severe and invasive carcinoma). LC-MRM-MS was performed on peptide samples and were analyzed in triplicate (4 peptides per protein and 5 transitions per peptides) on a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Fisher Scientific, Waltham MA) equipped with an Eksigent nanoLC Ultra 1D Plus pump and AS-2 autosampler (Eksigent Technologies, Dublin CA). A single isotopically labeled peptide was spiked into the samples which served as the normalization reference for all of the peptides in the analysis. All data analysis was performed using Skyline. Preliminary Results: A total of 373,673 spectra were confidently identified, which corresponded to 5,259 protein groups across all biological and technical replicates. An average of 3,534, 3,393, and 4,597 protein groups were identified in the adenomas, carcinomas in situ, and invasive carcinomas, respectively. Spectral count data indicated approximately 50 proteins differentially expressed between benign adenomas and carcinoma in situ and 176 proteins different between adenomas and invasive cancers. From these data, a list of 30 potential candidates was generated, which included proteins upregulated in carcinoma in situ and invasive cancer, as well as several down-regulated proteins, which included common digestive enzymes, such as amylase 2B and elastases. In addition, several MUC isoforms and CEACAM were also targeted, as these have been suggested to distinguish nonmucinous from mucinous cystic lesions. The preliminary MRM data are consistent with the trends observed in the spectral count data from shotgun analyses. The analyses of pancreatic cyst fluids using proteomics platforms may lead to biomarkers that can assist clinicians in differentiating benign from malignant cysts, thus leading to improved diagnosis and treatment. Citation Format: Lisa J. Zimmerman, Cristina R. Ferrone, Nipun Merchant, Robbert J. C. Slebos, Daniel C. Liebler. Proteomic analysis of cyst fluids from intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B8.