ACADEMIC TRAINEES ANNUAL EVENT 2011
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
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
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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
Crude centrifuged
sample:
water/ lipids/ bile
acids and pigments/
proteins
No cancer
Pancreatic
cancer
Slice gel into equal fragments containing
denatured proteins in size ranges
SDS-PAGE
Tryptic peptides
In-gel
protease
digestion
Extract from gel
slice
Label-free proteomics: LC-MS/MS
Intensity of detection
Mass/carge ration (m/z)
Peptide fragments
Retention time (min)
Retention time
(min)
Retention time (min)
Benign
Retention time (min)
11
CC
Retention time (min)
Mass/charge ration (m/z)
1
Mass/charge ration (m/z)
Mass/charge ration (m/z)
Label-free proteomics: Normalisation and
Quantification
1
8
PaCa
VPLQQNFQDNQFQGK
Retention time (min)
“196,454.71”
Progenesis, nonlinear dynamics
Normalised abundance profile
of single peptide spot
Normalised
abundance
of a
specific protein
in gel piece
Normalised abundance volume of
all peptide spots of an identified protein
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 gelatinaseassociated 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
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
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
Number
p-value
Western blot of NGAL in bile
Gel 1
Gel 2
P<0.0001
ELISA of NGAL in different body fluids
b)
p=0.007
NS
c)
NS
180
1800
160
600
140
NGAL [ng/ml]
500
NGAL [ng/ml]
a)
400
300
200
100
80
60
40
100
20
0
0
Benign
Bile
120
M alignant
Serum
Benign
M alignant
Urine
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
Cut-off NGAL in bile: 3000 ng/ml
Cut-off serum CA19-9: 110 U/ml
Below cut-off Above cut-off
Benign
13
3
Malignant
2
11
Sensitivity:
Specificity:
PPV:
NPV:
85%
82%
79%
87%
Validation cohort
Benign (n=14)
Malignant (n=7)
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)
NS
620 (350-917)
p-value
Cut-off >570ng/ml gives sensitivity 100%, specificity 55%
Biological functions of NGAL in malignancy
Schematic model of NGAL-mediated iron traffic.
[Kai M. Schmidt-Ott et al. J Am Soc Nephrol 18: 407–413, 2007]
A)
B)
Principal cellular mechanisms attributed to NGAL
in determining its pro- and anti-tumoral effects.
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]
[Bolignano D, et al. Cancer Lett. (2010) Vol. 288(1): pp 10-6.]
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
Patients!
HPB and Endoscopy teams at Imperial AHSC
Proteomics team at Imperial College
British Liver Trust
Alan Morement Memorial Fund
Thank you
Any questions?