Liquid Biopsy of Bile based on Targeted Mass Spectrometry for the Diagnosis of Malignant Biliary Strictures.

Bile holds biomarkers of malignant biliary strictures (MBS) but is unsuited for automated analyzers used in routine diagnostic laboratories. Selected reaction monitoring (SRM) is a flexible high-throughput analytical approach based on targeted mass spectrometry (MS) already implemented in clinical settings. We tested the hypothesis that SRM could be used to quantify cancer biomarkers in human bile. An SRM-based assay was developed to simultaneously quantify up to 37 peptides from 13 bile proteins in a developmental cohort of 15 patients (MBS, n = 8; benign biliary stricture or obstruction (BBS), n = 7). The most reliable biomarkers were then absolutely quantified by SRM in a verification cohort of 67 patients (MBS, n = 37; BBS, n = 30). The diagnostic performances of single and combined biomarkers were assessed. In the developmental cohort, SRM-based analysis revealed six protein biomarkers with significantly higher peptide ratios (endogenous vs. standard) in bile from MBS vs. BBS. In the verification cohort, five of these biomarkers proved good diagnostic ability (individual receiver operating characteristic-area under the receiver operating characteristic curve (ROC-AUC) up to 0.889, accuracies from 67.8% to 83.1%). Combining bile biomarkers and serum CA19-9 in 2 panels allowed differentiating MBS from BBS with up to 0.929 ROC-AUC and 89.8% accuracy. In this study, a newly developed SRM-based assay proved able to simultaneously quantify multiple biomarkers in bile samples. The combination of bile biomarkers with serum CA19-9 was highly accurate for the diagnosis of MBS. Liquid biopsy of bile based on targeted MS is eligible to support MBS diagnosis in clinical practice.

Extracellular Vesicles in Bile as Markers of Malignant Biliary Stenoses.

BACKGROUND & AIMS: Algorithms for diagnosis of malignant common bile duct (CBD) stenoses are complex and lack accuracy. Malignant tumors secrete large numbers of extracellular vesicles (EVs) into surrounding fluids; EVs might therefore serve as biomarkers for diagnosis. We investigated whether concentrations of EVs in bile could discriminate malignant from nonmalignant CBD stenoses. METHODS: We collected bile and blood samples from 50 patients undergoing therapeutic endoscopic retrograde cholangiopancreatography at university hospitals in Europe for CBD stenosis of malignant (pancreatic cancer, n = 20 or cholangiocarcinoma, n = 5) or nonmalignant (chronic pancreatitis [CP], n = 15) origin. Ten patients with CBD obstruction due to biliary stones were included as controls. EV concentrations in samples were determined by nanoparticle tracking analyses. The discovery cohort comprised the first 10 patients with a diagnosis of pancreatic cancer, based on tissue analysis, and 10 consecutive controls. Using samples from these subjects, we identified a threshold concentration of bile EVs that could best discriminate between patients with pancreatic cancer from controls. We verified the diagnostic performance of bile EV concentration by analyzing samples from the 30 consecutive patients with a diagnosis of malignant (pancreatic cancer or cholangiocarcinoma, n = 15) or nonmalignant (CP, n = 15) CBD stenosis. Samples were compared using the Mann-Whitney test and nonparametric Spearman correlation analysis. Receiver operating characteristic area under the curve was used to determine diagnostic accuracy. RESULTS: In both cohorts, the median concentration of EVs was significantly higher in bile samples from patients with malignant CBD stenoses than controls or nonmalignant CBD stenoses (2.41 × 1015 vs 1.60 × 1014 nanoparticles/L in the discovery cohort; P < .0001 and 4.00 × 1015 vs 1.26 × 1014 nanoparticles/L in the verification cohort; P < .0001). A threshold of 9.46 × 1014 nanoparticles/L in bile best distinguished patients with malignant CBD from controls in the discovery cohort. In the verification cohort, this threshold discriminated malignant from nonmalignant CBD stenoses with 100% accuracy. Serum concentration of EVs distinguished patients with malignant vs patients with nonmalignant CBD stenoses with 63.3% diagnostic accuracy. CONCLUSIONS: Concentration of EVs in bile samples discriminates between patients with malignant vs nonmalignant CBD stenosis with 100% accuracy. Further studies are needed to confirm these findings. Clinical Trial registration no: ISRCTN66835592.

Bile carcinoembryonic cell adhesion molecule 6 (CEAM6) as a biomarker of malignant biliary stenoses.

Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.