Current Biomarker Knowledge and Clinical Practice in Cystic Fibrosis

Current Biomarker Knowledge and Current Biomarker Knowledge and Clinical Practice in Cystic FibrosisClinical Practice in Cystic Fibrosis

Gap Analysis of Knowledge and Practice: Gap Analysis of Knowledge and Practice:

• • Prioritize CF Disease Aspects in Need of Prioritize CF Disease Aspects in Need of BiomarkersBiomarkers

• • Generate List of Patient Tissue/Fluid Sample Generate List of Patient Tissue/Fluid Sample Types Types and Relationship to Disease Aspects and Relationship to Disease Aspects

Overview:Overview:• Review of Cystic Fibrosis Emphasizing Review of Cystic Fibrosis Emphasizing BiomarkersBiomarkers

• Lung Disease Lung Disease

• Suggestions, RecommendationsSuggestions, Recommendations

F. Accurso, MD, CF Center Director and F. Accurso, MD, CF Center Director and Professor of Pediatrics, University of Colorado, Denver, USAProfessor of Pediatrics, University of Colorado, Denver, USA

(CF Foundation, NHLBI, NIDDK, NCRR, CF Community)(CF Foundation, NHLBI, NIDDK, NCRR, CF Community)

Cystic FibrosisCystic Fibrosis• Definition: Multisystem, genetic disorder leading Definition: Multisystem, genetic disorder leading to to early death, primarily from progressive lung early death, primarily from progressive lung disease.disease.• 30,000 individuals in US30,000 individuals in US• Median life expectancy - 35 years (CFF registry, Median life expectancy - 35 years (CFF registry, 2004) 2004) • Median age at death - 26 years (CFF registry, Median age at death - 26 years (CFF registry, 2001) 2001)

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• CFTR CFTR - Membrane glycoprotein - Membrane glycoprotein - ABC, c-AMP- ABC, c-AMP - Regulates ion flux - chloride, sodium, - Regulates ion flux - chloride, sodium,

bicarbonate bicarbonate - 1400 mutations (- 1400 mutations (www.genet.sickkids.on.ca/cftr/)www.genet.sickkids.on.ca/cftr/)

- Delta F508 (70% of alleles in US)- Delta F508 (70% of alleles in US)

• Diagnosis Diagnosis (CFF consensus conference, J (CFF consensus conference, J Pediatr,1998)Pediatr,1998) - Symptoms and Signs- Symptoms and Signs - Physiology - evidence of CFTR dysfunction- Physiology - evidence of CFTR dysfunction (sweat electrolytes, nasal potential (sweat electrolytes, nasal potential difference)difference) - - Genotype – Two defined mutationsGenotype – Two defined mutations

- Family History- Family History - “Positive Newborn Screen”- “Positive Newborn Screen”

((Protein BiomarkerProtein Biomarker -Immunoreactive -Immunoreactive Trypsinogen)Trypsinogen)

Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Transmembrane Conductance

Regulator (CFTR)Regulator (CFTR)

CFTR: Need for Protein CFTR: Need for Protein BiomarkersBiomarkers

• Diagnosis in Atypical CasesDiagnosis in Atypical Cases

• Clinical Trial - Proof of ConceptClinical Trial - Proof of Concept

Cellular Biomarkers Cellular Biomarkers of CFTR Presenceof CFTR Presenceand/or Functionand/or Function

Cells directly involved Cells directly involved in CFTR pathophysiologyin CFTR pathophysiology

(Airway Epithelium, Sweat Gland)(Airway Epithelium, Sweat Gland)

Dysfunctional CFTRDysfunctional CFTR

Organs/Tissue Secondarily involved Organs/Tissue Secondarily involved LungLung PancreasPancreas IntestineIntestine

LiverLiver

Fluid or Breath, Fluid or Breath, Biomarkers ofBiomarkers ofOrgan DysfunctionOrgan Dysfunctionand Injuryand Injury

Present inPresent inNewbornsNewborns

Lung Disease in CF: Clinical Lung Disease in CF: Clinical CourseCourse

• Decline in Lung functionDecline in Lung function

• Acute ExacerbationsAcute Exacerbations - personal, economic impact- personal, economic impact - ? decline in lung function. - ? decline in lung function. (Phase III trials, (Phase III trials, unpublished)unpublished)

• Structural Lung InjuryStructural Lung Injury

• Daily treatment Daily treatment - 3-25 medications per day- 3-25 medications per day - Oral, inhaled, injectable - Oral, inhaled, injectable - Chest Physical Therapy- Chest Physical Therapy - Nutritional - Nutritional supplementationsupplementation - Gastrostomy, central line- Gastrostomy, central line

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(CFF registry, 2001)

CF Pathophysiology CF Pathophysiology

NeutrophilsNeutrophils

LymphocytesLymphocytes(Hubeau et al, 2001)(Hubeau et al, 2001)

MacrophagesMacrophages(Durieu et al, 1998)(Durieu et al, 1998)

Epithelial Epithelial CellsCells

Lung Disease in CF: AssessmentLung Disease in CF: Assessment

AssessmentAssessment Clinical Clinical Research CommentResearch Comment HistoryHistory Yes Yes Needs Refinement Needs Refinement (CFFTDN)(CFFTDN)

Physical ExamPhysical Exam Yes Yes Not quantitativeNot quantitative

OO22 Saturation Saturation Yes Yes AcceptableAcceptable

Lung FunctionLung Function Yes Yes Rapidly Improving Rapidly Improving (CFFTDN)(CFFTDN)

ImagingImaging - x-ray- x-ray Yes Yes Scoring systemsScoring systems

- CT scan- CT scan Yes Yes Quantitation developing Quantitation developing

- VQ scan- VQ scan No No Not as outcome Not as outcome measuremeasure

- Mucociliary Cl. No- Mucociliary Cl. No Needs Needs standardizationstandardization

We do not have a biomarker (biochemical index) or We do not have a biomarker (biochemical index) or panel panel of biomarkers that is carefully defined for any of biomarkers that is carefully defined for any Pulmonary clinical use in CF. Pulmonary clinical use in CF.

AssessmentAssessment Clinical Clinical Research CommentResearch Comment

LaboratoryLaboratory

- CBC, ESR- CBC, ESR Yes Yes Acute Acute ExacerbationExacerbation

- CRP- CRP Yes Yes Acute ExacerbationAcute Exacerbation

- IgE- IgE Yes Yes Allergic Allergic BronchopulmonaryBronchopulmonary AspergillosisAspergillosis

- Pseudomonas - Pseudomonas No Uncertain utility No Uncertain utility AntibodiesAntibodies

- Quantitative - Quantitative Yes Yes Good UtilityGood Utility CulturesCultures

- Sputum - Sputum No No Cell Count, IL-8, Cell Count, IL-8, elastaseelastase InflammationInflammation (some research (some research utility,utility,

?sensitivity)?sensitivity)

Lung Disease in CF: AssessmentLung Disease in CF: Assessment

Protein Biomarkers for CF Lung DiseaseProtein Biomarkers for CF Lung DiseaseExistingExistingNeedNeed

Biomarker(s)Biomarker(s)

I. Risk for Rapid decline in lung functionI. Risk for Rapid decline in lung function No No

I. Ongoing Lung Structural Injury I. Ongoing Lung Structural Injury No No (Fibrosis, Elastolysis, Remodeling) (Fibrosis, Elastolysis, Remodeling)

I. Identification of InfectionI. Identification of Infection No No - - Pseudomonas aeruginosaPseudomonas aeruginosa - MRSA (Methicillin Resistant Staph.) - MRSA (Methicillin Resistant Staph.) - - Burkholderia cepaciaBurkholderia cepacia - NTM – - NTM – M. aviumM. avium, , M. abscessusM. abscessus - Fungal species- Fungal species

I. Clinical Trials of AntiinflammatoriesI. Clinical Trials of Antiinflammatories ? ? SputumSputum - Stratification- Stratification Elastase, Elastase, - Efficacy- Efficacy IL-8, IL-8, LTB4LTB4

Protein Biomarkers for CF Lung DiseaseProtein Biomarkers for CF Lung Disease

ExistingExistingNeedNeedBiomarker(s)Biomarker(s)

II. ExacerbationII. Exacerbation - Identification - Identification No No - Susceptibility to exacerbations- Susceptibility to exacerbations No No

II. Response to treatmentII. Response to treatment No No

II. Toxicity with treatment II. Toxicity with treatment No No

II. StagingII. Staging No No

II. Newborn ScreeningII. Newborn Screening Yes Yes

Protein Biomarkers for CF Lung DiseaseProtein Biomarkers for CF Lung Disease

ExistingExistingNeedNeedBiomarker(s)Biomarker(s)

Clues to PathogenesisClues to Pathogenesis

- Intracellular or Membrane- Intracellular or Membrane No? No? (CFTR Presence or Function)(CFTR Presence or Function)

- Key Pathway (Inflammation,- Key Pathway (Inflammation, No No Fibrosis, Antimicrobial Defense) Fibrosis, Antimicrobial Defense) in Organ Dysfunctionin Organ Dysfunction

- Diagnosis in Atypical Cases - Diagnosis in Atypical Cases No No (Disease Susceptibility)(Disease Susceptibility)

Search for Protein Biomarkers Search for Protein Biomarkers in CF Lung Disease: Summaryin CF Lung Disease: Summary

• Neutrophil Associated MoleculesNeutrophil Associated Molecules

• None have been explored in large populationsNone have been explored in large populations

• Panels of more than five markers are only now Panels of more than five markers are only now being investigated. (little multiplex data or “cluster”, being investigated. (little multiplex data or “cluster”, “ “network” or “pattern” analysis)network” or “pattern” analysis)

• Proteomic studies are appearingProteomic studies are appearing

• Bronchoalveolar Lavage Bronchoalveolar Lavage - Clinical Utility – Microbial diagnosis- Clinical Utility – Microbial diagnosis

- Clues to Pathogenesis- Clues to Pathogenesis

Protein Biomarkers of CF Lung Protein Biomarkers of CF Lung Disease: SputumDisease: Sputum

• Putative BiomarkersPutative Biomarkers

- Proteolytic- Proteolytic

- Cytokines, growth - Cytokines, growth factorsfactors

- Oxidant/antioxidant- Oxidant/antioxidant

- Surfactant proteins- Surfactant proteins

• Research Utility Research Utility - Azithromycin study- Azithromycin study

- Intravenous - Intravenous antibioticsantibiotics

- Interferon gamma - Interferon gamma studystudy

- Hypertonic saline - Hypertonic saline studystudy

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ControlControlAzithro.Azithro.

p=0.01 for changep=0.01 for change

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(Saiman et al., JAMA, 2003)

Protein Biomarkers of CF Lung Protein Biomarkers of CF Lung Disease: Blood/InflammationDisease: Blood/Inflammation

• Candidates: CRP, cytokines, growth factors, albumin, Candidates: CRP, cytokines, growth factors, albumin, oxidant/antioxidant, S-100, oxidant/antioxidant, S-100,

• Mostly exacerbation studiesMostly exacerbation studies

Biomarker DevelopmentBiomarker Development ValidationValidation ApplicationApplication

1. Proteomic Experiments Many Steps 1. Proteomic Experiments Many Steps

Choose MakeChoose Make Form ELISA Form ELISA

2. Multiplex Platforms2. Multiplex Platforms Many Steps Many Steps• Microsphere - LuminexMicrosphere - Luminex• Protein Arrays – RandoxProtein Arrays – Randox• Small Sample Volume, Parallel analysesSmall Sample Volume, Parallel analyses• 20 Mediators, Candidates can be added20 Mediators, Candidates can be added

Protein Biomarkers of CF Lung Protein Biomarkers of CF Lung Disease: ElastolysisDisease: Elastolysis

• Elastin Breakdown Products Elastin Breakdown Products - Urinary Desmosine, Isodesmosine (Stone et al. - Urinary Desmosine, Isodesmosine (Stone et al. AJRCCM, 1995)AJRCCM, 1995)

• Renewed interest (Ma et al, PNAS, 2003)Renewed interest (Ma et al, PNAS, 2003) - Demonstration in sputum, blood- Demonstration in sputum, blood - Measurement of Free and Polymerized Forms- Measurement of Free and Polymerized Forms • Mass Spec improves Mass Spec improves

sensitivity – 100 pgsensitivity – 100 pg

• 9 year old with CF 9 year old with CF well visitwell visit

Additional Biomarkers in CFAdditional Biomarkers in CF

• Breath – Proteins in very low abundance Breath – Proteins in very low abundance - Volatile gases, pH- Volatile gases, pH- Nitric oxide related compounds- Nitric oxide related compounds- - PseudomonasPseudomonas markers (PNAS, 2005) markers (PNAS, 2005)

• Nutrition – Very abnormal in CFNutrition – Very abnormal in CF- Albumin, Retinol Binding Protein- Albumin, Retinol Binding Protein- Fat soluble vitamins, Carotene- Fat soluble vitamins, Carotene- May confound interpretation of results- May confound interpretation of results

• Liver Disease – Focal sclerosisLiver Disease – Focal sclerosis- Cytokines, Growth Factors, Elastin - Cytokines, Growth Factors, Elastin

productsproducts- Confound Pulmonary analyses- Confound Pulmonary analyses

Protein Biomarker of Protein Biomarker of Pancreatic Disease in CF: Pancreatic Disease in CF:

TrypsinogenTrypsinogen

• Residual Trypsinogen at a year of age predicts Residual Trypsinogen at a year of age predicts Better Lung Function throughout childhoodBetter Lung Function throughout childhood

20042004 EndorsementEndorsement

19791979BenefitBenefitValidationValidation

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IRTIRTng/mlng/ml

N=288N=288

Samples for Protein Biomarker Samples for Protein Biomarker IdentificationIdentification

Sample Rapid Lung Pathogenesis Stratif Outcome Exacerb. Prog. Destruct. ( Clin Trials)

BALBAL + + ++++ + ++ +++ + ++++ + ++ ++

SputumSputum +++ +++ +++ +++ +++ ++++++ +++ +++ +++ +++ +++

Exhaled gases ?Exhaled gases ? ? ? ?? ? ? ? ? ? ?

Breath Cond.Breath Cond. ? ? ? ? ?? ? ? ? ? ? ?

Blood Blood ? ? ? ? ? ++ ? ? ? ? ? ++

Urine + ++ + ? ? ?Urine + ++ + ? ? ?

SalivaSaliva + + ? ? + ?+ ? ? ?? ?

Epithelium, Circulating Cells – protein expressionEpithelium, Circulating Cells – protein expression

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Current Protein Biomarker Current Protein Biomarker ProjectsProjects

LocalLocal SpecimenSpecimen FeaturesFeatures SupportSupport NNBiomarkers annualBiomarkers annual BloodBlood Multiplex Multiplex NCRR NCRR 300300 (Luminex)(Luminex) (Randox) (Randox) NHLBI NHLBI

Biomarkers annualBiomarkers annual Sputum Sputum Multiplex Multiplex NCRR NCRR 3535

Glutathione Glutathione Blood Blood MultiplexMultiplex CFF CFF 3030Clinical TrialClinical Trial Sputum Sputum Industry Industry

Elastin products Urine, othersElastin products Urine, others Exacerbations Exacerbations CFF CFF 100100

Novel biomarkers Urine Novel biomarkers Urine Several clinical CFF Several clinical CFF 150150

From proteomic studies From proteomic studies settingssettings

Inhaled NO Inhaled NO Breath, others MultiplexBreath, others Multiplex Industry 15 Industry 15 NCRRNCRR

AzithromycinAzithromycin Blood Blood S-100a12 S-100a12 CFF CFF 1515

MultiplexMultiplex Also, 6 CFF and/or Industry sponsored multicenter trials – 300 patientsAlso, 6 CFF and/or Industry sponsored multicenter trials – 300 patients

Future Clinical EvaluationFuture Clinical Evaluation

Enter Clinic (Ht, Wt, VS) Enter Clinic (Ht, Wt, VS)

Pulmonary Function Testing Lab Pulmonary Function Testing Lab

Sputum Induction LabSputum Induction Lab (Molecular Microbial Dx)(Molecular Microbial Dx)

Exam RoomExam Room- Clinical Evaluation- Clinical Evaluation- Lung Biomarker- Lung Biomarker ProfileProfile- Personalize Treatment- Personalize Treatment- Enrollment in Trial- Enrollment in Trial

Urine, BloodUrine, Blood

Point of Service TestingPoint of Service Testing