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Proteomics in Pediatrics

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Presentation on theme: "Proteomics in Pediatrics"— Presentation transcript:

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 © May 2012 Consensus in Pediatrics Moscow

5 Proteomics Platforms: Pros and Cons
2DE-MS SELDI-TOF LC-MS CE-MS May 2012 Consensus in Pediatrics Moscow

6 Capillary Electrophoresis coupled to Mass Spectrometry
SOP QM MIAPE guidelines* Urine profile *Taylor et al. (2007) Nat. Biotechnol. 25: May 2012 Consensus in Pediatrics Moscow

7 Low molecular weight urinary proteome profiling
May 2012 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 May 2012 Consensus in Pediatrics Moscow 9

10 DeToni-Debré-Fanconi Syndrome (FS)
FS Control CKD May 2012 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. May 2012 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 May 2012 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): May 2012 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 May 2012 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 May 2012 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.. May 2012 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. May 2012 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 May 2012 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 May 2012 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 May 2012 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 May 2012 Consensus in Pediatrics Moscow

24 Furthermore: Proteomics in adults
1 Coronary Artery Disease 2 Diabetic Nephropathy 1 2 3 Bladder cancer 4 Prostate cancer 3 4 May 2012 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: May 2012 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

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