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NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN IS ELEVATED IN BILE FROM

PATIENTS WITH MALIGNANT PANCREATOBILIARY DISEASE A. Zabron1, V. Horneffer-van der Sluis 2, C. Wadsworth 1, M. Gierula 2, A.

Thillainayagam 3, P. Vlavianos3, D. Westaby 3, S. Taylor-Robinson 1, R. Edwards 2, S. Khan 1

1Hepatology and Gastroenterology Section, Division of Diabetes Endocrinology and Metabolism, Department of Medicine, 2Centre of Pharmacology and Therapeutics , Imperial College London,3Gastroenterology 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

Content

• Focus on pancreatic adenocarcinoma

• Significance and current diagnostic difficulties

• Proteomic technique

• Results

• Possible clinical use and further work

• Summary

Pancreatic adenocarcinoma

• Imperial AHSC HPB tertiary referral centre

• 10th 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

Anatomy of biliary system

Endoscopic retrograde cholangiopancreatography : ERCP

Stenting across obstruction via ERCP

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)

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

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

Label-free proteomics: tryptic peptide production

No cancerPancreatic

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

Label-free proteomics: LC-MS/MS

Retention time (min)

Inte

nsi

ty o

f d

etec

tio

n

Peptide fragments

Retention time (min)

Retention time (min)M

ass/

carg

e ra

tio

n (

m/z

)

Normalised abundance profileof single peptide spot

Retention time (min)

Mas

s/ch

arg

e ra

tio

n (

m/z

)

Retention time (min)

Mas

s/ch

arg

e ra

tio

n (

m/z

)

Retention time (min)

Mas

s/ch

arg

e ra

tio

n (

m/z

)

Normalised abundance volume ofall peptide spots of an identified

protein

CCBenign PaCa1 1

118

Label-free proteomics: Normalisation and Quantification

VPLQQNFQDNQFQGK

“196,454.71”

Normalised abundance

of aspecific protein

in gel piece

Progenesis, nonlinear dynamics

Data analysis

• SEQUEST» human RefSeq protein sequence database (NCBI)

• Progenesis» Quantification by non-linear dynamics

• Statistical analysis

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

Volcano plot of identified proteins

Volcano plot of identified proteins

NGAL_HUMAN: Lipocalin 2 or Neutrophil gelatinase-associated

lipocalinNormalised 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

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

Immunoblotting : Clinical characteristics

Benign Malignant p-value

Number 22 16

Age [year] 59.83 ± 20.84 71.08 ± 11.62 0.043

Albumin [g/l] 33.84 ± 7.59 27.25 ± 7.7 0.013

Bilirubin [mmol/l] 35.33 ± 96.23 210.08 ± 155.3 <0.001

ALT [IU/l] 83.7 ± 101.8 298.17 ± 248.7 <0.001

ALP [IU/l] 346.8 ± 494.2 768.00 ± 602.9 0.006

CRP 15.14 ± 20.23 96.83 ± 100.0 0.008

urea 6.305 ± 4.116 5.59 ± 2.3 NS

creatinine 97.40 ± 45.17 86.86 ± 33.4 NS

Western blot of NGAL in bile

P<0.0001

Gel 1

Gel 2

p=0.007 NS NSa) b) c)

Bile Serum Urine

Benign Malignant0

100

200

300

400

500

600

1800

NG

AL

[n

g/m

l]

Benign Malignant

0

20

40

60

80

100

120

140

160

180

NG

AL

[n

g/m

l]

ELISA of NGAL in different body fluids

NGAL in bile as a diagnostic marker

Receiver Operator Characteristics

Area Under the Curve: 0.80

Cut-off level [ng/ml] Specificity% Sensitivity %

< 569.5 54.29 100.0

< 661.0 54.29 92.31

< 803.8 54.29 84.62

< 952.5 57.14 84.62

< 1015 60.00 84.62

< 1080 62.86 84.62

< 1142 62.86 76.92

< 1230 65.71 76.92

< 1319 68.57 76.92

< 1419 68.57 69.23

< 1549 71.43 69.23

Combination of NGAL in bile and serum CA19-9

Below cut-off Above cut-off

Benign 13 3

Malignant 2 11

Sensitivity: 85%Specificity: 82%PPV: 79%NPV: 87%

Cut-off NGAL in bile: 3000 ng/mlCut-off serum CA19-9: 110 U/ml

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:male 5:9 4:3

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%

Biological functions of NGAL in malignancy

Principal cellular mechanisms attributed to NGALin 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)

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

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.

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

Acknowledgements

British Liver Trust

Alan Morement Memorial Fund

Patients!

HPB and Endoscopy teams at Imperial AHSC

Proteomics team at Imperial College

Thank you

Any questions?