Current Biomarker Knowledge and Clinical Practice in Cystic Fibrosis

1. Current Biomarker Knowledge and Clinical Practice in Cystic Fibrosis • Gap Analysis of Knowledge and Practice: • • Prioritize CF Disease Aspects in Need of Biomarkers • • Generate List of Patient Tissue/Fluid Sample Types • and Relationship to Disease Aspects • Overview: • Review of Cystic Fibrosis Emphasizing Biomarkers • Lung Disease • Suggestions, Recommendations • F. Accurso, MD, CF Center Director and • Professor of Pediatrics, University of Colorado, Denver, USA • (CF Foundation, NHLBI, NIDDK, NCRR, CF Community)

2. 25 100 20 80 15 60 Number of Deaths Cumulative Percent 10 40 5 20 0 0 0 5 10 15 20 25 30 35 40 45 50+ Age at Death (n=409) Cystic Fibrosis • Definition: Multisystem, genetic disorder leading to • early death, primarily from progressive lung disease. • 30,000 individuals in US • Median life expectancy - 35 years (CFF registry, 2004) • Median age at death - 26 years (CFF registry, 2001)

3. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) • CFTR - Membrane glycoprotein • - ABC, c-AMP • - Regulates ion flux - chloride, sodium, bicarbonate • - 1400 mutations (www.genet.sickkids.on.ca/cftr/) • - Delta F508 (70% of alleles in US) • Diagnosis (CFF consensus conference, J Pediatr,1998) • - Symptoms and Signs • - Physiology - evidence of CFTR dysfunction • (sweat electrolytes, nasal potential difference) • - Genotype – Two defined mutations • - Family History • - “Positive Newborn Screen” • (Protein Biomarker -Immunoreactive Trypsinogen)

4. Organs/Tissue Secondarily involved Lung Pancreas Intestine Liver Fluid or Breath, Biomarkers of Organ Dysfunction and Injury CFTR: Need for Protein Biomarkers • Diagnosis in Atypical Cases • Clinical Trial - Proof of Concept Dysfunctional CFTR Cells directly involved in CFTR pathophysiology (Airway Epithelium, Sweat Gland) Cellular Biomarkers of CFTR Presence and/or Function Present in Newborns

5. 100 90 80 70 60 2001 50 40 30 6 18 Lung Disease in CF: Clinical Course (CFF registry, 2001) • Decline in Lung function • Acute Exacerbations • - personal, economic impact • - ? decline in lung function. • (Phase III trials, unpublished) • Structural Lung Injury • Daily treatment • - 3-25 medications per day • - Oral, inhaled, injectable • - Chest Physical Therapy • - Nutritional supplementation • - Gastrostomy, central line Years

6. CF Pathophysiology Neutrophils Epithelial Cells Lymphocytes (Hubeau et al, 2001) Macrophages (Durieu et al, 1998)

7. Lung Disease in CF: Assessment We do not have a biomarker (biochemical index) or panel of biomarkers that is carefully defined for any Pulmonary clinical use in CF. Assessment Clinical Research Comment History Yes Needs Refinement (CFFTDN) Physical Exam Yes Not quantitative O2 Saturation Yes Acceptable Lung Function Yes Rapidly Improving (CFFTDN) Imaging - x-ray Yes Scoring systems - CT scan Yes Quantitation developing - VQ scan No Not as outcome measure - Mucociliary Cl. No Needs standardization

8. Lung Disease in CF: Assessment Assessment Clinical Research Comment Laboratory - CBC, ESR Yes Acute Exacerbation - CRP Yes Acute Exacerbation - IgE Yes Allergic Bronchopulmonary Aspergillosis - Pseudomonas No Uncertain utility Antibodies - Quantitative Yes Good Utility Cultures - Sputum No Cell Count, IL-8, elastase Inflammation (some research utility, ?sensitivity)

9. Protein Biomarkers for CF Lung Disease Existing Need Biomarker(s) I. Risk for Rapid decline in lung function No I. Ongoing Lung Structural Injury No (Fibrosis, Elastolysis, Remodeling) I. Identification of Infection No - Pseudomonas aeruginosa - MRSA (Methicillin Resistant Staph.) - Burkholderia cepacia - NTM – M. avium, M. abscessus - Fungal species I. Clinical Trials of Antiinflammatories ? Sputum - Stratification Elastase, - Efficacy IL-8, LTB4

10. Protein Biomarkers for CF Lung Disease • Existing • Need Biomarker(s) • II. Exacerbation • - Identification No • - Susceptibility to exacerbations No • II. Response to treatment No • II. Toxicity with treatment No • II. Staging No • II. Newborn Screening Yes

11. Protein Biomarkers for CF Lung Disease • Existing • Need Biomarker(s) • Clues to Pathogenesis • - Intracellular or Membrane No? • (CFTR Presence or Function) • - Key Pathway (Inflammation, No • Fibrosis, Antimicrobial Defense) • in Organ Dysfunction • - Diagnosis in Atypical Cases No • (Disease Susceptibility)

12. Search for Protein Biomarkers in CF Lung Disease: Summary • Neutrophil Associated Molecules • None have been explored in large populations • Panels of more than five markers are only now • being investigated. (little multiplex data or “cluster”, • “network” or “pattern” analysis) • Proteomic studies are appearing • Bronchoalveolar Lavage • - Clinical Utility – Microbial diagnosis • - Clues to Pathogenesis

13. Elastase (log ug/ml) Control Azithro. p=0.01 for change 0 0 Months 6 Protein Biomarkers of CF Lung Disease: Sputum • Putative Biomarkers • - Proteolytic • - Cytokines, growth factors • - Oxidant/antioxidant • - Surfactant proteins • Research Utility • - Azithromycin study • - Intravenous antibiotics • - Interferon gamma study • - Hypertonic saline study 2 1 (Saiman et al., JAMA, 2003)

14. Protein Biomarkers of CF Lung Disease: Blood/Inflammation • Candidates: CRP, cytokines, growth factors, albumin, • oxidant/antioxidant, S-100, • Mostly exacerbation studies • Biomarker Development Validation Application • 1. Proteomic Experiments Many Steps • Choose Make • Form ELISA • 2. Multiplex Platforms Many Steps • Microsphere - Luminex • Protein Arrays – Randox • Small Sample Volume, Parallel analyses • 20 Mediators, Candidates can be added

15. Protein Biomarkers of CF Lung Disease: Elastolysis • Elastin Breakdown Products • - Urinary Desmosine, Isodesmosine (Stone et al. • AJRCCM, 1995) • Renewed interest (Ma et al, PNAS, 2003) • - Demonstration in sputum, blood • - Measurement of Free and Polymerized Forms • Mass Spec improves • sensitivity – 100 pg • 9 year old with CF • well visit

16. Additional Biomarkers in CF • Breath – Proteins in very low abundance • - Volatile gases, pH • - Nitric oxide related compounds • - Pseudomonas markers (PNAS, 2005) • Nutrition – Very abnormal in CF • - Albumin, Retinol Binding Protein • - Fat soluble vitamins, Carotene • - May confound interpretation of results • Liver Disease – Focal sclerosis • - Cytokines, Growth Factors, Elastin products • - Confound Pulmonary analyses

17. 600 400 200 0 0 24 48 72 96 120 144 Age (months) Protein Biomarker of Pancreatic Disease in CF: Trypsinogen N=288 IRT ng/ml • Residual Trypsinogen at a year of age predicts • Better Lung Function throughout childhood 2004 Endorsement 1979 Benefit Validation

18. Samples for Protein Biomarker Identification Sample Rapid Lung Pathogenesis Stratif Outcome Exacerb. Prog. Destruct. ( Clin Trials) BAL + + ++++ + ++ ++ Sputum +++ +++ +++ +++ +++ +++ Exhaled gases ? ? ? ? ? ? Breath Cond. ? ? ? ? ? ? Blood ? ? ? ? ? ++ Urine + ++ + ? ? ? Saliva + ? + ? ? ? Epithelium, Circulating Cells – protein expression .

19. Current Protein Biomarker Projects Local Specimen Features Support N Biomarkers annual Blood Multiplex NCRR 300 (Luminex) (Randox) NHLBI Biomarkers annual Sputum Multiplex NCRR 35 Glutathione Blood Multiplex CFF 30 Clinical Trial Sputum Industry Elastin products Urine, others Exacerbations CFF 100 Novel biomarkers Urine Several clinical CFF 150 From proteomic studies settings Inhaled NO Breath, others Multiplex Industry 15 NCRR Azithromycin Blood S-100a12 CFF 15 Multiplex Also, 6 CFF and/or Industry sponsored multicenter trials – 300 patients

20. Future Clinical Evaluation Enter Clinic (Ht, Wt, VS) Urine, Blood Pulmonary Function Testing Lab Sputum Induction Lab (Molecular Microbial Dx) Exam Room - Clinical Evaluation - Lung Biomarker Profile - Personalize Treatment - Enrollment in Trial Point of Service Testing