1. 1 Bringing Proteomics to the Clinic: From Discovery to Validation November 4, 2007 Support: NHLBI, NIDDK, NCRR, AHA, Cystic Fibrosis Foundation Frank J. Accurso, MD Professor of Pediatrics University of Colorado “Biomarkers of Pulmonary Arterial Hypertension” Acknowledgements – Dunbar Ivy, MD; Steve Abman, MD Disclosures : None

2. 2 Biomarkers of Pulmonary Arterial Hypertension (PAH): Overview PAH in adults and children PAH in adults and children PAH and biomarkers PAH and biomarkers Exploratory protein biomarker studies in PAH in Exploratory protein biomarker studies in PAH in children children Take home messages: 1.Improved biomarkers are needed to quickly “test” new treatment approaches. “test” new treatment approaches. 2. Biomarker studies should be incorporated into clinical trials and investigations in PAH. clinical trials and investigations in PAH.

3. 3 PAH - 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” components “fixed” (structural) and “reactive” components Better treatments are needed. Better treatments are needed. Rubin, Chest supplement, 2004

4. 4 Simonneau G, et al. J Am Coll Cardiol, Supplement, 2004. WHO Classification of PH (Venice, 2003) Primary Pulmonary Hypertension (PPH) – idiopathic or unknown cause OR Secondary Pulmonary Hypertension (SPH) – all other causes 1. Pulmonary arterial hypertension 2. Pulmonary hypertension with left heart failure 3. PH with lung diseases and/or hypoxemia 4. PH due to chronic thrombotic and/or embolic diseases 5. Miscellaneous Former Current WHO (Venice, 2003)

5. 5 PAH (WHO Group I) Idiopathic (IPAH)Idiopathic (IPAH) Familial (FPAH)Familial (FPAH) Associated with (APAH)Associated with (APAH) –Collagen vascular disease –Congenital systemic-to-pulmonary shunts –Portal hypertension Associated with significant venous or capillary involvementAssociated with significant venous or capillary involvement –Pulmonary veno-occlusive disease (PVOD) –Pulmonary capillary hemangiomatosis (PCH) Persistent pulmonary hypertension of the newbornPersistent pulmonary hypertension of the newborn –HIV infection –Drugs/toxins –Other Simonneau G, et al. J Am Coll Cardiol, Supplement, 2004.

6. 6 PAH: Diagnostic Evaluation History* History* Physical Physical Blood Blood EKG EKG ECHO ECHO Physiologic* Physiologic* - Lung function - Exercise testing Imaging Imaging Sleep study Sleep study Right Heart Catheterization Right Heart Catheterization * Difficult in young children * Difficult in young children ? Biochemical biomarkers of disease

7. 7 Idiopathic PAH: Survival Untreated - survival worse in children Adult median survival: 2.8 years (n=194) Pediatric median survival: 0.8 years(n=16) 0 20 40 60 80 100 00.511.522.533.544.55 Years of Follow-up % Survival D’Alonzo, et al. Ann Internal Med 1991 (n=194) 68% 48% 34%

8. 8 PAH: Acute vasodilator response depends on age Barst et al.

9. 9 PAH: Treatment Approaches Pathophysiology Pathophysiology - “fixed” – structural, associated with proliferation - “reactive” – amenable to vasodilator treatment - treatments based on known pathways of vasoactivity Calcium channel blockers – vasodilators Calcium channel blockers – vasodilators Prostacyclin – vasodilator, antiproliferative Prostacyclin – vasodilator, antiproliferative Nitric oxide – vasodilator, antiproliferative Nitric oxide – vasodilator, antiproliferative Endothelin – vasoconstrictor, proliferative – need antagonist Endothelin – vasoconstrictor, proliferative – need antagonist Vascular reactivity is a favorable prognostic sign Vascular reactivity is a favorable prognostic sign

10. 10 Years after Diagnosis Event Free Proportion Idiopathic PAH in Children: Survival and Treatment Success with Chronic Oral CCB in Acute Responders Yung, et al. Circulation 2004

11. 11 Complex, long-term PAH treatment strategies have been developed Trial of Calcium Channel Blocker Acute Vasodilator Response Yes CHF EpoprostenolTreprostinil Trial of : BosentanAmbrisentanSildenafilIloprostTreprostinilEpoprostenol Incomplete response Incompleteresponse Epoprostenol Nitric Oxide No Yes No Adapted from Rashid A, Ivy D. 2006: Current Paediatrics Biomarkers would be helpful in adding treatments.

12. 12 Biomarkers of Pulmonary Arterial Hypertension (PAH) PAH in Adults and Children PAH in Adults and Children Biomarkers and PAH Biomarkers and PAH Exploratory Biomarker Studies in PAH in Exploratory Biomarker Studies in PAH in Children Children

13. 13 Outcome Measures A characteristic or variable that reflects how a patient feels, functions, or survives. Clinical efficacy measures: A characteristic or variable that reflects how a patient feels, functions, or survives. Surrogate endpoint: A laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how a patient feels, functions, or survives and is expected to predict the effect of therapy. Biomarker: A characteristic that is objectively measured and evaluated as an indicator of normal biologic process, pathogenic process, or pharmacologic response to a therapeutic intervention. Pulmonary Hypertension Review : Snow and Kawut, 2007 ; Hamblett et al, 2007

14. 14 Biomarker Need in PAH Need Current biochemical biomarker Need Current biochemical biomarkerDiagnosis - Early identificationNo - StagingNo Prognosis - Rapid progression of disease No - Ongoing structural Injury No - Predict complicationsNo Treatment - Select treatmentNo - Response to treatmentNo - Toxicity with treatment No - Clinical Trials a. StratificationNo a. StratificationNo b. Proof of conceptNo b. Proof of conceptNo c. EfficacyNo c. EfficacyNo

15. 15 Value Added: Proof of Concept

16. 16 The specific goals of the Clinical Proteomics Program are to: (1) design panels of candidate proteins for disease areas (2) develop high throughput analytic methods (3) assess the predictive value of these proteomic measurements using biological specimens and clinical data from existing study populations (4) establish procedures and standards for quality control http://www.mc.vanderbilt.edu/root/vumc.php?site=proteomics

17. 17 Approaches to Protein BiomarkersProteomics(Discovery) Candidate - Single - Single - Panel - Panel Validation ValueAdded Single/Few Protein Assay(s)(Hypothesis) Multiplex Protein Assay(s)(Hypothesis/Discovery)

18. 18 Candidate Biomarker Pathways in PAH CandidateSource Natriuretic peptide (BNP) myocytes Troponin T myocytes Uric acid ubiquitous Endothelin-1 endothelium Serotonin platelets Angiotensin system vascular wall NO related products endothelium Cyclic nucleotidesvascular wall, smooth muscle Fibrinogen related productsfibrin Cytokines, Growth factors variety Acute Phase Reactantsliver Aubert, Swiss Med Weekly, 2007

19. 19 Caveat: Different Reagent Sources Brand X Brand X Brand Y Brand Y Serum Il-6 Serum Il-6 Standards Standards Agree Agree Controls Controls Agree Agree Duplicate Duplicate Clinical samples Clinical samples do not agree do not agree A second analytical A second analytical method is needed method is needed for validation. for validation.

20. 20 Biomarkers of Pulmonary Arterial Hypertension (PAH): Overview PAH in adults and children PAH in adults and children PAH and biomarkers PAH and biomarkers Exploratory protein biomarker studies in PAH in Exploratory protein biomarker studies in PAH in children children Take home messages: 1.Improved biomarkers are needed to quickly “test” new treatment approaches. “test” new treatment approaches. 2. Biomarker studies should be incorporated into clinical trials and investigations in PAH. clinical trials and investigations in PAH.

21. 21 Characteristics 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 the drug? How is the biomarker expected to change in response to the drug? How long will it take to see an expected change in response to the drug? How long will it take to see an expected change in response to the drug?