ESPN 2008 - Lyon Advances in urinary proteome analysis and biomarker discovery Joost Schanstra...
-
Upload
maximilian-warren -
Category
Documents
-
view
216 -
download
0
Transcript of ESPN 2008 - Lyon Advances in urinary proteome analysis and biomarker discovery Joost Schanstra...
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Joost SchanstraInserm U858, I2MRToulouse, France
ESPN 2008 - Lyon
Biomarkers in biofluids
Petricoin et al., Use of proteomic patterns in serum to identify ovarian cancer.Lancet. 2002
Irreproducible: - improper mass calibration, - technical flaws,- improper execution of the experimental protocol
- Serum versus plasma- Proteolysis
B L
O O
D
- Simple pre-analytic handling- Stable
U
R
I N
E
Reduction of sample variability
ESPN 2008 - Lyon
Urine – pool of biomarkers
Sources of urinary proteins1) Filtration and secretion of plasma proteins. 2) Secretion by various renal segments. 3) Proteolytic degradation products of extracellular matrix4) Secretion by the urinary tract. 5) Dead cells.
~70% of the urinary proteins/peptides originate from the kidney and urinary tract
Kidney “health” status~70%
Urine – pool for biomarkers of other diseases ?
~30%
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Advances in techniques
ESPN 2008 - Lyon
250150100 75
50
37
2520
15
Courtesy: C. Lacroix, Toulouse
- Dynamic range
- A few proteins make up the majority
Reduce complexity
A one-dimensional view of the urinary protein content
ESPN 2008 - Lyon
Reduce complexity
Fractionation Mass spectrometrySample
2D-PAGE SELDI
Capillary electrophoresis
ESPN 2008 - Lyon
250150100 75
50
37
2520
15
albumin
Ig kappa chain
microglobulin
Two-dimensional gel electrophoresis
Tho
ngbo
onke
rd,
2002
acidic basic
Mas
s (k
Da)
ESPN 2008 - Lyon
250150100 75
50
37
2520
15
SELDI-MS(surface enhanced laser desorption/ionisation-mass spectrometry)
Mas
s (k
Da)
3
100
ESPN 2008 - Lyon
250150100 75
50
37
2520
15
Capillary electrophoresis coupled to mass-spectrometry
Coo
n, in
pre
ss.
Migration on CE (time)
Mas
s (k
Da)
- 5000 frequently occurring peptides
- collagen fragments !!
ESPN 2008 - Lyon
Three techniques suitable for the analysis of the urinary proteome
More amendable for use in the clinic
2D-PAGE SELDI
CE-MS
Medium throughput
High throughput
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Individual markers:
- Lack of specificity (PSA, microalbuminuria…)- Sensitive to inter- and intra-individual variations
Multiple biomarkers or “panels” of biomarkers
proper statistics !!
Advances in concepts: panels rather than individual markers
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Advances in concepts: standards
ESPN 2008 - Lyon
A standard flow scheme for clinical proteomics
1. Define a clear clinical question- surgery or not ?- graft rejection or not ?
Clinicians
Mischak, Apweiler, Banks, Conaway, Coon, Dominiczak, Ehrich, Fliser, Hermjakob, Hochstrasser, Jankowski, Julian, Kolch, Massy, Neusuess, Novak, Peter, Rossing, Schanstra, Semmes, Theodorescu, Thongboonkerd, Weissinger, Van Eyk, Yamamoto Proteomics Clin. Appl. 1: 148 (2007).
4. Validate in separate blinded population
Statisticians/bioinformatics
3. Training population
extract biomarkers/ proper statistics!!
Proteomics core platforms
5b. Identify
pathophysiology
5a. Large scale validation
clinic
2. Select appropriate patient groups /clinical data/samples/proteomic analysis
Biochemists
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discoveryAge matching
young old
Biomarkers selected for this age range
young old
? ?
ESPN 2008 - Lyon
324 individuals: 2-73 year
Analysis of the urinary proteome by CE-MS of 324 individuals
Secretion of 325 out of 5000 urinary peptides is modified during aging
Advances in urinary proteome analysis and biomarker discoveryAge matching
ESPN 2008 - Lyon
Renal aging = CKD ?
Disease% of age markers
Diabetic nephropathy
22.4
IgAN 4.1
FSGS 14.3
MGN 8.2
Vasculitis 8.2
MCD 16.3
Zurbig et al., submitted
Age match !!!!!
ESPN 2008 - Lyon
Outliers !!
x y
age
mar
kers
19-30 31-40 41-50 51-73
?? Zurbig et al., submitted
age
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Advances: examples
1. Renal allograft rejection
2. Ureteropelvic junction obstruction
3. Bladder Cancer (BCa)
ESPN 2008 - Lyon
Prediction of acute rejection in renal allografts
Training
20 healthy 22 stable transplants23 acute rejection
SELDI
O’Riordan et al., 2004. JASN 15: 3240
Year 2004
Acute rejection versus stable: - 4 markers (sens 90,5%, spec 83,3%)
healthy stable transplantsacute rejection
Random forrest
Classification:
ESPN 2008 - Lyon
Prediction of acute rejection in renal allografts
Year 2007
Sensitivity 91.3 %Specificity 77.3 %
AUC 0.912
Stable versus acute
Validation (n=45)
Two markers
MS-based
O’Riordan et al., 2007. Am J Transpl. 7:930
Validation of -defensin-1 in immunoassay
- -defensin-1 - -1-antichymotrypsin
AUC 0.749
Loss of sensitivity !
Immunoassay based
ESPN 2008 - Lyon
Ureteropelvic junction (UPJ) obstruction
Spontaneousresolution
pyeloplasty
?Intermediateobstruction
Spontaneous resolution
pyeloplasty
Obstructive nephropathy
birth 1 to 2 years
scintigraphies
Objective: identify early urinary biomarkers indicative for surgery
ESPN 2008 - Lyon
Healthy controls (n=13)
Sp. Resolution (n=19)
Pyeloplasty (n=19)
1
13
Patient groups:
Individual CE-MS Profiles:
Reference (compliled)Group profile:
Training
1
19
1
19
ClassificationSupport vector machines
Extraction of group-specific markers
Set of specific biomarkers separating different groups:
ESPN 2008 - Lyon
Small scale (n=36) prospective blinded validation
Spontaneousresolution
pyeloplasty
IntermediateObstruction (n=36)
CE-MS based prediction
Clinical outcome
birth 1 to 2 years1 month
Biomarkers obtained during « training »
prediction
9 months: 34 out of 36 patients correctly predicted15 months: 35 out of 36 patients correctly predicted
Decramer et al., 2006. Nat Med. 12: 398.
Large scale multicenter validation of markers started in 2008: 358 patients
ESPN 2008 - Lyon
Bladder cancer
Transitional cell carcinomas
80% superficial (Tis, Ta, T1)
Reoccurrence - cystoscopy (invasive)- urine cytology (low-sensitivity)
Urinary proteomics to search for biomarkers of BCa
ESPN 2008 - Lyon
Transitional cell carcinoma detection: Training
group Number of patients
Training set
Bladder cancer
46
controls 73
various Renal diseases 281
Prostate cancer 21
Renal cancer 24
Confounding disease
ESPN 2008 - Lyon
Controls + confounding
Bladder cancer
Training set
22 biomarkers
ESPN 2008 - Lyon
Theodorescu et al., Lancet Oncol. 7:230-240 (2006)
Transitional cell carcinoma detection: Validation
32/32 Bladder cancer Sensitivity 100 %
12/12 Controls Specificity 100 %124/131 Various renal diseases Specificity 95 %6/7 Nephrolithiasis Specificity 86 %
Controls (n=12)Various renal diseases (n=131)Nephrolithiasis (n=7)
Bladder cancer (n=32)Controls + confounding diseases
versus
Suitable biomarkers in the presence of confounding diseases
Clinical setting
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Advances: examples
Coronary artery disease
Urine as a source of biomarkers for non-urogenital diseases?
ESPN 2008 - Lyon
Urinary proteomics for biomarkers of Coronary artery disease
Training set
CAD (Glasgow) n = 30Controls (Glasgow) n = 20Additional controls: Ramipril (medication controls) n = 18 Hannover (center specific biais) n = 232
Validation set
CAD n = 47Controls n = 12
Study Setup
ESPN 2008 - Lyon
Downregulated in comparison
upregulated in comparison
Training: 15 biomarkers differentiate between CAD and non-CAD controls
non-CAD controlspatients with CAD
ESPN 2008 - Lyon
46/47 CAD Sensitivity 98%
10/12 controls Specificity 83%
Validation of urinary CAD biomarkers
Zimmerli et al., 2008 Mol. Cell. Proteomics 7, 290-298
Urinary markers for disease from more distant organs
Low molecular weight proteins: glomerular filtration
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
From biomarkers to pathophysiology?
m/z
600 800 1000 1200 1400 1600
10
20
30
40
50
60
70
80
90
100
Inte
nsi
ty
609.256
b6
755.422
y8
882.357
b9
852.476
y9
995.435
b10
1092.506
b11
1181.252
y12
1318.578
b13
1587.759
b16
1715.817
b18
858.408
b18 ++
794.380
b16 ++
0
Sequence identification
tandem mass-spectrometry
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
From biomarkers to pathophysiology?
Biomarkers:
Abundant plasma proteins and fragments. (albumin, beta2-macroglobulin, alpha1-antitrypsin etc…).
Abundant (structural) kidney proteins and fragments. (collagens, uromodullin).
Not highly informative
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Chasing low abundance proteins
A few proteins and their fragments make up the major partof the urinary proteome
Although we fractionate with proteomics these abundant proteins still “hide” the less abundant ones
ESPN 2008 - Lyon
- Depletion of human serum albumin + 815 other protein species- Depletion of IgGs + 2091 other protein species
Human plasma
Shen et al., 2005, Proteomics 5: 4034
Immunodepletion of high abundance proteins
Advances in urinary proteome analysis and biomarker discovery
Chasing low abundance proteins
ESPN 2008 - Lyonbefore after
Advances in urinary proteome analysis and biomarker discovery
Chasing low abundance proteins
Ligand peptide library(millions of different ligands) Flow-Through:
Highly abundant proteins
REDUCEDDYNAMICRANGE
+
LARGE DYNAMIC
RANGE
Albumin
IgGs
ESPN 2008 - Lyon
Advances in urinary proteome analysis and biomarker discovery
Conclusion
* Urine has emerged as a stable « reservoir » of biomarkers for both urogenital and non-urogenital diseases.
* The variability in single biomarkers is counteracted by patterns that tolerate instability and inconsistency of individual polypeptides/biomarkers
* First studies using adequate statistics and validation have been published.
Large scale validation
* Studies including confounding diseases are being published.
One step closer to use in the clinic
* The contribution of proteomics to the understanding of pathophysiology is still limited
ESPN 2008 - Lyon
Thank you
ESPN 2008 - Lyon
Beyond urine
Bilateral developmental nephropathy
Post-natal renal function ?
Early renal lesions invisible by sonography
Responsible for the modification of post-natal renal function
Presence of specific peptides/proteins in amniotic fluid
Clinical question: can we define AF biomarkers predicting post-natal renal function?
22/24
ESPN 2008 - Lyon
Beyond urine
CE-MS
1000-1200 peptides/sample
2D-PAGELC-MS
842 non-redundant proteins
SELDI
Identification of intra-amniotic inflammation(n=169, blinded, high spec/sens)
Buhimschi et al., 2007, PLoS Med 4(1): 84-94.
Amniotic fluid is great source of biomarkers !!
Amniotic fluid !!