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Bringing Proteomics to the Clinic:Bringing Proteomics to the Clinic:From Discovery to Validation From Discovery to Validation

November 4, 2007November 4, 2007

Support: NHLBI, NIDDK, NCRR, AHA, Cystic Fibrosis FoundationSupport: NHLBI, NIDDK, NCRR, AHA, Cystic Fibrosis Foundation

Frank J. Accurso, MDFrank J. Accurso, MDProfessor of Pediatrics Professor of Pediatrics University of ColoradoUniversity of Colorado

““Biomarkers of Pulmonary Arterial Hypertension”Biomarkers of Pulmonary Arterial Hypertension”

Acknowledgements – Dunbar Ivy, MD; Steve Abman, MDAcknowledgements – Dunbar Ivy, MD; Steve Abman, MD

DisclosuresDisclosures: None: None

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Biomarkers of Pulmonary Arterial Biomarkers of Pulmonary Arterial Hypertension (PAH): OverviewHypertension (PAH): Overview

• PAH in adults and childrenPAH in adults and children

• PAH and biomarkersPAH and biomarkers

• Exploratory protein biomarker studies in PAH in Exploratory protein biomarker studies in PAH in children children

Take home messages:Take home messages:

1.1. Improved biomarkers are needed to quickly Improved biomarkers are needed to quickly “ “test” new treatment approaches.test” new treatment approaches.

2. Biomarker studies should be incorporated into2. Biomarker studies should be incorporated into clinical trials and investigations in PAH. clinical trials and investigations in PAH.

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PAH - definitionPAH - definition

• Sustained elevation of mean pulmonary arterial Sustained elevation of mean pulmonary arterial pressure to > 25 mm Hg at rest or >30 mm Hg pressure to > 25 mm Hg at rest or >30 mm Hg with exercise, with a mean pulmonary capillary with exercise, with a mean pulmonary capillary and left atrial pressure < 15 mm Hg at rest.and left atrial pressure < 15 mm Hg at rest.

• “ “fixed” (structural) and “reactive” componentsfixed” (structural) and “reactive” components

• Better treatments are needed.Better treatments are needed.

Rubin, Chest supplement, 2004Rubin, Chest supplement, 2004

4Simonneau G, et al. Simonneau G, et al. J Am Coll CardiolJ Am Coll Cardiol , Supplement, 2004. , Supplement, 2004.

WHO Classification of PH (Venice, 2003)WHO Classification of PH (Venice, 2003)

Primary Pulmonary Hypertension (PPH) Primary Pulmonary Hypertension (PPH) – – idiopathic or unknown causeidiopathic or unknown cause

ORORSecondary Pulmonary Hypertension (SPH) Secondary Pulmonary Hypertension (SPH)

– – all other causesall other causes

1. Pulmonary arterial hypertension1. Pulmonary arterial hypertension2. Pulmonary hypertension with left heart failure2. Pulmonary hypertension with left heart failure3. PH with lung diseases and/or hypoxemia3. PH with lung diseases and/or hypoxemia4. PH due to chronic thrombotic and/or embolic diseases4. PH due to chronic thrombotic and/or embolic diseases5. Miscellaneous 5. Miscellaneous

FormerFormer

CurrentCurrent WHO (Venice, 2003)WHO (Venice, 2003)

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PAH (WHO Group I)PAH (WHO Group I)

• Idiopathic (IPAH) Idiopathic (IPAH)

• Familial (FPAH)Familial (FPAH)

• Associated with (APAH)Associated with (APAH)

– Collagen vascular diseaseCollagen vascular disease

– Congenital systemic-to-pulmonary shuntsCongenital systemic-to-pulmonary shunts

– Portal hypertensionPortal hypertension

• Associated with significant venous or capillary Associated with significant venous or capillary involvementinvolvement

– Pulmonary veno-occlusive disease (PVOD)Pulmonary veno-occlusive disease (PVOD)

– Pulmonary capillary hemangiomatosis (PCH)Pulmonary capillary hemangiomatosis (PCH)

• Persistent pulmonary hypertension of the newbornPersistent pulmonary hypertension of the newborn

– HIV infectionHIV infection

– Drugs/toxins Drugs/toxins

– OtherOther

Simonneau G, et al. Simonneau G, et al. J Am Coll CardiolJ Am Coll Cardiol , Supplement, 2004. , Supplement, 2004.

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PAH: Diagnostic EvaluationPAH: Diagnostic Evaluation

• History*History*

• PhysicalPhysical

• BloodBlood

• EKGEKG

• ECHOECHO

• Physiologic*Physiologic*- Lung function- Lung function- Exercise testing- Exercise testing

• ImagingImaging

• Sleep studySleep study

• Right Heart CatheterizationRight Heart Catheterization

* Difficult in young children* Difficult in young children

? Biochemical biomarkers of disease? Biochemical biomarkers of disease

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Idiopathic PAH: SurvivalIdiopathic PAH: SurvivalUntreated - survival worse in childrenUntreated - survival worse in children

Adult median survival: 2.8 years (n=194)Adult median survival: 2.8 years (n=194)Pediatric median survival: 0.8 yearsPediatric median survival: 0.8 years (n=16)(n=16)

00

2020

4040

6060

8080

100100

00 0.50.5 11 1.51.5 22 2.52.5 33 3.53.5 44 4.54.5 55

Years of Follow-upYears of Follow-up

% S

urv

ival

% S

urv

ival

D’Alonzo, et al. D’Alonzo, et al. Ann Internal MedAnn Internal Med 1991 1991

(n=194)(n=194)

68%68%

48%48%

34%34%

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PAH: Acute vasodilator responsePAH: Acute vasodilator responsedepends on agedepends on age

Barst et al.

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PAH: Treatment Approaches PAH: Treatment Approaches

• PathophysiologyPathophysiology- “fixed” – structural, associated with proliferation- “fixed” – structural, associated with proliferation- “reactive” – amenable to vasodilator treatment- “reactive” – amenable to vasodilator treatment- treatments based on known pathways of vasoactivity- treatments based on known pathways of vasoactivity

• Calcium channel blockers – vasodilatorsCalcium channel blockers – vasodilators

• Prostacyclin – vasodilator, antiproliferativeProstacyclin – vasodilator, antiproliferative

• Nitric oxide – vasodilator, antiproliferativeNitric oxide – vasodilator, antiproliferative

• Endothelin – vasoconstrictor, proliferative – need antagonistEndothelin – vasoconstrictor, proliferative – need antagonist

• Vascular reactivity is a favorable prognostic signVascular reactivity is a favorable prognostic sign

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Years after DiagnosisYears after Diagnosis

Ev

ent

Fre

e P

rop

ort

ion

Ev

ent

Fre

e P

rop

ort

ion

Idiopathic PAH in Children:Survival and Treatment Success with

Chronic Oral CCB in Acute Responders

Yung, et al. Yung, et al. CirculationCirculation 2004 2004

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Complex, long-term PAH treatment Complex, long-term PAH treatment strategies have been developedstrategies have been developed

Trial of Trial of Calcium Channel Calcium Channel BlockerBlocker

Acute Vasodilator ResponseAcute Vasodilator ResponseYesYes

CHFCHF

EpoprostenolEpoprostenolTreprostinilTreprostinil

Trial of :Trial of :BosentanBosentanAmbrisentanAmbrisentanSildenafilSildenafilIloprostIloprostTreprostinilTreprostinilEpoprostenolEpoprostenol

Incomplete responseIncomplete response

IncompleteIncompleteresponseresponse

EpoprostenolEpoprostenolNitric OxideNitric Oxide

NoNo

YesYesNoNo

Adapted from Rashid A, Ivy D. 2006: Adapted from Rashid A, Ivy D. 2006: Current Paediatrics Current Paediatrics

Biomarkers would be helpful in adding treatments.Biomarkers would be helpful in adding treatments.

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Biomarkers of Pulmonary Arterial Biomarkers of Pulmonary Arterial Hypertension (PAH)Hypertension (PAH)

• PAH in Adults and ChildrenPAH in Adults and Children

• Biomarkers and PAHBiomarkers and PAH

• Exploratory Biomarker Studies in PAH in Exploratory Biomarker Studies in PAH in Children Children

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Outcome MeasuresOutcome Measures

Clinical efficacy measures: A characteristic or variable that A characteristic or variable that reflects how a patient feels, functions, or survives.reflects how a patient feels, functions, or survives.

Surrogate endpoint:Surrogate endpoint: A laboratory measurement or physical A laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how clinically meaningful endpoint that is a direct measure of how a patient feels, functions, or survives and is expected to a patient feels, functions, or survives and is expected to predict the effect of therapy. predict the effect of therapy.

Biomarker: Biomarker: A characteristic that is objectively measured and A characteristic that is objectively measured and evaluated as an indicator of normal biologic process, evaluated as an indicator of normal biologic process, pathogenic process, or pharmacologic response to apathogenic process, or pharmacologic response to atherapeutic intervention.therapeutic intervention.

Pulmonary Hypertension Review : Snow and Kawut, 2007 ; Hamblett et al, 2007

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Biomarker Need in PAHBiomarker Need in PAH NeedNeed Current biochemical Current biochemical

biomarkerbiomarker

DiagnosisDiagnosis

- Early identification- Early identification NoNo

- Staging- Staging NoNo

PrognosisPrognosis

- - Rapid progression of diseaseRapid progression of disease NoNo

- Ongoing structural Injury - Ongoing structural Injury NoNo

- Predict complications- Predict complications NoNo

TreatmentTreatment

- Select treatment- Select treatment NoNo

- Response to treatment- Response to treatment NoNo

- Toxicity with treatment - Toxicity with treatment NoNo

- Clinical Trials - Clinical Trials

a. Stratificationa. Stratification NoNo

b. Proof of conceptb. Proof of concept NoNo

c. Efficacyc. Efficacy NoNo

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Value Added: Proof of ConceptValue Added: Proof of Concept

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The specific goals of the Clinical Proteomics The specific goals of the Clinical Proteomics Program are to:Program are to:

(1) design panels of candidate proteins for disease (1) design panels of candidate proteins for disease areasareas

(2) develop high throughput analytic methods(2) develop high throughput analytic methods

(3) assess the predictive value of these proteomic (3) assess the predictive value of these proteomic measurements using biological specimens and measurements using biological specimens and clinical data from existing study populationsclinical data from existing study populations

(4) establish procedures and standards for quality (4) establish procedures and standards for quality control control http://www.mc.vanderbilt.edu/root/vumc.php?site=proteomicshttp://www.mc.vanderbilt.edu/root/vumc.php?site=proteomics

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Approaches to Protein Biomarkers

ProteomicsProteomics(Discovery)(Discovery)

CandidateCandidate - Single - Single - Panel- Panel

ValidationValidation Value Value AddedAdded

Single/Few Single/Few Protein Protein Assay(s)Assay(s)(Hypothesis)(Hypothesis)

Multiplex Protein Multiplex Protein Assay(s)Assay(s)(Hypothesis/ (Hypothesis/ Discovery)Discovery)

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Candidate Biomarker Pathways in PAHCandidate Biomarker Pathways in PAH

CandidateCandidate SourceSource

Natriuretic peptide (BNP)Natriuretic peptide (BNP) myocytes myocytes

Troponin T Troponin T myocytes myocytes

Uric acidUric acid ubiquitous ubiquitous

Endothelin-1 Endothelin-1 endothelium endothelium

Serotonin Serotonin platelets platelets Angiotensin systemAngiotensin system vascular wall vascular wall

NO related productsNO related products endotheliumendothelium

Cyclic nucleotidesCyclic nucleotides vascular wall,vascular wall,smooth muscle smooth muscle

Fibrinogen related productsFibrinogen related products fibrinfibrin

Cytokines, Growth factors Cytokines, Growth factors varietyvariety

Acute Phase ReactantsAcute Phase Reactants liverliverAubert, Swiss Med Weekly, 2007

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Caveat: Different Reagent SourcesCaveat: Different Reagent Sources

Brand XBrand X

B

ran

d

Bra

nd

YY

Serum Il-Serum Il-66

• StandardsStandards AgreeAgree

• Controls Controls AgreeAgree

• Duplicate Duplicate Clinical samplesClinical samples do do notnot agree agree

• A second analyticalA second analytical method is neededmethod is needed for validation.for validation.

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Biomarkers of Pulmonary Arterial Biomarkers of Pulmonary Arterial Hypertension (PAH): OverviewHypertension (PAH): Overview

• PAH in adults and childrenPAH in adults and children

• PAH and biomarkersPAH and biomarkers

• Exploratory protein biomarker studies in PAH in Exploratory protein biomarker studies in PAH in children children

Take home messages:Take home messages:

1.1. Improved biomarkers are needed to quickly Improved biomarkers are needed to quickly “ “test” new treatment approaches.test” new treatment approaches.

2. Biomarker studies should be incorporated into2. Biomarker studies should be incorporated into clinical trials and investigations in PAH. clinical trials and investigations in PAH.

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Characteristics of biomarkersCharacteristics of biomarkers

• Is the outcome measure specific to a process occurring Is the outcome measure specific to a process occurring either early or late in the disease process?either early or late in the disease process?

• Is the biomarker in the causal pathway of the drug?Is the biomarker in the causal pathway of the drug?

• • How is the biomarker expected to change in response to How is the biomarker expected to change in response to the drug?the drug?

• • How long will it take to see an expected change in How long will it take to see an expected change in response to the drug? response to the drug?