17.-20. May 2012 Consensus in Pediatrics Moscow Proteomics in Pediatrics Disclosure ES is an employee of mosaiques diagnostics GmbH. Presentation does.

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


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


Moscow

Concentration of analyte

Compliance Degree of invasion

Proteolysis Proximity to disease

Tissue

Bile

Blood

Urine

Sources of Protein Biomarkers


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


Moscow

Proteomics Platforms: Pros and Cons

2DE-MS SELDI-TOF LC-MS CE-MS


Moscow

Capillary Electrophoresis coupled to Mass

Spectrometry

Urine profile

SOP

QM

MIAPE guidelines*

*Taylor et al. (2007) Nat. Biotechnol. 25: 887-893.


Moscow

Low molecular weight urinary proteome profiling


Moscow

Normal Control

Amp 2,0Frequency 28/267

CardiovascularDisease


DiabeticNephropathy


Bladder Cancer


Prostate Cancer


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


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


Moscow

DeToni-Debré-Fanconi Syndrome(FS)

FS Control CKD


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.


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


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


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)


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


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


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..


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.


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.


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


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


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


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


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


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’


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)


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.

17.-20. May 2012 Consensus in Pediatrics Moscow Proteomics in Pediatrics Disclosure ES is an employee of mosaiques diagnostics GmbH. Presentation does.

Documents

fs slide

pediatrics moscow urine

pediatrics moscow upjo

pediatrics moscow genomics

pediatrics moscow detonidebr

median fs

mscems slide

pediatrics disclosure