Transcript Current Biomarker Knowledge and Clinical Practice

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)
Cystic Fibrosis
25
100
20
80
15
60
10
40
5
20
0
0
5
10
15
20
25
30
35
Age at Death (n=409)
40
45
0
50+
Cumulative Percent
Number of Deaths
• 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)
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)
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
Fluid or Breath,
Present in
Pancreas
Biomarkers of
Newborns
Intestine
Organ Dysfunction
Liver
and Injury
Lung Disease in CF: Clinical Course
• Decline in Lung function 100
(CFF registry, 2001)
90
• Acute Exacerbations
80
70
- personal, economic impact
60
- ? decline in lung function.
50
(Phase III trials, unpublished)
40
6
• Structural Lung Injury
• Daily treatment
- 3-25 medications per day
- Oral, inhaled, injectable
- Chest Physical Therapy
- Nutritional supplementation
- Gastrostomy, central line
2001
18
Years
3
CF Pathophysiology
Neutrophils
Epithelial
Cells
Lymphocytes
(Hubeau et al, 2001)
Macrophages
(Durieu et al, 1998)
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
History
Clinical
Yes
Research Comment
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
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
Antibodies
No
Uncertain utility
- Quantitative
Cultures
Yes
Good Utility
- Sputum
Inflammation
No
Cell Count, IL-8, elastase
(some research utility,
?sensitivity)
Protein Biomarkers for CF Lung Disease
Need
Existing
Biomarker(s)
I. Risk for Rapid decline in lung function
No
I. Ongoing Lung Structural Injury
(Fibrosis, Elastolysis, Remodeling)
No
I. Identification of Infection
- Pseudomonas aeruginosa
- MRSA (Methicillin Resistant Staph.)
- Burkholderia cepacia
- NTM – M. avium, M. abscessus
- Fungal species
No
I. Clinical Trials of Antiinflammatories
- Stratification
- Efficacy
? Sputum
Elastase,
IL-8, LTB4
Protein Biomarkers for CF Lung Disease
Need
Existing
Biomarker(s)
II. Exacerbation
- Identification
- Susceptibility to exacerbations
No
No
II. Response to treatment
No
II. Toxicity with treatment
No
II. Staging
No
II. Newborn Screening
Yes
Protein Biomarkers for CF Lung Disease
Need
Existing
Biomarker(s)
Clues to Pathogenesis
- Intracellular or Membrane
(CFTR Presence or Function)
No?
- Key Pathway (Inflammation,
Fibrosis, Antimicrobial Defense)
in Organ Dysfunction
No
- Diagnosis in Atypical Cases
(Disease Susceptibility)
No
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
Protein Biomarkers of CF Lung
Disease: Sputum
Elastase
(log ug/ml)
• Putative Biomarkers
- Proteolytic
- Cytokines, growth factors
- Oxidant/antioxidant
- Surfactant proteins
• Research Utility
- Azithromycin study
- Intravenous antibiotics
- Interferon gamma study
- Hypertonic saline study
2
Control
Azithro.
1
p=0.01 for change
0
0
Months
6
(Saiman et al., JAMA, 2003)
Protein Biomarkers of CF Lung
Disease: Blood/Inflammation
• Candidates: CRP, cytokines, growth factors, albumin,
oxidant/antioxidant, S-100,
• Mostly exacerbation studies
Biomarker Development
1. Proteomic Experiments
Choose
Form
Validation
Many Steps
Make
ELISA
2. Multiplex Platforms
Many Steps
• Microsphere - Luminex
• Protein Arrays – Randox
• Small Sample Volume, Parallel analyses
• 20 Mediators, Candidates can be added
Application
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
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
Protein Biomarker of Pancreatic
Disease in CF: Trypsinogen
IRT
ng/ml
N=288
600
400
200
0
0
24 48 72 96 120 144
Age (months)
• Residual Trypsinogen at a year of age predicts
Better Lung Function throughout childhood
1979
Validation
Benefit
2004
Endorsement
Samples for Protein Biomarker
Identification
Sample
.
Rapid
Prog.
Lung
Pathogenesis
Destruct.
BAL
+
+
++++
Sputum
+++
+++
+++
Exhaled gases
?
?
Breath Cond.
?
Blood
Stratif
Outcome
( Clin Trials)
Exacerb.
+
++
++
+++
+++
+++
?
?
?
?
?
?
?
?
?
?
?
?
?
?
++
Urine
+
++
?
?
?
Saliva
+
?
?
?
?
+
+
Epithelium, Circulating Cells – protein expression
Current Protein Biomarker Projects
Local
Biomarkers annual
Biomarkers annual
Specimen
Blood
Features
Support
N
Multiplex
(Luminex) (Randox)
NCRR
NHLBI
300
Multiplex
NCRR
35
Sputum
Glutathione
Clinical Trial
Blood
Sputum
Multiplex
CFF
Industry
30
Elastin products
Urine, others
Exacerbations
CFF
100
Several clinical
settings
CFF
150
Industry
NCRR
15
CFF
15
Novel biomarkers
Urine
From proteomic studies
Inhaled NO
Azithromycin
Breath, others Multiplex
Blood
S-100a12
Multiplex
Also, 6 CFF and/or Industry sponsored multicenter trials – 300 patients
Future Clinical Evaluation
Enter Clinic (Ht, Wt, VS)
Urine, Blood
Pulmonary Function Testing Lab
Sputum Induction Lab
(Molecular Microbial Dx)
Point of Service Testing
Exam Room
- Clinical Evaluation
- Lung Biomarker
Profile
- Personalize Treatment
- Enrollment in Trial