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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
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
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
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