Biomarker Development and Disease Pathology in Modern

Healthcare

Gyorgy Marko-Varga

Division of Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and

Industrial Electrical Engineering,Lund University, BMC C13, SE-221 84 Lund, Sweden,

and First Department of Surgery, Tokyo Medical University,

Tokyo, Japan

Outline

• Disease Biology - Peronalized Medicine• Drug & Clinical Work Flow• IRESSA Study (Asia)• Biomarker Discovery• Biobanking• Technology Platforms• Drug Imaging• Summary

Linking Disease Pathology to

Treatment-Diagnostic Land Marks

Toward Precision Medicine: Building Knowledge Network for BiomedicalResearch and a New Taxonomy of

Disease

New data network Integrates emerging researchOn the molecular makeup of diseases With clinical data on individual patients

Taxonomy” that defines disease Based on underlying molecular And environmental causes

A Century with Progression View;Long-term Transition of Elderly Population Rate

FranceGermany

England

Italia

Sweden

Korea

USA

Japan

Data: National Institute of Population and Social Security Research (2009)

21/09/2004 15

We have the tools and platforms in place for a Personalised Medicine approach

Technology Platforms•Genetics•Epidemiology•Omics•Imaging•Histolopgy•Pathology•Physiology

Data Bases and Bioinformatics

Clinical Data

Target Link to Disease Mechanism Disease Understanding

Sharing information key to future increase in productivity

Personalised Medicine

tumor

Section of FFPETissue Specimen by a MicrotomeArchived Blocks of FFPE Tissues

De-paraffined and Stained by Hematoxylin on aDIRECTOR Slide

Laser MicroDissection (LMD)

Collect Specific Tumor Cells

Solubilized by Liquid Tissueand Digest by Trypsin

Peptide Mixture Sample

LC-MS/MS “Shotgun” Analysis

Patient

Side view

Resection

BIOMARKERS in Targeted Disease Cell isolation

Medical History – Drug Treatment

Computed Tomography

Lung CancerTumor

Chronic ObstructivePulmonary Disease ”COPD”Emphysema/Inflammation

Time, min

Inte

nsi

ty,

cps

44 proteins182 MRM transitions

MRM Panels for NSCLC-Grade III

Localization with Hospital Accesss - both Clinical & Pathology

MRM in pooled plasma samples

0

10000

20000

30000

40000

50000

60000

Apo A1 ApoA4 Apo B Apo C-I Apo C-III Apo D Apo E Apo H

pe

ak

are

a M-I

M-II

F-I

F-II

Validation of Assay in Sex/Age Matched Blood Samples

Biobanking – Lung Cancer/ Cardiovascular Diseases/COPD

10 Million Samples – 5 Year Longitudinal Collection

Sample Processing

IS/IT

BirthBirth

FamilyFamily

Medical HistoryMedical History

DeathDeath

RepositoryServer/Database

Bar code identifierBar code identifier

Global ProjectGlobal Project

Access code

Access code

Chromosome AnnotationsChromosome Annotations

Reported ValueReported Value

Clinical Delivery & Disease LinkClinical Delivery & Disease Link

Patient BenefitPatient Benefit

Targeted Treatment in Asia

• Asian mutations in the EGFR are found to be as high as 40-60%

• At 10-15% in the western population, a very interesting• Observation that is proving to have medical

consequences for cancer frequency, based on a genetic background

• It is evident in Asian populations that the majority of theNSCLC treated patients with activated mutations achieveda durable and effective response with EGFR TKI-treatment –

Gefitinib (IRESSA) and Erlotinib (TARCEVA)

Case Control Study in Japan• Case-control study in Japan - Observational study of ILD • In a cohort of advanced NSCLC patients receiving treatment either with

gefitinib or other chemotherapy• Planned cohort recruitment about 4000 patients

52 Clinics involved throughout JapanProteomics performed on gefitinib treated patients that develop ILD and controls

Probably the Largest Clinical Proteomics Biomarker Activity

EGF-R Pathway – Drug Intervention

Vegvari and Marko-Varga, Chem Rev, 2010

In Vitro StudiesIn Vivo animal models

In Japan

Gefitinib : a small-moleculeEGFR-TK inhibitor

• Orally bioavailable• Selective inhibitor of

epidermal growth factor receptor tyrosine kinase (EGFR-TK)

• Competitive inhibitor of ATP bindingApoptosis

Angiogenesis

R

K

R

GefitinibGefitinib

Ligand

Membrane

Extracellular

IntracellularK

Signalling

Growth factors

Proliferation

MetastasisChemotherapy/radiation sensitivity

D N A

Patient complains of:• worsening of breathlessness• increasing cough• fever

On examination, patient has:• ‘crackles’ on chest• increased respiratory rate• increased temperature• reduced arterial oxygen

saturations <90% on air

Check chest X-ray

Check high-resolutionCT scan

Close monitoring of lungfunction, signs and

symptomsIf FEV1 or PEF isreduced, treat asexacerbation of

COPD or asthma

No parenchymalshadows

Parenchymalshadows

Investigate cause of typicalparenchymal shadows with

the following tests

Suspected orprobable ILD

Confirmed typical parenchymal shadows(especially ground glass shadows and/or linear opacities)

Fibre-opticbronchoscopy withlavage and possibly

transbronchial biopsy

Echocardiogram Contrast thoracicCT scan or test

for thrombophilia

Sputum exam and culture,blood and urine culture(bacterial antigens &

PCR)

Blood tests:WBC, Hb, urea

and/or

No evidence ofcarcinomatosis,

pathogens orgranulomas

Left ventricularfailure and/or

ischemic heartdisease

Pulmonaryembolus

seen

ElevatedWBC

Identification ofa pathogen

Failedresponse

Failedresponse

Failedresponse

Highly probableILD diagnoses

Treat withbroad-spectrum

antibiotics

Treat withheart failure

therapy Treat as apulmonary embolus

p:\proteo_data\...\case\ccs_cs01_01285 2004/11/08 17:23:10 SC1285B

RT: 5.00 - 75.00

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

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50

100

0

50

100

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100

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19.45529.7 47.53

807.4 58.87786.5

20.62575.7

59.82816.2

27.51570.2

67.93757.0

34.93671.5

19.30529.6

38.69881.5

73.33735.2

49.63946.3

57.14853.4

47.45807.8

12.06608.7

9.86580.2

34.68671.5

47.63807.4

65.701248.5

19.34529.6 66.74

725.044.02945.9

42.47808.4

20.39575.7

48.56785.4

59.79816.2

58.87786.5

26.91708.5

34.58671.6

19.22529.7

65.46821.6

10.75485.6

29.37838.728.23

603.043.25807.632.75

613.015.81529.8

23.11570.2

60.73822.1

54.62694.7

63.181248.5

34.60725.4

67.091293.0

46.60837.5

14.23620.7

7.89608.8

36.74725.4

34.69612.9

31.00671.6 45.66

807.569.601292.9

18.09529.8

36.88725.3

26.78688.0

16.63465.8

57.60694.5

65.411086.8

48.16946.4

50.39834.0

12.93462.7

29.09859.5

33.24838.6

39.30725.3 48.18

807.219.34620.5

28.07596.8

40.32585.8

72.041292.8

66.811248.550.86

837.360.18694.4

59.37786.5

17.29530.013.98

608.56.21

638.3

31.77838.7 38.27

725.327.61859.4 31.97

838.839.19585.6

26.28596.8

17.34465.7

62.44984.5

65.29821.8

50.01837.3

71.65894.8

10.37485.6

52.27859.3

58.21776.5

NL: 7.43E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS CCS_CT01_05921

NL: 5.60E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS ccs_ct01_05479

NL: 4.48E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS ccs_ct01_02176

NL: 9.66E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS ccs_ct01_03034

NL: 7.38E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS ccs_ct01_04729

NL: 5.58E6Base Peak F: ITMS + c NSI Full ms [ 450.00-2000.00] MS ccs_cs01_01285

Patient Profiles – Shotgun Sequencing

Personalized Medicine and Proteomics Lessons from Non-Small Cell Lung CancerJ.Proteome Res. 2007, 6, 2925-2935

Diagnostic Biomarkers in the ClinicProof of Mechanism/ Proof of Principle/ Proof of Concept

Rezeli...Marko-Varga, Clin Bioinf, 2011

Compound Tissue Imaging

Targeting the “HEART” of the interaction

Drug Target & Drug Compound

25

Today Optimise lung retention and correlate drug PK in lung with effect and toxicity when only total concentration of drug in lung tissue homogenate can be measured.

Business benefit of tissue imagingLocalisation of unlabelled drugs and metabolites and peptides/proteins in tissue• PK/PD – does compound distribute to target site? • Toxicity – does compound distribute specifically to affected tissue/cell type?• Drive chemistry towards

compounds with optimal distribution – FIC & BIC• Applicable to

Lung/Liver/Kidney/Brain Pathophysiology

Today Optimise lung retention and correlate drug PK in lung with effect and toxicity when only total concentration of drug in lung tissue homogenate can be measured.

Business benefit of tissue imagingLocalisation of unlabelled drugs and metabolites and peptides/proteins in tissue• PK/PD – does compound distribute to target site? • Toxicity – does compound distribute specifically to affected tissue/cell type?• Drive chemistry towards

compounds with optimal distribution – FIC & BIC• Applicable to

Lung/Liver/Kidney/Brain Pathophysiology

Tissue imaging for compound and metabolite distribution in tissues – Disease Staging

Fehniger, Marko-Varga, J. Prot.Res., 2011,11,3

GRADINGof Disease

PtN_sample1_slide6A_100629_100um_FTMS_ITMS2_332 #176 RT: 4.87 AV: 1 NL: 1.21E3T: ITMS + c MALDI w Full ms2 332.33@cid50.00 [90.00-500.00]

100 150 200 250 300 350 400m/z

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Re

lativ

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bu

nd

an

ce

240.27298

273.16840

166.21658

212.31976290.12024147.19325

331.37045124.21567 177.21432

PtN_sample1_slide6A_100629_100um_FTMS_ITMS2_332 #387 RT: 10.19 AV: 1 NL: 6.02E6T: FTMS + p MALDI Full ms [100.00-1000.00]

100 150 200 250 300 350 400m/z

0

10

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Re

lativ

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bu

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184.07297

228.26810

146.98134198.08853

237.01282 304.29901332.32956169.04671115.03611 373.13678

MS/MS: 166.2

Muscarinic Receptor Antagonist

Ipratropium

Patient K - sample 2

Parent ion (m/z 332.22)localization

Fragment ion (m/z 166.2)

Fragment ion (m/z 290.2)

The Path of Drugs & Pathology

Human Disease

Drug Treatment

Delivery: BioMarker DiagnosisBioBanking

ClinicalCharacterization

Validation

Target Biomarker

Identification Measurements

EvaluatedHuman Disease

Clinical Association

Pathology Index

In Summary

• Precision Medicine – Novel Direction• MultiPlex Protein Sequencing Assay

Platform• Personalized Medicine – Mass Spec Drug

Imaging• BioBank – High quality sample archives• Biomarkers – Biology Guidance and

prediction