Objectives: Accurate differentiation between benign and malignant causes of biliary obstruction remains challenging and reliable biomarkers are urgently needed. Bile is a potential source of such biomarkers. Our aim was to apply a proteomic approach to identify a potential biomarker in bile that differentiates between malignant and benign disease, and to assess its diagnostic accuracy. Neutrophil gelatinase-associated lipocalin (NGAL) is multi-functional protein, released from activated neutrophils, with roles in inflammation, immune function, and carcinogenesis. It has not previously been described in bile.
Methods: Bile, urine, and serum were collected prospectively from 38 patients undergoing endoscopic retrograde cholangiopancreatography ("discovery" cohort); 22 had benign and 16 had malignant pancreatobiliary disease. Initially, label-free proteomics and immunoblotting were performed in samples from a subset of these patients. Enzyme-linked immunosorbent assay was then performed for NGAL as a potential biomarker on all samples in this cohort. The diagnostic performance of biliary NGAL was then validated in a second, independent group ("validation" cohort) of 21 patients with pancreatobiliary disease (benign n=14, malignant n=7).
Results: NGAL levels were significantly raised in bile from the malignant disease group, compared with bile from the benign disease group in the discovery cohort (median 1,556 vs. 480 ng/ml, P=0.007). Biliary NGAL levels had a receiver operating characteristic area under curve of 0.76, sensitivity 94%, specificity 55%, positive predictive value 60%, and negative predictive value 92% for distinguishing malignant from benign causes. Biliary NGAL was independent of serum biochemistry and carbohydrate antigen 19-9 (CA 19-9) in differentiating between underlying benign and malignant disease. No significant differences in serum and urine NGAL levels were found between benign and malignant disease. Combining biliary NGAL and serum CA 19-9 improved diagnostic accuracy for malignancy (sensitivity 85%, specificity 82%, positive predictive value 79%, and negative predictive value 87%). The diagnostic accuracy of biliary NGAL was confirmed in the second independent validation cohort.
Conclusions: NGAL in bile is a novel potential biomarker to help distinguish benign from malignant biliary obstruction.