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. 2017 Aug 9;17(1):110.
doi: 10.1186/s12890-017-0455-x.

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker

Affiliations

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker

S N Kehlet et al. BMC Pulm Med. .

Abstract

Background: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders.

Methods: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls.

Results: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively.

Conclusion: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

Keywords: Cancer; Cathepsin-S; Decorin; Extracellular matrix; Idiopathic pulmonary fibrosis; Serum biomarker.

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Conflict of interest statement

Ethics approval and consent to participate

For the clinical IPF study (CTgov reg. NCT00786201) the following ethic committee approved the study: Sterling Institutional Review Board, Sterling Independent Services, Inc. (Atlanta). The healthy control serum samples (cohort 2) were obtained from a Danish study population approved by The National Committee on Health Research Ethics (Denmark). According to Danish law additional ethical approval for measuring biochemical biomarkers in previously collected samples is not required. For the samples obtained from the commercial vendors Proteogenex, Asterand and Valley Biomedical, appropriate Institutional Review Board/Independent Ethical Committee approved sample collection and all patients filed informed consent.

Consent for publication

Not applicable.

Competing interests

C.L. Bager, N. Willumsen, D.J. Leeming and M.A. Karsdal are employed at Nordic Bioscience A/S which is a company involved in discovery and development of biochemical biomarkers. M.A. Karsdal owns stocks at Nordic Bioscience. M. Curran, B Dasgupta and C. Brodmerkel are employees of Janssen R&D LLC. and own stock in Johnson & Johnson. S.N. Kehlet and S. Brix reports no conflict of interest.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Specificity of the DCN-CS monoclonal antibody. Monoclonal antibody reactivity towards the standard peptide (VPKDLPPDTT), the elongated peptide (KVPKDLPPDTT), a non-sense peptide (DSSAPKAAQA) and a non-sense coating peptide (biotin-DSSAPKAAQA) was tested for in the competitive DCN-CS ELISA assay. Signals are shown as optical density (OD) at 450 nm (subtracted the background at 650 nm) as a function of peptide concentration
Fig. 2
Fig. 2
Cleavage of decorin by Cathepsin-S. Degraded decorin levels were measured after 1 h and 24 h incubation of human recombinant decorin with Cathepsin-S. Data were normalized by subtracting the background measured in buffer alone. The experiment was repeated twice and data are shown as the mean of the two replicates with standard deviation
Fig. 3
Fig. 3
Serum DCN-CS levels in patients with fibrotic lung disorders. Serum DCN-CS was assessed in three independent cohorts: Cohort 1 included patients with NSCLC (n = 8), IPF (n = 8), COPD (n = 8), colonoscopy-negative controls (CNC) (n = 8) and a panel of healthy controls (HC) (n = 20). Data were compared using one-way ANOVA adjusted for Tukey’s multiple comparisons test. Cohort 2 consisted of patients with NSCLC (n = 12), SCLC (n = 8) and healthy controls (HC) (n = 43). Data were compared using Kruskal-Wallis adjusted for Dunn’s multiple comparisons test. Cohort 3 comprised serum samples from patients diagnosed with IPF (n = 116) and healthy controls (HC) (n = 38). Groups were compared using unpaired, two-tailed Mann-Whitney test. Data are shown as Tukey box plots. Significance levels: ***: p < 0.001 and ****: p < 0.0001
Fig. 4
Fig. 4
ROC curve analysis. Roc curve analysis was used to evaluate the ability of DCN-CS to discriminate between patients and healthy controls. The preliminary estimated cut-off values for the reported sensitivity/specificity are marked with a red asterix

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