Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts
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Published OnlineFirst April 19, 2017; DOI: 10.1158/1078-0432.CCR-16-2987 Biology of Human Tumors Clinical Cancer Research Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts Sam L. Ivry1,2, Jeremy M. Sharib3, Dana A. Dominguez3, Nilotpal Roy4, Stacy E. Hatcher3, Michele T. Yip-Schneider5, C. Max Schmidt5, Randall E. Brand6, Walter G. Park7, Matthias Hebrok4, Grace E. Kim8, Anthony J. O'Donoghue9, Kimberly S. Kirkwood3, and Charles S. Craik1 Abstract Purpose: Pancreatic cysts are estimated to be present in 2%–3% sion was associated with regions of low-grade dysplasia, whereas of the adult population. Unfortunately, current diagnostics do not cathepsin E expression was independent of dysplasia grade. accurately distinguish benign cysts from those that can progress Gastricsin activity differentiated mucinous from nonmucinous into invasive cancer. Misregulated pericellular proteolysis is a cysts with a specificity of 100% and a sensitivity of 93%, whereas hallmark of malignancy, and therefore, we used a global approach cathepsin E activity was 92% specific and 70% sensitive. Gastricsin to discover protease activities that differentiate benign nonmu- significantly outperformed the most widely used molecular bio- cinous cysts from premalignant mucinous cysts. marker, carcinoembryonic antigen (CEA), which demonstrated Experimental Design: We employed an unbiased and global 94% specificity and 65% sensitivity. Combined analysis of gas- protease profiling approach to discover protease activities in 23 tricsin and CEA resulted in a near perfect classifier with 100% cyst fluid samples. The distinguishing activities of select proteases specificity and 98% sensitivity. was confirmed in 110 samples using specific fluorogenic sub- Conclusions: Quantitation of gastricsin and cathepsin E strates and required less than 5 mL of cyst fluid. activities accurately distinguished mucinous from nonmuci- Results: We determined that the activities of the aspartyl nous pancreatic cysts and has the potential to replace current proteases gastricsin and cathepsin E are highly increased in fluid diagnostics for analysis of these highly prevalent lesions. from mucinous cysts. IHC analysis revealed that gastricsin expres- Clin Cancer Res; 23(16); 4865–74. Ó2017 AACR. Introduction plasms (MCN), pseudocysts, and serous cystadenomas (SCA; ref. 4). Both IPMNs and MCNs, which are collectively referred The detection of pancreatic cysts has increased dramatically due to as mucinous cysts, may develop foci of high-grade dysplasia or to the rising use of high-resolution abdominal imaging. Pancre- cancer (5). At the time of resection, approximately 30% of IPMNs atic cysts are incidentally detected in 13%–45% of patients and approximately 15% of MCNs contain invasive cancer (6, 7). evaluated by MRI and 2% of patients evaluated by CT (1–3). The Pseudocysts and SCAs, which are both nonmucinous, rarely most frequently detected pancreatic cysts include intraductal undergo malignant degeneration and are considered benign papillary mucinous neoplasms (IPMN), mucinous cystic neo- lesions that typically do not require resection or continued surveillance. Clinical decision making for pancreatic cysts relies 1Department of Pharmaceutical Chemistry, University of California, San Fran- largely on radiographic and clinical features, augmented by anal- cisco, San Francisco, California. 2Pharmaceutical Sciences and Pharmacoge- ysis of cyst fluid collected by endoscopic ultrasound with fine- nomics Graduate Program, University of California, San Francisco, San Francisco, needle need aspiration (EUS-FNA; ref. 8). Unfortunately, with California. 3Department of Surgery, University of California, San Francisco, San current clinical guidelines, distinguishing nonmucinous from 4 Francisco, California. Diabetes Center, Department of Medicine, University of mucinous cysts remains a challenge. The preoperative diagnosis California, San Francisco, San Francisco, California. 5Department of Surgery, of mucinous cysts is incorrect in up to 30% of cases and benign Indiana University School of Medicine, Indianapolis, Indiana. 6Division of Gas- troenterology, Hepatology, and Nutrition, University of Pittsburgh Medical cysts are often resected, exposing patients to an unnecessary risk Center, Pittsburgh, Pennsylvania. 7Department of Medicine, Stanford University for morbidity (9–12). School of Medicine, Stanford, California. 8Department of Pathology, University As abdominal imaging remains unable to accurately differ- of California, San Francisco, San Francisco, California. 9Skaggs School of Phar- entiate pancreatic cyst types, there has been considerable effort macy and Pharmaceutical Chemistry, University of California, San Diego, La towards developing improved diagnostic biomarkers. Most of Jolla, California. these biomarkers utilize cyst fluid collected by EUS-FNA. CEA is Note: Supplementary data for this article are available at Clinical Cancer the most widely investigated biomarker and is 60%–80% Research Online (http://clincancerres.aacrjournals.org/). accurate for differentiating mucinous from nonmucinous cysts Corresponding Author: Charles S. Craik, University of California, San Francisco, (13, 14). KRAS mutations occur in more than 90% of pancreatic San Francisco, S-512, 600 16th Street, San Francisco, CA 94143. Phone: 415-476- cancers and are frequently observed in mucinous cysts (15, 16). 8146; Fax: 415-502-8298; E-mail: Charles.Craik@ucsf.edu Analysis of cyst fluid DNA revealed that KRAS mutations are doi: 10.1158/1078-0432.CCR-16-2987 100% specific, but only 50% sensitive for diagnosing a mucin- Ó2017 American Association for Cancer Research. ous cyst (17). Similarly, analysis of mutations in the oncogene www.aacrjournals.org 4865 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 19, 2017; DOI: 10.1158/1078-0432.CCR-16-2987 Ivry et al. and outperformed CEA for differentiating mucinous from non- Translational Relevance mucinous pancreatic cystic lesions. With advances in abdominal imaging technologies, the incidental detection of pancreatic cysts continues to rise. Materials and Methods However, there remains a lack of accurate molecular diagnos- tics for differentiating benign cystic lesions from those that can Patients and sample acquisition progress into pancreatic cancer. This has led to a dramatic Pancreatic cyst fluid samples were collected from preconsented increase in the number of potentially unnecessary pancreatic patients under Institutional review board–approved protocols resections, which are associated with high rates of morbidity. and in accordance with U.S. Common Rule at the University of Using a global and unbiased protease-activity profiling California San Francisco (San Francisco, CA), the University of approach and patient cyst fluid, we determined that the Pittsburgh Medical Center (Pittsburgh, PA), Indiana University activities of the aspartyl proteases gastricsin and cathepsin E School of Medicine (Indianapolis, Indiana), and Stanford Uni- accurately differentiate premalignant mucinous cysts from versity School of Medicine (Stanford, CA). Patient information is benign nonmucinous cysts. In particular, analysis of gastricsin summarized in Supplementary Table S1. All patients included in activity demonstrated 93% sensitivity and 100% specificity for our study underwent surgical resection of their cystic lesion and differentiating mucinous lesions. Our simple and direct fluo- have a pathologically confirmed diagnosis. The highest grade of rescence-based approach for stratification of pancreatic cysts dysplasia observed during pathologic evaluation of each cystic significantly outperformed the most widely used molecular lesion is reported. Samples were collected either at the time of biomarker, carcinoembryonic antigen (CEA), and can be surgical resection or during diagnostic endoscopic ultrasound readily translated into an actionable diagnostic assay to help prior to resection of the cystic lesion. Cyst fluid samples were improve clinical management of these challenging lesions. split into 100-mL aliquots and frozen to À80 C within 60 minutes of collection. Samples underwent at most two freeze–thaw cycles prior to experimental analysis. Total cyst fluid protein concentra- tion was determined by the bicinchoninic acid assay. CEA levels GNAS are specific for diagnosing IPMNs, but suffer from low were evaluated for the majority of samples, but were unavailable sensitivity (18). A variety of other cyst fluid biomarkers have in 21 cases due to limited cyst fluid volume. also been explored (19–23); however, CEA remains the only widely applied molecular biomarker for differentiating mucin- Multiplex substrate profiling by mass spectrometry assay ous from nonmucinous cysts. The multiplex substrate profilingbymassspectrometry Proteases mediate a variety of critical processes in cancer, (MSP-MS) assay was performed as described previously (34). including invasion of the basement membrane via cleavage of Cyst fluidwasdilutedto100mg/mL in assay buffer (either extracellular matrix proteins and promotion of oncogenic sig- pH 7.5 phosphate buffer or pH 3.5 acetate buffer) and pre- naling pathways through activation of growth factors and incubated for 10 minutes. For analysis of protease inhibitor receptor tyrosine kinases (24, 25). In pancreatic cancer, mem- sensitivity, 1 mmol/L AEBSF (Sigma, A8456), 2 mmol/L E-64 bers of the cathepsin family of endolysosomal proteases are (Sigma,