Serum proteome profiling identifies novel and powerful markers of cystic fibrosis liver disease.

Cystic Fibrosis associated liver disease (CFLD) develops in approximately 30% of CF patients. However, routine sensitive diagnostic tools for CFLD are lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CFLD. 45 CF patients were included in the study and received transient elastography. Differential regulation of 220 different serum proteins was assessed in a subgroup of patients with and without CFLD. Most interesting candidate proteins were further quantified and validated by ELISA in the whole patient cohort. To assess a potential relation of biomarker expression to the degree of hepatic fibrosis, serum biomarkers were further determined in 18 HCV patients where liver histology was available. 43 serum proteins differed at least 2-fold in patients with CFLD compared to those without liver disease as identified in proteome profiling. In ELISA quantifications, TIMP-4 and Endoglin were significantly up-regulated in patients with CFLD as diagnosed by clinical guidelines or increased liver stiffness. Pentraxin-3 was significantly decreased in patients with CFLD. Serum TIMP-4 and Endoglin showed highest values in HCV patients with liver cirrhosis compared to those with fibrosis but without cirrhosis. At a cut-off value of 6.3 kPa, transient elastography compassed a very high diagnostic accuracy and specificity for the detection of CFLD. Among the biomarkers, TIMP-4 and Endoglin exhibited a high diagnostic accuracy for CFLD. Diagnostic sensitivities and negative predictive values were increased when elastography and TIMP-4 and Endoglin were combined for the detection of CFLD. Serum TIMP-4 and Endoglin are increased in CFLD and their expression correlates with hepatic staging. Determination of TIMP-4 and Endoglin together with transient elastography can increase the sensitivity for the non-invasive diagnosis of CFLD

Identification of neutrophil activation markers as novel surrogate markers of CF lung disease.

Cystic Fibrosis (CF) lung disease is characterized by progressively declining lung function and represents a major factor contributing to the high morbidity and mortality associated with CF. However, apart from spirometry, respiratory disease surrogate markers reliably indicating CF lung disease and the occurrence of pulmonary exacerbations (PEx) are still lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CF lung disease.54 adult and 26 pediatric CF patients were included in the study and serum concentrations of MMP-1, -2, -8, -9, -13, TIMP-1, TIMP-2, YKL-40, hyaluronic acid, procollagen III peptide were quantified by ELISA. CF lung disease was diagnosed by lung function test, PEx was defined based on a clinical scoring established by Rosenfeld in 2001.Adults and children with moderate to severe CF lung disease exhibited significantly increased serum expression of MMP-8, MMP-9, YKL-40 and TIMP-1. Further, MMP-8, MMP-9 and YKL-40 were significantly increased in adult CF patients suffering from PEx compared to those without clinical signs of respiratory exacerbation. MMP-8, MMP-9, YKL-40, and TIMP-1 serum levels were unaffected by the presence or absence of CF liver disease or pancreatic insufficiency.MMP-8, MMP-9, and YKL-40 might serve as novel non-invasive biomarkers of CF lung disease and PEx

Serum expression of matrix and neutrophil markers in adult CF patients without (n = 22) and with (n = 32) CFLD.

<p>Serum expression of matrix and neutrophil markers in adult CF patients without (n  = 22) and with (n  = 32) CFLD.</p

Serum expression of MMP-8 and MMP-9 in adult CF patients with CF lung disease.

<p>In adult CF patients serum MMP-8 levels were consistently increased in patients with moderate to severe CF lung disease, as assessed by declined %FEV1 and %VC below 80% and a declined ratio of FEV1/VC below 70%, compared to those with mild CF lung disease (FEV1&VC: ≥80%; FEV1/VC ratio: ≥70%). Serum MMP-9 was also significantly increased in CF patients with decreased %VC and decreased FEV1/VC. Upper and lower hinge: 75th and 25th percentile, respectively; Line: median value; Error bars: minimum and maximum (*p<0.05, **p<0.01).</p

Serum expression of matrix and neutrophil markers in pediatric CF patients without (n = 16) and with (n = 10) CFLD.

<p>Serum expression of matrix and neutrophil markers in pediatric CF patients without (n  = 16) and with (n  = 10) CFLD.</p

Concordance between clinical markers and novel diagnostic markers for CFLD.

<p>Measurement agreement between AST, ALT, γGT and ALP as commonly used clinical markers of CFLD and TIMP-4, Endoglin and transient elastography as novel diagnostic modalities was assessed in Bland-Altman Analyses. TIMP-4 and Endoglin showed very good agreement with ALT (A) and AST (B) levels. Comparison of the concordance of TE and levels of ALT (A), AST (B) and γGT (C) showed good agreement of the different methods although a greater bias was observed with higher average values. A proportional error was observed in the agreement of γGT and the biomarkers TIMP-4 and Endoglin (C) and in the agreement of ALP and TE (D). The dashed line shows 95% limits of agreement, the solid line indicates the systemic error (bias).</p

Demographic and clinical data of the CF patient cohort.

*<p>significantly different, P <0.05</p>**<p>significantly different, P <0.01</p><p>UDCA: Ursodeoxycholic Acid</p

Serum expression of YKL-40 and TIMP-1 in pediatric CF patients with CF lung disease.

<p>Serum levels of YKL-40 were significantly increased in children with reduced %VC and increased by trend in children with a reduced FEV1/VC ratio. TIMP-1 was increased in CF children with a reduced FEV1/VC ratio below 70% compared to those with a ratio above 70%. Upper and lower hinge: 75th and 25th percentile, respectively; Line: median value; Error bars: minimum and maximum. (*p<0.05, **p<0.01).</p

Increased serum levels of Endoglin and decreased concentration of pentraxin-3 in CF patients with increased liver stiffness. CF patients with liver stiffness values above a threshold value of 6.3 kPa, indicative for CFLD, exhibited significantly higher serum levels of Endoglin and significantly lower serum levels of pentraxin-3 compared to CF patients with liver stiffness values below 6.3 kPa.

<p>TIMP-4 was also increased in patients with a liver stiffness above the threshold of 6.3 kPa, although these elevations did not reach statistical significance. The cut-off value of 6.3 kPa for the identification of patients with CFLD was derived from ROC analyses as value at which the sum of diagnostic sensitivity and specificity for the detection of CFLD was maximal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058955#pone-0058955-t003" target="_blank">Table 3</a>).</p