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

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2520

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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

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albumin

Ig kappa chain

microglobulin

Two-dimensional gel electrophoresis

Tho

ngbo

onke

rd,

2002

acidic basic

Mas

s (k

Da)

ESPN 2008 - Lyon

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SELDI-MS(surface enhanced laser desorption/ionisation-mass spectrometry)

Mas

s (k

Da)

3

100

ESPN 2008 - Lyon

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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:

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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

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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

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Downregulated in comparison

upregulated in comparison

Training: 15 biomarkers differentiate between CAD and non-CAD controls

non-CAD controlspatients with CAD

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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

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- 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

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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 !!