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. 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) 5 10 15 20 25 30 35 40 45
50+
Age at Death (n=409)
Number of Deaths 60 80
100
Cumulative Percent

3. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
CFTR - Membrane glycoprotein
- ABC, c-AMP
- Regulates ion flux - chloride, sodium, bicarbonate
mutations (
- 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. 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
Organs/Tissue Secondarily involved
Lung
Pancreas
Intestine
Liver
Fluid or Breath,
Biomarkers of
Organ Dysfunction
and Injury
Present in
Newborns

5. 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(Phase III trials, unpublished)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
medications per day
- Oral, inhaled, injectable
- Chest Physical Therapy
- Nutritional supplementation
- Gastrostomy, central line 40 50 60 70 80 90
100 6 18 30
2001
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. 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
(log ug/ml)
Elastase 6
Months
Control
Azithro.
p=0.01 for change 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. Protein Biomarker of Pancreatic Disease in CF: Trypsinogen
200
400
600
Age (months) 24 48 72 96
120
144
N=288
IRT
ng/ml
Residual Trypsinogen at a year of age predicts
Better Lung Function throughout childhood
2004
Endorsement
1979
Validation
Benefit

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
Glutathione Blood Multiplex CFF
Clinical Trial Sputum Industry
Elastin products Urine, others Exacerbations CFF 100
Novel biomarkers Urine Several clinical CFF
From proteomic studies settings
Inhaled NO Breath, others Multiplex Industry
NCRR
Azithromycin Blood S-100a CFF
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