1. Proteomics in Pediatrics
Eric Schiffer1,2, Jens Drube3, Lars Pape3, Jochen HH. Ehrich3
1mosaiques diagnostics GmbH, Hannover (Germany),
2Department of Biomarkers and Systems Medicine, University of Glasgow, Glasgow (UK).
3Clinic of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany
Disclosure
ES is an employee of mosaiques diagnostics GmbH. Presentation does not include discussion of off-label or investigational use
May 2012
Consensus in Pediatrics Moscow

2. Genomics and Proteomics
approx. 20,000 genes
relatively invariant / static
Explains vulnerability for a disease
static
Proteomics
approx. 1,000,000 proteins
highly dynamic
Explains environmental influences
dynamic
May 2012
Consensus in Pediatrics Moscow

3. Sources of Protein Biomarkers
Tissue
Bile
Blood
Urine
Compliance
Degree of invasion
Concentration of analyte
Proteolysis
Proximity to disease
May 2012
Consensus in Pediatrics Moscow

4. The concept of urinary biomarker patterns
Urine
Obtained non invasive in large quantities
Low protease activity compared to blood
Urinary polypeptides are stable, yielding comparable proteomic profiles
Biomarker pattern
Currently single biomarkers
are analyzed in clinical routine
Single biomarkers often cannot
display the complexity of a disease
Biomarker patterns may compensate
inconsistencies of single markers ©
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Consensus in Pediatrics Moscow

5. Proteomics Platforms: Pros and Cons
2DE-MS
SELDI-TOF
LC-MS
CE-MS
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Consensus in Pediatrics Moscow

6. Capillary Electrophoresis coupled to Mass Spectrometry
SOP QM
MIAPE guidelines*
Urine profile
*Taylor et al. (2007) Nat. Biotechnol. 25:
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Consensus in Pediatrics Moscow

7. Low molecular weight urinary proteome profiling
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Consensus in Pediatrics Moscow

8. Statistical biomarker definition
Normal Control
Amp 2,0
Frequency 28/267
Cardiovascular
Disease
Amp 3,0
Frequency 22/40
Diabetic
Nephropathy
Amp 4,3
Frequency 25/34
Bladder Cancer
Amp 3,5
Frequency 20/30
Prostate Cancer
Amp 2,9
Frequency 49/153
Statistics for Marker ID 90840
ID 90840
Mass Da
CE time min
Sequence MIEGNTKSPLFMGKVVNPTQK
Peptide alpha-1-Antitrypsin [aa ]
Protein ID: 90840 2 4 6 8 1 3 5
Amp P NC
CVD DN BC PC
May 2012
Consensus in Pediatrics Moscow
Schiffer et al., Proteomics 2006, 6:

9. Sensitivity and Specificity
Clinical Study Design
CASE
diseased
treated (Drug)
CONTROL
healthy
not treated (Placebo)
discriminatory pattern
compiled pattern
Unblinding
Sensitivity and Specificity
94% %
individual analyses
discriminatory biomarkers
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Consensus in Pediatrics Moscow 9

10. DeToni-Debré-Fanconi Syndrome (FS)
FS Control CKD
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Consensus in Pediatrics Moscow

11. FS: Clinical Validation
Blinded: FS
9 Controls
AUC=0.86
Open-label: 11 FS
294 CKD
AUC=0.84
dashed
FS Control CKD
N= N= N=294
Drube et al. (2009) Nephrol. Dial. Transplant. 24: 2161–2169.
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Consensus in Pediatrics Moscow

12. Sequenced marker proteins
Peptide ID
Mass
(Da)
CE-time
(min)
Median FS
(counts)
Median HC (counts)
maxT
(p-value)
Sequence
Protein
11413
981.59
24.8 94
116
0.008
VLNLGPITR
Uromodulin
36769
20.1 92
509
0.002
DGPpGRDGQpGHKG
Collagen alpha-2 (I)
47855
19.5
186
861
0.001
YKRKANDESNEHS
Osteopontin
55143
30.9
438
2498
0.041
PpGEAGKpGEQGVPGDLG
Collagen alpha-1 (I)
60355
30.3
951
125
0.012
GEpGSpGENGApGQMGPRG
67217
21.6
339
516
0.027
GDDGEAGKPGRpGERGPpGP
67911
21.7
220
GDDGEAGkPGRpGERGPpGP
76839
27.0
179
158
0.044
DGKTGpPGPAGQDGRPGPpGppG
124886
22.6
2305
1173
0.013
PpGESGREGAPGAEGSpGRDGSpGAKGDRGETGP
In FS decreased / increased urinary excretion
The marker peptides are fragments derived from osteopontin, uromodulin and collagens
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Consensus in Pediatrics Moscow

13. FS: Conclusions Biomarkers suggest fibrosis as an early event
CE-MS can be used to specifically diagnose FS in pediatric patients
CE-MS might be a future tool for the non-invasive diagnosis of FS.
Reduced levels of osteopontin and uromodulin might indicate loss of tubular function regardless of underlying cause
Fragments of the collagen alpha-1 (I) might hint to change of proteases in collagen degradation as observed in interstitial fibrosis.
Biomarkers suggest fibrosis as an early event
in the development of renal insufficiency in FS
May 2012
Consensus in Pediatrics Moscow

14. Ureteropelvic junction obstruction (UPJO)
spontaneous
remission
Surgery ?
Prognosis
Surgery yes or no
Sensitivity 94% (21/23)
Specificity 100% (13/13)
Decramer et al. (2006) Nat. Med. 12(4):
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Consensus in Pediatrics Moscow

15. UPJO: Clinical Validation
Application of the Decramer et al. pattern to older children
Prospective cohort with hydronephrosis enroled at Hannover
Diuretic renal scan to identify urodynamically relevant UPJO
N=19 ≤ 1 year of age
N=8 > 1 year of age
Sensitivity 20% (1/5)
Specificity 66% (2/3)
AUC=57%, P=0.7655
Sensitivity 83% (5/6)
Specificity 92% (12/13)
AUC=88%, P<0.0001
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Consensus in Pediatrics Moscow

16. UPJO: Cost-effectiveness
Marcov process decision tree model
Comparison of watchful waiting / imaging to urinary proteomics
Incremental gain of $8,000 per QUALY
Results insensitive to any included cost parameter
Application to 2,000 newborns would save $US 16 Mio. per year while improving quality of life
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Consensus in Pediatrics Moscow

17. Drube et al. (2010) Pediatr. Nephrol. 25:1673–1678..
UPJO: Conclusions
Confirmation of results reported by Decramer et al. in infants ≤ 1 year of age,
Urinary proteome analysis predicted obstruction with 83% sensitivity and 92% specificity
In older patients sensitivity decreased to 20% and specificity to 66%
The proteome pattern established by Decramer and coworkers predicts the need for surgery in infants but not in older children with UPJO
Drube et al. (2010) Pediatr. Nephrol. 25:1673–1678..
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Consensus in Pediatrics Moscow

18. Vesicoureteral reflux (VUR)
High grade VUR (grade IV or V) is a risk factor for renal scarring, impaired renal function and arterial hypertension.
Voiding cystourethrography (VCUG) is the gold standard for detecting the severity of VUR.
High grade VUR is present in the minority of children with urinary tract infection (UTI), thus exposing the majority to invasive diagnostics which have no surgical consequence.
We therefore aimed at establishing a non-invasive test to identify children with high grade VUR.
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Consensus in Pediatrics Moscow

19. High grade VUR (grade IV or V)
Case-control study to establish specific urinary proteome pattern by CE-MS
18 cases with primary VUR grade IV or V
19 controls without VUR after UTI.
VUR
Controls
CE Time (min)
Mass (Da)
Drube et al. (2012) Pediatrics 129(2):e
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Consensus in Pediatrics Moscow

20. VUR: Blinded Clinical Validation
The test’s accuracy was independent of age, gender and grade of VUR in the contra-lateral kidney.
The odds ratio of suffering from VUR grade IV or V when tested positive was 28 (95%CI: 4.5 to 176)
Sensitivity
1-Specificity
Sensitivity 88% (15/17)
Specificity 79% (15/19)
AUC=80%, P<0.0001
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Consensus in Pediatrics Moscow

21. VUR: Sequencing of Markers
Peptide ID
Mass (Da)
CE-Time (min)
VUR
UTI
Sequence
Protein
16773
27.8 19
119
DRGEpGPpGPA
Collagen alpha-1(I) ( )
17968
28.2 31
152
DGGGSPKGDVDP
Sodium/potassium-transporting ATPase, subunit gamma (7-18)
19773
33.6 68
302
DFDDFNLED
CD99 antigen-like protein 2 (26-34)
24944
36.2 17 58 -
33973
25.6
481
710
KGEAGLpGApGSPGQ
Collagen alpha-1(XIX) ( )
39607
19.5
797
1423
41654
31.4 10 34
60357
31.8
397
967
NDGAKGDAGApGApGSQGApG
Collagen alpha-1(I) ( )
72153
25.2 88
235
In VUR decreased / increased urinary excretion
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Consensus in Pediatrics Moscow

22. VUR: Pathophysiology of Markers
CD99 is strongly expressed in normal urinary mucosa, which is known to show histological changes in animal models for VUR
Collagen alpha-1 (I) fragments suggest alterations of extra cellular matrix turnover
Na/K-transporting ATPase plays a key role in the active transportation of Na+ and K+ across basolateral membranes of nephron epithelial cells
Biomarkers might indicate
early onset of reflux nephropathy
May 2012
Consensus in Pediatrics Moscow

23. Proteomics in Pediatrics: Conclusion
Proteomic patterns tolerate instability and inconsistency of individual biomarkers
CE-MS platform allows biomarker discovery and validation as a pattern in large patient cohorts
Proteomic biomarkers generate novel hypothesis for potential pathological processes during disease development
CE-MS enables transfer of urinary proteome analysis from bench side to bed side
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Consensus in Pediatrics Moscow

24. Furthermore: Proteomics in adults1
Coronary Artery Disease 2
Diabetic Nephropathy 1 2 3
Bladder cancer 4
Prostate cancer 3 4
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Consensus in Pediatrics Moscow

25. Furthermore: Treatment Monitoring
Δ CADScore
Irbesartan
300 mg/day
Placebo
P=0.0066
CE-time
Mass
CAD negative
CAD positive
Long-term treatment effects.
Patients with type 2 diabetes
irbesartan 300 mg once daily (n=11) or placebo (n=11)
At baseline and after 2 years of
Decrease in CADScore toward ‘healthier’
Delles et al. (2010) J. Hypertension 28:
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Consensus in Pediatrics Moscow

26. Acknowledgment Mosaiques: Partners: Harald Mischak Mohammed Dakna
Igor Golovko
Annika Dorban
Justyna Siwy
Jochen Metzger
Petra Zürbig
Partners:
Markus J. Kemper (Hamburg, Germany)
Thomas Neuhaus (Zurich, Switzerland)
Ralf Lichtinghagen (Hannover, Germany)
Esther Lau (Hannover, Germany)
Benno Ure (Hannover, Germany)
Sylvia Glüer (Hannover, Germany)
Martin Kirschstein (Celle, Hannover)
Stéphane Decramer (Toulouse, France)
Jean-Loup Bascands (Toulouse, France)
Joost P. Schanstra (Toulouse, France)
Claus Petersen (Hannover, Germany)
The Physician
Gerrit Dou
Leiden ( )
May 2012
Consensus in Pediatrics Moscow

27. Implementation of Proteomics
The number of publications on proteomics has increased tremendously, however, the implementation of urinary proteomics into routine nephrology diagnostics is awaiting further progress.
Combined efforts should focus on implementing clinical proteomics after identification of disease specific proteome patterns by providing guidance for further analysis of samples from biobanks, and providing guidance for clinical study design concerning intervention studies.
Feedback mechanisms to evaluating cost-effectiveness and clinical adoption are urgently needed to allow a timely introduction of proteomics into every day clinical care.
May 2012
Consensus in Pediatrics Moscow