Abstract
BackgroundDistal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. Diagnostic and prognostic biomarkers for distal cholangiocarcinoma are lacking. The aim of the present study was to identify differentially expressed proteins between distal cholangiocarcinoma and normal bile duct samples.MethodsA workflow utilizing discovery mass spectrometry and verification by parallel reaction monitoring was used to analyze surgically resected formalin-fixed, paraffin-embedded samples from distal cholangiocarcinoma patients and normal bile duct samples. Bioinformatic analysis was used for functional annotation and pathway analysis. Immunohistochemistry was performed to validate the expression of thrombospondin-2 and investigate its association with survival.ResultsIn the discovery study, a total of 3057 proteins were identified. Eighty-seven proteins were found to be differentially expressed (q < 0.05 and fold change ≥ 2 or ≤ 0.5); 31 proteins were upregulated and 56 were downregulated in the distal cholangiocarcinoma samples compared to controls. Bioinformatic analysis revealed an abundance of differentially expressed proteins associated with the tumor reactive stroma. Parallel reaction monitoring verified 28 proteins as upregulated and 18 as downregulated in distal cholangiocarcinoma samples compared to controls. Immunohistochemical validation revealed thrombospondin-2 to be upregulated in distal cholangiocarcinoma epithelial and stromal compartments. In paired lymph node metastases samples, thrombospondin-2 expression was significantly lower; however, stromal thrombospondin-2 expression was still frequent (72%). Stromal thrombospondin-2 was an independent predictor of poor disease-free survival (HR 3.95, 95% CI 1.09–14.3; P = 0.037).ConclusionSeveral proteins without prior association with distal cholangiocarcinoma biology were identified and verified as differentially expressed between distal cholangiocarcinoma and normal bile duct samples. These proteins can be further evaluated to elucidate their biomarker potential and role in distal cholangiocarcinoma carcinogenesis. Stromal thrombospondin-2 is a potential prognostic marker in distal cholangiocarcinoma.
Highlights
Cholangiocarcinoma (CCA), an epithelial tumor arising along the biliary tree, is a low- incidence malignancy accounting for approximately 3% of all gastrointestinal cancers [1]
Discovery study A workflow including discovery liquid chromatography (LC)–mass spectrometry (MS)/MS followed by Parallel reaction monitoring (PRM) verification using macrodissected archived FFPE samples was used to identify differentially expressed proteins (DEPs) between Distal cholangiocarcinoma (dCCA) and controls
The clinicopathological data of the patients whose samples were analyzed are presented in Bioinformatic analysis Two-way unsupervised hierarchical clustering was applied to the 87 DEPs, and the results were visualized in a heat-map (Fig. 2)
Summary
Cholangiocarcinoma (CCA), an epithelial tumor arising along the biliary tree, is a low- incidence malignancy accounting for approximately 3% of all gastrointestinal cancers [1]. CCA is a notably aggressive malignancy with a current overall 5-year survival rate of less than 5% [2]. Based on its anatomical location along the biliary tree, CCA is classified as intrahepatic (iCCA) or extrahepatic (eCCA) which is further divided into the perihilar (pCCA) and distal (dCCA) subtypes [4]. DCCA is located between the insertion of the cystic duct and the ampulla of Vater [4, 5]. Distal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. The aim of the present study was to identify differentially expressed proteins between distal cholangiocarcinoma and normal bile duct samples
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