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Introduction Pancreatobiliary malignancies are amongst the commonest causes of cancer-related death worldwide and usually present with biliary obstruction. Accurate differentiation between benign and malignant causes of obstruction is likely to improve outcome in pancreatobiliary disease and reliable biomarkers are urgently needed. Bile is a potential source of such biomarkers due to its anatomical proximity to the site of pathology. Our aim was to apply a proteomic approach to identify potential biomarkers in bile which could differentiate between malignant and benign disease. Methods Bile, urine and serum were collected prospectively from 59 patients undergoing ERCP (endoscopic retrograde cholangiopancreatography) for benign (n = 36) or malignant (n = 23) pancreatobiliary disease at our centre. Initially, label-free proteomics and immunoblotting were performed in a sub-set of patients. Candidate biomarkers were selected on the basis of increased abundance of peptide fragments, and immunoblotting performed on an expanded cohort. ELISA was then performed for our potential biomarker on all samples in bile, blood and urine. Results Neutrophil gelatinase associated lipocalin (NGAL) levels were significantly raised in bile from the malignant disease group, compared to bile from the benign disease group (median 1839 ng/mL vs 472 ng/mL, p Conclusion NGAL in bile is a novel potential biomarker to distinguish benign from malignant biliary obstruction, particularly in combination with serum tumour markers.

Introduction MicroRNAs (miRNAs) are short, non-coding RNAs with a key role in post-transcriptional regulation, and regulate multiple pathways of tumorogenesis. Expression profiling of malignant cell lines, fresh and formalin-fixed tumour have identified potential miRNA signatures for pancreatic ductal adenocarcinoma (PDAC), and RNA deep sequencing has demonstrated that miRNAs can be quantified from bile, in bilary tract cancers 1 . Here, we report the first study of biliary miRNAs in PDAC. Methods 3 target miRNAs known to be overexpressed in PDAC were selected. Bile was collected at endoscopic retrograde cholangiopancreatography (ERCP) from patients with PDAC (n = 10) or gallstones (n = 8; used as a benign control). Bile was prepared as described1 and total RNA isolated using TRIzol (Invitrogen, Paisley, UK). Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was performed using Taqman mature miRNA primers and probes (Applied Biosystems, Cheshire, UK). Expression of oncomiR-21, miR-155 and miR-106a was measured. Cycle passing threshold (Ct) was recorded and normalised to RNU6B expression. Relative expression was calculated as 2Ct_miRNA-Ct_RNU6B. PCR reactions were carried out in duplicate. Results miR-106a and oncomiR-21 were both highly expressed in the bile of patients with PDAC, compared to those with gallstones. miR-155 was not significantly upregulated in PDAC bile. Using miR-106a (cut-off level > 8.02), we discriminated PDAC from benign disease with a sensitivity and specificity of 80% [95% CI 44–98] and 100% [95% CI 63–100], respectively with an area under the curve (AUC) value of 0.88. OncomiR-21 was also upregulated in bile, but was not as reliable in discriminating diagnosis. Conclusion This study demonstrates that bile miRNAs are well-suited analytes for the development of sensitive and specific tests in PDAC. Although tumour breach of the biliary epithelium was not always suspected in our cohort from clinical investigations, malignancy-specific miRNAs were found intraluminally. Of the 3 miRNAs tested, ROC analysis identified miR-106a as the best potential PDAC biomarker, with reliable diagnostic specificity and sensitivity. However, further study may also identify miRNAs that also discriminate prognoses and response to therapy, guiding individualised treatment. Disclosure of Interest None Declared Reference Shigehara K, Yokomuro S, Ishibashi O, et al. Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer. PloS one. 2011; 6(8):e23584.

Introduction Cholangiocarcinoma (CCA) is a primary biliary ductal cancer which is often difficult to diagnose and which carries a poor prognosis. New diagnostic tests are urgently needed to improve patient outcome. Bile is a rich source of potential novel biomarkers for CCA, due to its intimate proximity to the malignant lesion. However, there are no biliary biomarkers for CCA currently available. MicroRNAs (miRNAs) are key post-transcriptional regulators, and influence tumorogenesis. Studies on cell lines and tissue have identified several potential miRNA signatures for CCA, and recently miR-9 was identified as elevated in bile from CCA compared with benign disease 1 . In this study we aimed to measure specific miRNA expression in bile from patients with CCA, gallstone disease and pancreatic adenocarcinoma (PA) and to assess their performance in differentiating these causes of biliary obstruction. Methods Bile was collected at endoscopic retrograde cholangiopancreatography (ERCP) from patients with CCA (n = 6), PA (n = 10) and gallstones (n = 8; benign control). Bile was prepared as previously described1 and total RNA was isolated using TRIzol (Invitrogen, Paisley, UK). Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was performed using Taqman mature miRNA primers and probes (Applied Biosystems, Cheshire, UK). Expression of oncomiR-21, miR-155 and miR-106a was measured. Cycle passing threshold (Ct) was recorded and normalised to RNU6B expression. Relative expression was calculated as 2Ct_miRNA-Ct_RNU6B. PCR reactions were carried out in duplicate. Results MiR-106a and oncomiR-21 were highly expressed in bile from patients with CCA compared to those with gallstones. MiR-155 was not significantly upregulated in malignancy. In discriminating CCA from benign disease, miR-106a had sensitivity and specificity of 83.3% [95% CI 36–100] and 88% [95% CI 47–100] respectively, with an AUC of 0.83 (cut-off level > 6.05). OncomiR-21 was also upregulated in the bile of both tumour types, but was not as reliable. Neither miRNA discriminated significantly between PA and CCA. Conclusion MiRNA can be reliably isolated from bile pellets. Both miR-106a and oncomiR-21 are potential novel biliary biomarkers for CCA, and could improve differentiation of benign from malignant disease at ERCP. Further work is required to validate these findings in a larger cohort and against other disease groups, and to investigate possible correlations between miRNA profile and disease course. Disclosure of Interest None Declared Reference Shigehara K, Yokomuro S, Ishibashi O, et al. Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer. PloS one . 2011; 6(8):e23584.

Introduction Cholangiocarcinoma (CC) is a malignant neoplasm of the bile duct. Diagnosis of CC is hampered by the inadequate performance of current plasma markers of disease, particularly in patients with preexisting primary sclerosing cholangitis (PSC). We aimed to identify potential new protein biomarkers of CC Methods In an initial discovery study, blood plasma samples from 18 subjects with CC, 17 with PSC and 10 healthy controls were subjected to SELDI-TOF MS. Comparisons of m/z peak intensity were made between groups using the Mann-Whitney U test. Differentiating m/z peaks were then confirmed in a further validation study of 81 subjects with CC, 54 with PSC and 90 healthy controls. Pearson´s correlation was used to investigate the relationship of each m/z peak´s intensity to routine laboratory indices. Diagnostic performance was investigated using receiver operator characteristic area-under-the curve (ROC-AUC) analyses. Multiple linear regression was used to investigate the performance of combinations of differentiating m/z peaks, as well as the combination of m/z peaks with routine laboratory markers (including CA19–9). Results Seven differentially expressed m/z peaks were identified in the CC group and these were subsequently confirmed in the validation study (p = 2.6 × 10–4 to 9.4 × 10–13). The intensity of the seven m/z peaks of interest did not correlate with creatinine, ALP, bilirubin, CRP, white cell count or CA19–9. A panel of three peaks discriminated CC from PSC subjects with ROC-AUC of 0.76 (sensitivity 75%, specificity 64%). A panel of five peaks discriminated CC subjects from healthy controls with ROC-AUC of 0.90 (sensitivity 95%, specificity 74%). Addition of routine laboratory indices did not change the diagnostic performance of these models significantly. Conclusion SELDI-TOF has been used to successfully identify seven m/z peaks that are differentially intense in CC subjects (total n = 99), when compared to PSC subjects (n = 64) and healthy controls (n = 107). These peaks appear to be independent of standard markers of renal impairment, cholestasis, sepsis and inflammation, as well as CA19–9. Individually, and more so in combination, these peaks exceed the expected diagnostic performance of CA19–9, particularly in discriminating CC from PSC. Work to identify the proteins represented by these m/z peaks is ongoing. Disclosure of Interest None Declared

Introduction Better biomarkers are urgently needed to assist accurate diagnosis and appropriate treatment of malignant biliary obstruction, as, although malignancy is a common cause of obstructive jaundice, current diagnostic techniques often fail to differentiate benign from malignant disease. Molecular analysis of bile has recently produced promising candidate biomarkers. Previous work from our group found that biliary neutrophil gelatinase-associated lipocalin (NGAL), a small extracellular 25-kDa protein with several biological functions, differentiates obstructive jaundice from malignancy from that in benign disease. The mechanism of NGAL in hepatopancreatobiliary (HPB) malignancy is unknown, although in other systems it promotes neoplastic diffusion by complexing and stabilising matrix metalloproteinase-9 (MMP9), enabling local invasion. Aims (1) To investigate possible biliary complexing of MMP9 and NGAL as a mechanism of tumorigenesis. (2) To validate our previous findings of biliary NGAL as a novel biomarker of malignancy in biliary obstruction. Methods Bile samples were collected from 77 patients undergoing ERCP (n=77, 22 with malignant disease and 55 with benign disease) at Imperial College London. ELISA was used to quantify levels of MMP9/NGAL complexes and of NGAL and MMP9 occurring independently in bile. Pearson9s correlation analysis was used to determine the relationship between NGAL, MMP9 and NGAL/MMP9 complex levels, and statistical significance assessed by the Mann–Whitney U test. Results Biliary NGAL levels were significantly higher in malignant biliary obstruction compared to benign disease (median 1555 ng/ml vs 402 ng/ml, p=0.003), giving a ROC AUC of 0.74. Biliary MMP9 and NGAL/MMP9 complex levels were not different between these groups (p=0.527, p=0.760). Unbound biliary NGAL and MMP9 levels correlated poorly (r 2 =0.03, p>0.05). Unbound NGAL correlated poorly with complex (r 2 =0.07, p>0.05) whereas unbound MMP9 correlated with NGAL/MMP9 complex level (r 2 =0.73, p Conclusion This study is novel in confirming the presence of MMP9 in bile, alone and in complex with NGAL. However, although NGAL was increased in malignancy, MMP9 and MMP9/NGAL complex were not, suggesting that NGAL acts independently of MMP9 in endobiliary HPB malignancy. Mechanisms remain to be elucidated. This study also supports previous reports of NGAL as a novel and independent bile biomarker of malignant biliary obstruction. Competing interests None declared.

Introduction Obstruction of the biliary tree has a variety of aetiologies. The correct diagnosis is crucial for appropriate definitive therapy. A major differential diagnosis of biliary obstruction is pancreatic adenocarcinoma (PA). PA is the 10th most common cancer in the UK with mortality similar to incidence. Diagnosis of PA relies on a combination of cross-sectional imaging and serum biomarkers, but only a minority of cases are diagnosed correctly and early enough for curative resection. Earlier diagnostic certainty would be likely to improve outcome and guide therapeutic strategy. Proteomic profiling is a promising technique for new biomarker discovery, but has rarely been successfully carried out in human bile. This project (1) analyses and compares the proteomes of bile from PA patients vs. bile from patients with benign biliary disease and (2) seeks to identify potential protein biomarkers that can differentiate PA from benign biliary disease. Methods Bile was aspirated at ERCP from patients with benign or malignant biliary obstruction. Particulate matter was removed by centrifugation and equal volumes subjected to SDS-PAGE. In-gel trypsin digestion was performed and the resulting peptides analysed by tandem mass spectrometry. Proteins were identified using SEQUEST to search the human RefSeq protein sequence database. Progenesis software was used to compare relative levels of proteins between patient groups. Results Proteomic analysis of bile from four patients with PA and four with benign biliary obstruction was performed. Over 100 different proteins were identified, including pancreatic gastric triacylglycerol lipase, carboxypeptidase B and bile salt-activated lipase, confirming that the profile of proteins represents the biliary proteome. Several differentially expressed proteins were identified including upregulation of carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6) in the malignant group. Conclusion This work confirms that bile is amenable to proteomic analysis and that differences in the biliary proteome can be detected in malignant and non-malignant cases. We confirmed earlier reports of CEACAM6 overexpression in PA. We are currently analysing the data further to identify additional candidate biomarker proteins. Future work will extend this approach to the study of bile in other diseases, such as cholangiocarcinoma.