Symposium 18 Tools for Measuring Early Lung Disease Proteomic biomarkers of Lower Airway Disease Frank Accurso, MD Professor of Pediatrics CF Center Director University of Colorado Denver, Colorado, USA

Lung Disease in CF: Clinical Course Decline in Lung function Decline in Lung function Acute Exacerbations Acute Exacerbations Structural Lung Injury Structural Lung Injury Years (CFF registry, 2001)

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

Proteomic Biomarkers of Lower Airway Disease: Overview Protein Biomarkers of disease Protein Biomarkers of disease Proteolytic and Oxidative Injury in the CF airway Proteolytic and Oxidative Injury in the CF airway Should the lower airway be sampled in clinic? Should the lower airway be sampled in clinic? Take home message Take home message – There is compelling evidence that proteolytic and – There is compelling evidence that proteolytic and oxidative damage occur early in CF indicating oxidative damage occur early in CF indicating the need for early treatment. the need for early treatment.

Biomarkers and Proteomics Biomarker Biomarker A characteristic that is objectively measured and A characteristic that is objectively measured and evaluated as an Indicator of normal biologic or evaluated as an Indicator of normal biologic or pathogenic process or Pharmacological responses pathogenic process or Pharmacological responses to a Therapeutic intervention ( to a Therapeutic intervention ( Proteomics The comprehensive, complete qualitative and The comprehensive, complete qualitative and quantitative description of proteins in a biological quantitative description of proteins in a biological sample. sample.

Protein Biomarkers in CF Need Biomarker(s) 1. Clues to Pathogenesis? 2. Rapid decline in lung function No 3. Ongoing Lung Structural Injury No 4. Identification of Infection No 5. Exacerbation a. Identification No a. Identification No b. Susceptibility No b. Susceptibility No 6. Response to treatment No 7. Clinical Trials a.StratificationNo a.StratificationNo b. Efficacy ?Elastase, IL-8, LTB4 b. Efficacy ?Elastase, IL-8, LTB4 8. Toxicity with treatment No 9. StagingNo 10. Newborn ScreeningYes

Protein Biomarkers from Proteomics Potential Protein Biomarkers - Western Blot - ELISA - activity cytokines proteases Proteome Proteins identified ElastaseCathepsinMMPs Proteinase K Proteomic Experiment Mass Spectrometry Limitations - protein size - protein size - abundance - abundance Panels of Protein Biomarkers - Statistical Techniques are improving. are improving. - Difficult to apply laboratory validation laboratory validation techniques to panels. techniques to panels. Ollero et al., Proteomics, 2006

Cytokines are plentiful in the CF airway Armstrong et al, 2005

Clustering distances between two observations Euclidean distances are roots of sums of squares distances (the distance between two observations in n- dimensional space with n variables) Manhattan distance is not a straight line & larger R. Deterding and S. Heltshe

Linkage Methods in clustering Single Linkage: Individuals are merged with another individual that has the least distance. That cluster is then merged with the most similar cluster, etc.. Complete Linkage: At each merge, the distance between clusters is determined by the distance between the two elements (one from each cluster) that are most distant. Average Linkage: At each merge, the distance between clusters is determined by the average distance between all pairs of items (where one member of a pair belongs to each cluster) R. Deterding and S. Heltshe

Proteases are greatly increased in the early CF airway Armstrong, 2005

Proteolytic Damage in the CF airway Complement receptor – M. Berger, 1989 Complement receptor – M. Berger, 1989 Surfactant Protein A – Rubio, 2004, Von Bredow, Griese, 2003, Surfactant Protein A – Rubio, 2004, Von Bredow, Griese, 2003, Alpha one antitrypsin – Sloane, 2005 Alpha one antitrypsin – Sloane, 2005 IgG - Sloane, 2005 IgG - Sloane, 2005 Phosphaphosphatidylserine receptor, CD36, - apoptosis Phosphaphosphatidylserine receptor, CD36, - apoptosis - Vandivier,2002 Sloane, 2005 Alpha one antitrypsin

Oxidative Damage in the CF Airway Chlorinated tyrosines in BAL Kettle, 2004 Protein carbonyls - Starosta, Griese, 2006 Protein carbonyls - Starosta, Griese, 2006 Lipid Hydroperoxides – Hull, 1997 Lipid Hydroperoxides – Hull, 1997 Active myeloperoxidase – Kettle, 2004 Active myeloperoxidase – Kettle, 2004

Antibiotic treatment does not necessarily decrease inflammation in CF Tobi treatment eradicated Pseudomonas but did not change airway inflammation (Gibson, 2003)

Microbial Diversity in an 8 year old with CF (culture negative) Culture neg Treatable Treatable 61 patients – 7 cx negative (6 identified) - 4 additional microbes - 4 additional microbes

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

Proteomic Biomarkers of Lower Airway Disease: Overview Protein Biomarkers of disease Protein Biomarkers of disease Proteolytic and Oxidative Injury in the CF airway Proteolytic and Oxidative Injury in the CF airway Should the lower airway be sampled in clinic? Should the lower airway be sampled in clinic? Take home message Take home message – There is compelling evidence that proteolytic and – There is compelling evidence that proteolytic and oxidative damage occur early in CF indicating oxidative damage occur early in CF indicating the need for early treatment. the need for early treatment.