ROLE OF COMPOSITE INDICES AND

SYSTEMIC BIOMARKERS IN COPD

Prof. NIKOS SIAFAKAS MD,PhD,FRCP

MEDICAL SCHOOL, UNIVERSITY OF CRETE, GREECE

President of ERS 2009-2010

CONTENTS

Spirometry (FEV1)Spirometry (FEV1)

BODE indexBODE index

Biomarkers a) simpleBiomarkers a) simple

b) geneticb) genetic

Patient-centered biomarkersBODE index

Celli BR et al NEJM 2004

The BODE index, a simple multidimensional grading system, is better than the FEV1 at predicting the risk of death from any cause and from respiratory causes among patients with COPD.

Celli BR et al NEJM 2004

BODE index can be used to determine the magnitude of the effect of exacerbations on patients with COPD.

The frequency of exacerbations negatively impacted on the progression of disease severity as measured by the BODE index.

Chest 2007

The BODE index helps to stratify disease severity and its changes related to the progression of disease.

How a Biomarker is defined?

• The use of biomarkers in medicine lies in their ability to detect disease and support diagnostic and therapeutic decisions.

• As Biomarker, is defined any biochemical feature, or molecule, or material (cells, tissue), that can be measured in the body or its products, and which could reflect the disease process or predict its outcome.

• A suitable biomarker should be reproducible, sensitive, specific, easy and cheap to determine, and should present a high predictive value.

Barnes PJ et al AJRCCM 2006

Biomarkers in COPD• Several attempts to identify biomarker(s) have been made

that are related to disease pathophysiology, the

inflammatory and destructive processes .

• However, there are still significant questions:

-How the above biomarkers relate to COPD severity?

-How reproducible they are?

-How they may be affected by concurrent therapies?

-How they relate to other disease measurements, such as

FEV1 decline, exacerbation frequency, and mortality.

In INDUCED SPUTUM

• Sputum induction is a practical and safe procedure and an important research tool of local inflammation in the lung.

• Repeatability of induced sputum cellular counts and inflammatory mediators is satisfactory, even over extended periods of time and normal ranges derived from a relatively large adult population have been published.

• Induced sputum is sampled predominantly from large airways, thus, may not reflect the peripheral inflammation that may be important in COPD outcome.

Barnes PJ et al AJRCCM 2006,Chung KF ERJ 2001, Beeh KM et al CHEST 2003,

Nightingale J et al Thorax 1998.

Sputum Cellular-phase Subpopulations

↑↑↑ Neutrophils

↑↑ CD8+ T-Lymphocytes

?↑ Eosinophils

↑↑↑ Macrophages

Sputum Fluid-phase Mediators

↑ MPO, HNL, NE, ECP, IL-8, LTB-4, GRO-a, MCP-1, GM-CSF, TNF-a

↑ nitrogen oxides

↑ sputum proteases [metalloproteinases -1, -9 (MMP-1, -9)]↓ sputum anti-proteases [TIMP-1]

Biomarkers of COPD in induced sputum

Tzortzaki E, et al. Curr Med. Chem 2007

• Exhaled breath condensate (EBC) is a non-invasive method for obtaining samples from the lung.

• EBC contains several volatile chemicals (nitric oxide, carbon monoxide, hydrocarbons) and many non-volatile molecules (mediators, oxidation nitration products, peptides and cytokines), which have been used to quantify inflammation and oxidative stress in the respiratory trac

• However, there are significant methodological considerations related to variability in the degree of dilution, which does not allow precise assessment of individual solute concentrations within native airway lining fluid.

In EXHALED AIR CONDENSATE

Barnes, P.J. Pharmacol. Rev., 2004Horvath I et al ERJ 2005.

↑ CO

↑ NO

↑ pentane

↑ ethane

↑ H2O2

↑ 8-isoprostane

↑ Inteleukin-6

↑ nitrogen,↑ nitrosothiols

↑ LTB-4, PGE-2

Biomarkers of COPD, in exhaled air condensate

Tzortzaki E, et al. Curr Med. Chem 2007

In PERIPHERAL BLOOD

• Circulating neutrophils from COPD patients show enhanced respiratory burst compared with smokers with normal lung function.

• Peripheral blood mononuclear cells from COPD patients demonstrate increased chemotactic responses compared to healthy smokers and receptor studies using flow cytometry have shown that monocyte expression of CXCR2 is regulated differently in COPD patients than in healthy smokers.

• Monocyte TGF-beta production has been also found increased Circulating T-lymphocytes from COPD patients demonstrate increased apoptosis, along with up-regulation of the apoptotic pathways, TGF-beta, TNF-a and Fas.

• Peripheral blood T-lymphocyte abnormalities might be different depending on the disease phenotype, as increased CD8+ve cell numbers and decreased CD4/CD8 ratios have been associated with small airway disease.

Traves SL, et al. J Leukoc Biol, 2004Kim WD et al. Chest, 2002

Hodge SJ et al. Am J Physiol Lung Cell Mol Physiol, 2003

PERIPHERAL BLOOD

↑neutrophilic respiratory burst

↑monocytic CXCR2

↑monocytic TGF-b

↓ apoptosis of neutrophils, ↑ apoptosis of T-lymphocytes,

↓Trolox equivalent antioxidant capacity

↑ Elastin-derived peptides (EDP)

↑ fibrinogen

↑ C-reactive protein (CRP)

Biomarkers of COPD, in peripheral blood

Tzortzaki E, et al. Curr Med. Chem 2007

In URINE

• Urine sample analysis is an easy method to measure elastin breakdown and collagen degradation products (urinary desmosine: a marker of mature elastin degradation,)

• Markers of oxidative stress, like 8-hydroxydeoxyguanosine Also, 8- Isoprostane, a lipid peroxidation product was increased.

• Thromboxane levels were increased in urine samples of COPD patients, compared to controls and correlated with the degree of hypoxia.

• Issues to be considered:-methodological differences between laboratories, -minimum number of manipulations would avoid indeterminate losses of analytes, -urine markers respond to variations in the clinical conditions of the subjects.

Annovazi et al, Electrophoresis 2004

URINE

↑ urinary desmosine

↑ 8-hydroxydeoxygouanosine

↑ 8- Isoprostane

↑ Thromboxane 11-dehydro-TxB2

Biomarkers of COPD, in urine

Tzortzaki E, et al. Curr Med. Chem 2007

In BAL & BRONCHIAL BIOPSIES

Bronchoscopic biopsy provides airway wall tissue from which the morphology can be assessed, inflammatory cells can be quantified, and gene products identified.

Such studies increase our understanding of the disease process in COPD, and can contribute to the development of new treatments.

Hattotuwa K et al. Chest, 2002

↑ alveolar macrophages

↑ neutrophils

↑ CD8 lymphocytes

↑ eosinophils

↑ apoptosis mediators

↓ Toll-like recognition receptors, TLR2, TLR4 and CD14

↓ Histone deacetylases (HDACs)

↑ IL-6, IL-8, MPO (myeloperoxidase), GRO-a, GSH (glutathione),

↑ Collagenase 1 and 2 (MMP-1, MMP-8) and gelatinases A and B (MMP-2 , MMP-9)

↓ TGF- beta 1 and tissue inhibitors of MMP (TIMP)

↑ Transcription factor signal transducer and activator of transcription (STAT)-4

↓ thiol

↑ Nuclear factor- κB (NF-κB)

↑ Epithelial-derived neutrophil attractant-78 (CXCL5), interleukin-8(CXCL8), CXR receptor 1 and 2

Overview of Biomarkers of COPD, in bronchial biopsies and BALF.

Genetic Markers

Pathway Candidate Gene Chromosomal region

Inflammatory orchestration TNF-αIL-8Vitamin-D-binding proteinIL 11IL-1 familyImmunoglobulinsHLA

6p2114q4q11-q139p132q12-2q1414q326p

Parenchymal destruction protease / antiprotease imbalance

AAT, AACTSerpine2MMP-1,-2, -9, -12Proteinase 3TIMP1-4Alpha2-macroglobulinSLPIADAM33PAR-2TGF-B1

14q32.12q 11q2219p13.33p254p16.320q12-13. 220p135q1319q13.13

Mucociliary clearance CFTRMucins

77q22 and 11p15.5

Oxidative stress EC-SODGlutathione S TransferaseMicrosomal epoxide hydrolaseCYP1A1

21q22.16p12.21q42.115q22-q24

Pathway and candidate genes, by chromosomal region associated with COPD.

Sampsonas F. et al. Curr. Med. Chem 2006; Tzortzaki et al, CCA 2006; Hersch et al, AJRCMB 2005

COPD & Markers of GENETIC INSTABILITY

Cell Death Cascades

Oxidative DNA damage of LEBCs

Somatic mutations of LEBCs

Aberrant Host Immune Response Towards “damaged” LEBCs

Oxidative stress

Figure 1.

Inactivation of DNA mismatch repair

THE PROPOSED MODEL FOR THE INITIATION OF COPD

COPD (59) 23%

Healthy (60) 0%

MSI

Sag

aGT

MSI143bp 151bp

141bp 147bp

BL DNA

D6S344

SP DNABL SP

Microsatellite DNA instability in COPD

MSI

0

10

20

30

40

50

60

70

80

%

po

sit

ive

MS

I

COPD ASTHMA

p<0.001

COPD vs Asthma COPD vs Asthma :: MSI MSI

Zervou et al, ERJ 2006

0

10

20

30

40

50

60

70

%

po

sit

ive

MS

I

COPD ASTHMA

Mild Moderate Severe Very Severe

49.2

22.2

Makris et al, ERJ 2008

MSI positive MSI negative0

1

2

3

4

5

Exacerbations among COPD patients exhibited MSI in sputum cells

p= 0.02

Exa

cerb

atio

ns

(n)

• The annual rate of

severe exacerbations

was greater for those

exhibited MSI (p=0.02).

• The rate was

significantly greater in

those subjects exhibited

MSI in perforin related

G29802 marker [p=0.03].

MSI & COPD EXACERBATIONS: Conclusions

Makris et al, ERJ 2008

Conclusions• COPD lacks established and viable biomarkers to

monitor disease progression, severity, clinical subtypes, or response to therapy.

• Many candidate biomarkers were evaluated revealing a high degree of redundancy.

• A recent meta-analysis of over 652 published studies, including 146.255 COPD patients, suggested that few of these biomarkers have been validated, and there is little information about reproducibility and the relationship to disease development, severity, or progression (Luigi G et al. Pulm Pharm Therap 2006)

University Hospital, Heraklion Crete

Prof. N. M. Siafakas

E. TzortzakiN. Tzanakis

E. VlachakiM. SiganakiM. TsoumakidouM. ParaskakisM. ZervouK. SamaraA. KaratzanisD. Makris

Molecular Pulmonology Research Laboratory