1. sdfgafgafga NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN IS ELEVATED IN BILE FROM PATIENTS WITH MALIGNANT PANCREATOBILIARY DISEASE A. Zabron 1, V. Horneffer-van der Sluis 2, C. Wadsworth 1, M. Gierula 2, A. Thillainayagam 3, P. Vlavianos 3, D. Westaby 3, S. Taylor-Robinson 1, R. Edwards 2, S. Khan 1 1 Hepatology and Gastroenterology Section, Division of Diabetes Endocrinology and Metabolism, Department of Medicine, 2 Centre of Pharmacology and Therapeutics, Imperial College London, 3 Gastroenterology Unit, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom Previously presented at UEGW and BSG. Accepted for publication in the American Journal of Gastroenterology ACADEMIC TRAINEES ANNUAL EVENT 2011

2. Content Focus on pancreatic adenocarcinoma Significance and current diagnostic difficulties Proteomic technique Results Possible clinical use and further work Summary

3. Pancreatic adenocarcinoma Imperial AHSC HPB tertiary referral centre 10 th commonest cancer in the UK Mortality similar to incidence Diagnosis by cross-sectional imaging, histology / cytology and serum biomarkers imperfect Frequently present with biliary obstruction

4. Anatomy of biliary system

5. Endoscopic retrograde cholangiopancreatography : ERCP

6. Stenting across obstruction via ERCP

7. Bile as a diagnostic sample in malignant pancreatobiliary disease Current cytological analysis of brushings – low sensitivity Spy-glass Metabonomic interest Recent proteomic interest IGF1 and pancreatic elastase-3B higher in CC bile(Alvaro D. et al., 2009) CEACAM-6 and MUC1(CA 19-9) higher in PA bile (Farina et al., 2009)

8. Current Study Aims: To investigate the potential of bile as a source of novel biomarkers in pancreatic adenocarcinoma Design: Cohort study Discovery phase - label-free proteomics to unfractionated sample, antibody-based techniques Validation cohort

9. Sample collection and preparation Bile aspirated at ERCP in patients with benign or malignant disease prior to introduction of contrast Routine blood results/ serum markers, demographics etc recorded Blood and urine collected at same event

10. Label-free proteomics: tryptic peptide production No cancer Pancreatic cancer In-gel protease digestion Crude centrifuged sample: water/ lipids/ bile acids and pigments/ proteins SDS-PAGE Slice gel into equal fragments containing denatured proteins in size ranges Extract from gel slice Tryptic peptides

11. Label-free proteomics: LC-MS/MS Retention time (min) Intensity of detection Peptide fragments Retention time (min) Mass/carge ration (m/z)

12. Normalised abundance profile of single peptide spot Retention time (min) Mass/charge ration (m/z) Retention time (min) Mass/charge ration (m/z) Retention time (min) Mass/charge ration (m/z) Normalised abundance volume of all peptide spots of an identified protein CCBenignPaCa 1111818 Label-free proteomics: Normalisation and Quantification VPLQQNFQDNQFQGK “196,454.71” Normalised abundance of a specific protein in gel piece Progenesis, nonlinear dynamics

13. Data analysis SEQUEST »human RefSeq protein sequence database (NCBI) Progenesis »Quantification by non-linear dynamics Statistical analysis

14. Results of label-free proteomics Bile analysed from patients with pancreatic adenocarcinoma (n=4) and benign biliary disease (n=4) Over 200 different proteins identified including S100A6, LCN2, CEACAM6, REG1α, PRDX6 10 proteins varied significantly in abundance between benign and malignant groups

15. Volcano plot of identified proteins

17. NGAL_HUMAN: Lipocalin 2 or Neutrophil gelatinase- associated lipocalin Normalised abundance volume of all peptide spots of lipocalin 2 P = 0.029, protein fold change 13.9 10 unique tryptic peptides 52% protein sequence coverage

18. NGAL as a novel biomarker: Biological plausibility “stress protein” Novel urinary biomarker in for acute kidney injury Overexpression in malignancy e.g. breast, ovarian, colon and pancreatic tissue

19. Immunoblotting : Clinical characteristics BenignMalignantp-value Number2216 Age [year] 59.83 ± 20.8471.08 ± 11.62 0.043 Albumin [g/l] 33.84 ± 7.5927.25 ± 7.7 0.013 Bilirubin [mmol/l] 35.33 ± 96.23210.08 ± 155.3 <0.001 ALT [IU/l] 83.7 ± 101.8298.17 ± 248.7 <0.001 ALP [IU/l] 346.8 ± 494.2768.00 ± 602.9 0.006 CRP 15.14 ± 20.2396.83 ± 100.0 0.008 urea 6.305 ± 4.1165.59 ± 2.3 NS creatinine97.40 ± 45.1786.86 ± 33.4NS

20. Western blot of NGAL in bile P<0.0001 Gel 1 Gel 2

21. p=0.007NS a) b) c) BileSerumUrine ELISA of NGAL in different body fluids

22. NGAL in bile as a diagnostic marker Receiver Operator Characteristics Area Under the Curve: 0.80 Cut-off level [ng/ml]Specificity%Sensitivity % < 569.554.29100.0 < 661.054.2992.31 < 803.854.2984.62 < 952.557.1484.62 < 101560.0084.62 < 108062.8684.62 < 114262.8676.92 < 123065.7176.92 < 131968.5776.92 < 141968.5769.23 < 154971.4369.23

23. Combination of NGAL in bile and serum CA19-9 Below cut-offAbove cut-off Benign133 Malignant211 Sensitivity:85% Specificity:82% PPV:79% NPV:87% Cut-off NGAL in bile:3000 ng/ml Cut-off serum CA19-9:110 U/ml

24. Validation cohort Benign (n=14)Malignant (n=7)p-value Diagnoses (n)Stone (6), Chronic Pancreatitis (3), PSC (3), Leak (2) Pancreatic Adenocarcinoma (5), Gallbladder Cancer (1), HCC (1) Female:male5:94:3 Age [year]61 (36-79)75 (64-78)NS Albumin [g/l]35.5 (27.5-42.0)31 (26-36)NS Bilirubin [mmol/l]15.0 (6.5-26.0)121 (14-421)0.004 ALT [IU/l]60.0 (28.0-156.5)168 (126-357)NS ALP [IU/l]181 (122-1176)620 (350-917)NS Cut-off >570ng/ml gives sensitivity 100%, specificity 55%

25. Biological functions of NGAL in malignancy Principal cellular mechanisms attributed to NGAL in determining its pro- and anti-tumoral effects. [Bolignano D, et al. Cancer Lett. (2010) Vol. 288(1): pp 10-6.] Schematic model of NGAL-mediated iron traffic. [Kai M. Schmidt-Ott et al. J Am Soc Nephrol 18: 407–413, 2007] General structure of A) the ferrichromes, prototypical hydroxamate type siderophores, and B) ferric enterobactin, a prototypical catechol-type siderophore. [Neilands J B, et al. J. Biol. Chem. 1995;270:26723-26726] A)B)

26. Summary Valuable HPB patient cohort and scientific facilities available at Imperial AHSC This study identified over 200 proteins in bile Confirmed that proteomic analysis of body fluids allows identification of potential biomarkers in pancreatobiliary malignancy There are significant differences in the proteome of bile in malignant and benign pancreatic disease

27. Summary 2 This study highlights the increased abundance of bile LCN2/ NGAL in pancreatic adenocarcinoma. Further work is required to elucidate the possible use of biliary LCN2/NGAL as a clinical marker of disease.

28. Future work Increasing collaborations with Imperial HPB teams to expand recruitment Validation of biliary NGAL as a diagnostic and/ or prognostic marker in pancreatic cancer Exploration of its biological mechanism e.g. role stabilising MMP-9 Extension of this approach to other biliary tract disease

29. Acknowledgements British Liver Trust Alan Morement Memorial Fund Patients! HPB and Endoscopy teams at Imperial AHSC Proteomics team at Imperial College

30. Thank you Any questions ?