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, 27 (1), 13

Bile and Urine Peptide Marker Profiles: Access Keys to Molecular Pathways and Biological Processes in Cholangiocarcinoma

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Bile and Urine Peptide Marker Profiles: Access Keys to Molecular Pathways and Biological Processes in Cholangiocarcinoma

Torsten Voigtländer et al. J Biomed Sci.

Abstract

Background: Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers.

Methods: Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression.

Results: For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events.

Conclusions: The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

Keywords: Biomarkers; Biomolecular pathways; Cholangiocarcinoma; Proteomics.

Conflict of interest statement

Harald Mischak is founder and co-owner and Jochen Metzger, Martin Pejchinovski, Agnieszka Latosinska and Maria Frantzi are employees of the biotechnology company mosaiques-diagnostics GmbH located in Hannover, Germany, which developed the bile and urine peptide marker patterns for cholangiocarcinoma diagnosis. All other authors have no competing interests.

Figures

Fig. 1
Fig. 1
Overall survival of patients with a positive or negative test result in combined bile (BPA) and urine (UPA) proteome analysis during a 1-year follow-up starting from the date of endoscopy at which bile and urine were obtained from the patients
Fig. 2
Fig. 2
Group-specific CE-MS peptide marker profiles for primary sclerosing cholangitis (PSC, n = 57), benign biliary diseases (BBD, n = 19) other than PSC, cholangiocarcinoma (CCA, n = 33) alone or concomitant to PSC (CCA on top of PSC, n = 19) in bile (BPA) and urine (UPA) proteome analysis. The molecular mass on a logarithmic scale (0.8–20 kDa) is plotted against normalized capillary electrophoresis (CE) migration time (18–50 min). Mean signal intensities are encoded by peak height
Fig. 3
Fig. 3
Functional association of proteins from which the cholangiocarcinoma (CCA) peptide markers included in the bile and urine proteomic models are derived together with the proteases predicted to be responsible for the generation of these peptides. Proteins were analyzed by functional Gene Ontology biological process group-clustering using CytoScape’s ClueGO plug-in (CytoScape v2.8.3, ClueGO v1.5). Enriched GO-terms are represented as circles, and lines denote the relationship between these terms as functional groups. Line thickness and font-size are directly correlated with the statistical significance of terms and relationships (all with p <  0.05 after Bonferroni-adjustment for multiple testing correction). Statistically significant molecules primarily identified and modulated in bile are marked by a small red circle, whereas urinary proteins are small green circles. The larger circles are associated with GO terms and the coloration is either urine (green) or modulated in both sources (grey)

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