17.-20. may 2012 consensus in pediatrics moscow proteomics in pediatrics disclosure es is an...
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17.-20. May 2012 Consensus in Pediatrics
Moscow
Proteomics in Pediatrics
DisclosureES is an employee of mosaiques diagnostics GmbH. Presentation does not include discussion
of off-label or investigational use
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
17.-20. May 2012 Consensus in Pediatrics
Moscow
Genomics and Proteomics
Genomics
• approx. 20,000 genes
• relatively invariant / static
• Explains vulnerability for a disease
static
Proteomics
• approx. 1,000,000 proteins
• highly dynamic
• Explains environmental influencesdynamic
17.-20. May 2012 Consensus in Pediatrics
Moscow
Concentration of analyte
Compliance Degree of invasion
Proteolysis Proximity to disease
Tissue
Bile
Blood
Urine
Sources of Protein Biomarkers
17.-20. May 2012 Consensus in Pediatrics
Moscow
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
The concept of urinary biomarker patterns
© http://www.sciencemag.org
17.-20. May 2012 Consensus in Pediatrics
Moscow
Proteomics Platforms: Pros and Cons
2DE-MS SELDI-TOF LC-MS CE-MS
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Moscow
Capillary Electrophoresis coupled to Mass
Spectrometry
Urine profile
SOP
QM
MIAPE guidelines*
*Taylor et al. (2007) Nat. Biotechnol. 25: 887-893.
17.-20. May 2012 Consensus in Pediatrics
Moscow
Low molecular weight urinary proteome profiling
17.-20. May 2012 Consensus in Pediatrics
Moscow
Normal Control
Amp 2,0Frequency 28/267
CardiovascularDisease
Amp 3,0Frequency 22/40
DiabeticNephropathy
Amp 4,3Frequency 25/34
Bladder Cancer
Amp 3,5Frequency 20/30
Prostate Cancer
Amp 2,9Frequency 49/153
Statistics for Marker ID 90840
ID 90840
Mass 2389.24 Da
CE time 22.40 min
Sequence MIEGNTKSPLFMGKVVNPTQK
Peptide alpha-1-Antitrypsin [aa 398-418]
Protein ID: 90840
0 2 4 6 80
1
2
3
4
5
Amp
P
NCCVDDNBCPC
2
3
4
5
1
Schiffer et al., Proteomics 2006, 6: 5615-5627
Statistical biomarker definition
17.-20. May 2012 Consensus in Pediatrics
Moscow
Unblinding
Sensitivity and Specificity94% 89%
Clinical Study Design
CASE diseased treated (Drug)
CONTROL healthy not treated (Placebo)discriminatory pattern discriminatory pattern
compiled patterncompiled pattern
discriminatory biomarkers
individual analyses
17.-20. May 2012 Consensus in Pediatrics
Moscow
DeToni-Debré-Fanconi Syndrome(FS)
FS Control CKD
17.-20. May 2012 Consensus in Pediatrics
Moscow
1,5
1,0
0,5
0,0
-0,5
-1,0
-1,5
-2,0
-2,5
SV
M S
co
re
FS control CKD0
25
50
75
100
0 25 50 75 100
100-Specificity
Sen
siti
vity
FS Control CKD N=11 N=9 N=294
FS: Clinical Validation
Blinded: 11 FS9 ControlsAUC=0.86
Open-label:11 FS294 CKDAUC=0.84dashed
Drube et al. (2009) Nephrol. Dial. Transplant. 24: 2161–2169.
17.-20. May 2012 Consensus in Pediatrics
Moscow
Peptide IDMass(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 1405.64 20.1 92 509 0.002 DGPpGRDGQpGHKG Collagen alpha-2 (I)
47855 1576.74 19.5 186 861 0.001 YKRKANDESNEHS Osteopontin
55143 1692.80 30.9 438 2498 0.041 PpGEAGKpGEQGVPGDLG Collagen alpha-1 (I)
60355 1798.76 30.3 951 125 0.012 GEpGSpGENGApGQMGPRG Collagen alpha-1 (I)
67217 1933.88 21.6 339 516 0.027 GDDGEAGKPGRpGERGPpGP Collagen alpha-1 (I)
67911 1949.89 21.7 0 220 0.001 GDDGEAGkPGRpGERGPpGP Collagen alpha-1 (I)
76839 2128.98 27.0 179 158 0.044DGKTGpPGPAGQDGRPGPpGppG
Collagen alpha-1 (I)
124886 3193.38 22.6 2305 1173 0.013PpGESGREGAPGAEGSpGRDGSpGAKGDRGETGP
Collagen alpha-1 (I)
In FS decreased / increased urinary excretion
Sequenced marker proteins
The marker peptides are fragments derived from osteopontin, uromodulin and collagens
17.-20. May 2012 Consensus in Pediatrics
Moscow
FS: Conclusions
• 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
17.-20. May 2012 Consensus in Pediatrics
Moscow
spontaneousremission
Surgery
?
PrognosisSurgery yes or no
Sensitivity 94% (21/23)Specificity 100% (13/13)
Decramer et al. (2006) Nat. Med. 12(4): 398-400
Ureteropelvic junction obstruction (UPJO)
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Moscow
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
Sensitivity 83% (5/6)Specificity 92% (12/13)AUC=88%, P<0.0001
N=8 > 1 year of age
Sensitivity 20% (1/5)Specificity 66% (2/3)AUC=57%, P=0.7655
17.-20. May 2012 Consensus in Pediatrics
Moscow
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
17.-20. May 2012 Consensus in Pediatrics
Moscow
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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Moscow
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.
17.-20. May 2012 Consensus in Pediatrics
Moscow
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)
CE Time (min)
Mas
s (D
a)
Mas
s (D
a)
Drube et al. (2012) Pediatrics 129(2):e356-63.
17.-20. May 2012 Consensus in Pediatrics
Moscow
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)
Sen
sit
ivit
y
1-Specificity
Sensitivity 88% (15/17)Specificity 79% (15/19)AUC=80%, P<0.0001
0
17.-20. May 2012 Consensus in Pediatrics
Moscow
VUR: Sequencing of Markers
Peptide IDMass(Da)
CE-Time(min)
VUR UTI Sequence Protein
16773 1080.48 27.8 19 119 DRGEpGPpGPA Collagen alpha-1(I) (801-811)
17968 1099.49 28.2 31 152 DGGGSPKGDVDPSodium/potassium-transporting ATPase, subunit gamma (7-18)
19773 1128.39 33.6 68 302 DFDDFNLEDCD99 antigen-like protein 2 (26-34)
24944 1209.56 36.2 17 58 - -
33973 1353.66 25.6 481 710KGEAGLpGApGSPG
QCollagen alpha-1(XIX) (291-305)
39607 1447.70 19.5 797 1423 - -
41654 1470.65 31.4 10 34 - -
60357 1798.78 31.8 397 967NDGAKGDAGApGAp
GSQGApGCollagen alpha-1(I) (705-725)
72153 2038.92 25.2 88 235 - -
In VUR decreased / increased urinary excretion
17.-20. May 2012 Consensus in Pediatrics
Moscow
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
17.-20. May 2012 Consensus in Pediatrics
Moscow
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
17.-20. May 2012 Consensus in Pediatrics
Moscow
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
1 Coronary Artery Disease
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
2 Diabetic Nephropathy
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
3 Bladder cancer20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
20 25 30 35 40 45
0.8
1.0
2.0
6.0
8.010.0
20.0
4.0
4 Prostate cancer
Furthermore: Proteomics in adults
1
2
3
4
17.-20. May 2012 Consensus in Pediatrics
Moscow
CE-time CE-time
Mass
CAD negative CAD positive
Δ C
AD
Score
Irbesartan 300 mg/day
Placebo
P=0.0066
Delles et al. (2010) J. Hypertension 28:2316-22.
Furthermore: Treatment Monitoring
• 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’
17.-20. May 2012 Consensus in Pediatrics
Moscow
The Physician
Gerrit DouLeiden (1613-1675)
Acknowledgment
Mosaiques:Harald MischakMohammed DaknaIgor GolovkoAnnika DorbanJustyna SiwyJochen MetzgerPetra 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)
17.-20. May 2012 Consensus in Pediatrics
Moscow
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.