Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts

Abstract

Purpose Pancreatic cysts are estimated to be present 2–3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Missregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts. Experimental Design We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 µL of cyst fluid. Results We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. Immunohistochemical analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity. Conclusions Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions.

Early Detection of Pancreatic Cancer: Opportunities and Challenges

“…98 Promising biomarker results with PCF have also been reported for differentially methylated DNA, telomerase activity, protease expression, and the overexpression of Das-1. [99][100][101][102] However, the majority of these biomarkers have not been rigorous validated in a diverse cohort of pancreatic cysts. Hence, this is the goal of the Pancreatic Cyst Biomarker Validation Study, an ongoing double-blinded PCF biomarker study that is sponsored by the National Cancer Institute Early Detection Research Network.…”

Activatable zymography probes enable in situ localization of protease dysregulation in cancer

“…biospecimens (21)(22)(23)(24). Recent approaches leveraging synthetic substrates have used dropletbased microfluidics (25) or highly multiplexed peptide libraries (26,27) to profile protease activity in patient-derived biospecimens. However, these assays do not provide insight on spatial localization of protease activity within the tissue.…”

Global substrate specificity profiling of post‐translational modifying enzymes

“…The MSP‐MS assay has been used with protease inhibitors, gene deletions, and immunodepletion in combination with traditional proteomic methods to identify component proteases that are highly active in complex biological samples. This has enabled the “deconvolution” of proteolytic signatures from fungal pathogens, parasitic organisms, cancer cell lines, and patient samples, and allowed for the prioritization of proteases based on their functional contribution to the global substrate specificity profile. For example, the MSP‐MS assay was used to analyze the global activity signatures of the opportunistic fungal pathogen Candida albicans in the planktonic and biofilm states .…”

“…Comparison of the activity signatures from each state coupled to inhibitor and proteomic analysis revealed that two specific secreted aspartyl proteases (Saps) are upregulated during biofilm growth and are critical to biofilm formation in vitro and in vivo. MSP‐MS was also recently used to identify two human aspartyl proteases that are selectively upregulated in cystic precursor lesions of pancreatic cancer . This strategy enabled the development of a highly accurate diagnostic assay using fluorogenic substrates for differentiating benign and premalignant lesions within a cohort of patient cyst fluid samples.…”

“…MSP-MS was also recently used to identify two human aspartyl proteases that are selectively upregulated in cystic precursor lesions of pancreatic cancer. 73 This strategy enabled the development of a highly accurate diagnostic assay using fluorogenic substrates for differentiating benign and premalignant lesions within a cohort of patient cyst fluid samples.…”