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Future of Pathology and

Laboratory Medicine

Michael Oellerich

Department of Clinical Chemistry

Georg-August-University Göttingen

Germany

www.clinchem.med.uni-goettingen.de

German universities in the 17th – 18th century

Göttingen

1737

George II, King of England and Elector of Hanover

Universities founded:

Göttingen

1737

Princeton

1746

Columbia

1754

Robert KochPhysician and Bacteriologist

1843 - 1910

Nobel Prize 1905

Physiology and Medicine

Friedrich Gustav Jacob HenleAnatomist and Pathologist

1809 - 1885

Evolution of laboratory testing in the USA

1. Hospital based labs (1920 – 1960)

2. Private labs by pathologists (1950 – 1975)

3. Commercial reference labs by industry, with sell out by pathologists, and diminution in work in hospitals (1975 – 2000)

Raslavicus P. (unpublished)

4. Commercial labs by academic centers and by consortia of entrepreneurial pathologists

(2000 - --)

5. Single purpose commercial labs to provide services on pattern lab procedures, especially in genomics (2000 - --)

6. Reincorporation of physician practices into hospital control with economic effects on independent lab revenue, and resurgence of hospital laboratory work (2005 - --)

Evolution of laboratory testing in the USA

Raslavicus P. (unpublished)

Evolution of laboratory testing at University Medical Center Göttingen (UMG)

Clinical Laboratory 1935

Clinical Laboratory 2010

Clinical Chemistry at UMG

Teaching

Department of Clinical Chemistry

ResearchCore laboratory

(routine and stat service)Proteomics

Clinical trialsUMG laboratoriesMPI

Biophysical

chemistry

External

hospitals

Multidisciplinary diagnostic network at UMG

Centre for

Bioanalytics

Cli

nic

al

Ch

emis

try

UMG – laboratories incl. TLA-platform

Hem

ato

logy

/ O

nco

logy

Med

ica

l M

icro

bio

logy

/

Vir

olo

gy

Nep

hro

logy

/ R

heu

mato

logy

Tra

nsf

usi

on

Med

icin

e

Pa

tholo

gy

(in

fu

ture

)

Laboratory Medicine is currently undergoing a rapid concentration process in Germany

Bobrowski 2008

Lab 1

Lab 2

Lab 3

Partnerships

Lab 1

Lab 2

Lab 3

Core Lab with satellite labs

Lab 2

Lab 3

Lab 1Core Lab

Market shares of leading private laboratory chains in Europe

Expenses for laboratory tests in Germany

private insurance

compulsory insurance (out-pts)

compulsory insurance (hosp. pts)

Expenses: ~6.1 bn € per year

Börmann, Asklepios, Medilys 2010

Market shares of laboratory chains in Germay

Börmann, Asklepios, Medilys 2010

Con

soli

dati

on

Total Laboratory Automation (TLA)

Information management

Preanalytics Analytics Postanalytics

• Sorting

• Centrifugation

• Distribution

• Plasma

• Blood

• CSF

• Validation

• Interpretation

• Transmission

Integration

R. Michel [email protected] 2010Trillium-Report 2008

Trend is toward increased use of automation, particularly modular and task-targeted solutions, by medical laboratories across the globe.

Integrated diagnostics

Information management

Prevention

Early diagnosis

Diagnostics Therapy

In vitro

(Laboratory medicine, Pathology)

In vivo

(Radiology, Ultrasonography)

Trillium-Report 2008

Multidisciplinary networks useful to best promote personalized medicine

Laboratory Medicine Physicians - Chemical Pathologists -in Germany

Bobrowski 2008

Practising

942

Private laboratories

516

Other

113

Hospitals

313

Hospitals with Physicians for Laboratory Medicine / Microbiology in Germany

-> Tendency to merge hospital laboratories

Bobrowski 2008

Total number of hospitals 2197

- with division for laboratory medicine 493 (22.4%)

- with laboratory medicine physicians 160 ( 7.3%)

Laboratory Medicine Physicians newly registered by the German Regional Medical Boards

Specialisation 2004 2005 2006 2007 2008 2009

Laboratory Medicine

49 47 32 51 57 42

Microbiology 27 50 13 24 28 24

Statistik Bundesärztekammer 2010

Work force crisis in laboratory medicine?

So far there is no relevant shortage of young

laboratory medicine physicians.

However, the loss of training positions at

hospitals is worrying.

Future trends and challenges in lab testing

�Heightened interest in diagnostics and laboratory testing among patients and investors

�Nevertheless health policymakers in the public and private sector continue to handle clinical pathology laboratory testing as a commodity

�Under - reimbursement for lab tests is a threat to the integrity of laboratory medicine

Robert Michel, 2010; [email protected]

Future of Pathology

Will Pathology end up in the Mausoleum of Medical Research?

A traditional discipline is bleeding dry: How to survive competition with

molecular biologists and laboratory physicians

26.09.2007

Pathology – future challenges

Major structural risks

- Pathology may be viewed by university committees primarily as a service unit rather than a high profile scientific discipline

- University chairs may no longer be guaranteed

Frankfurter Allgemeine Zeitung, 26.09.2007

Required activities to overcome these problems

• Scientific innovations in the field of pathology

• Rapid integration of new molecular and genetic analyses into diagnostics

• Development of appropriate networks to promote personalized medicine


Frankfurter Allgemeine Zeitung, 26.09.2007


Economic and social pressure is causing:

� increasing competition between disciplines for:

- diagnostic fields

- funds for equipment and personnel

- grants for research projects

� rapidly changing conditions in health care systems

and university structures

External change demands internal change

„The circumstances in our environment,

the pace of innovation,

the extraordinary flood of new technologies

– these factors are driving our

transformation.“

Pathology – need for transformation

J.N. Schwartz, CAP TODAY 2009

Sophisticated diagnostic technologies

• Mass spectrometry platforms

−e.g. Q-TOF, MALDI-TOF, Orbitrap

-> proteomics

−e.g. LC-MS/MS

-> steroids, drugs, amino acids, peptides

• Genome technologies

−gene chip platforms (e.g. Affymetrix)

−real time PCR

−next generation sequencing

(e.g. Genome Sequencer FLX)

• Cell sorting platforms

−fluorescence activated cell sorting (FACS)

−laser capture microdissection (LCM)

Mass spectrometry represents a vision forward

- a future where a revolution in clinical diagnostics

could lead to the widespread use of mass

spectrometry

Adapted from Petricoin et al., Clin. Chem. 2003; 49 : 1228 - 1229

Expanding role of tandem mass spectrometry in the clinical laboratory

LC-MS/MS

• TDM (e.g immunosuppressants, antiretroviral drugs, antidepressants)

•Drugs of abuse

•Endocrinology (e.g. steroid profiles, FT3, FT4)

•Screening of pheochromocytoma (e.g. free metanephrines)

•Newborn screening (e.g. acylcarnitines, amino acids, steroids)

•Vitamin D (25-OH-D2, 25-OH-D3)

• Peptidomics (Angiotensins, Oxytocin, ADH)

MALDI-TOF; Q-TOF

• Proteomics (research, Biomarker discovery)

Streit et al, Clin. Chem. 2002; 48:955-958

* with SPE

TDM - Simultaneous determination of immunosuppressants by LC-MS/MS

Steroid Profiles using Liquid

Chromatography–Tandem Mass Spectrometry

with Atmospheric Pressure Photoionization Source

Tiedong Guo, MS; Michael Chan, PhD; Steven J. Soldin, PhD

Arch Pathol Lab Med 2004; 128:469-475

Application of LC-MS/MS in endocrinology

Conclusion:

The improved specificity and simultaneous quantification features afforded by this method represent distinct advantages over current immunoassays.

Correlation between Tandem Mass Spectrometry and Immunoassays

nSteroid Equations

DHEAS1 y= 1.15x + 43.18 50 0.971

Cortisol2 y= 1.036x + 18.28 50 0.983

Androstenedione3 y= 1.051x + 0.769 50 0.905

Estriol4 y= 1.132x + 0.079 13 0.959

Progesterone5 y= 1.236x – 0.502 50 0.988

DHEA3 y= 1.973x + 2.063 27 0.886

11-Deoxycortisol6 y= 0.795x + 1.176 15 0.908

Testosterone6 y= 0.919x – 0.064 50 0.971

17α-Hydroxyprogesterone7 y= 1.587x + 0.123 46 0.988

Estradiol1 y= 1.436 + 0.252 43 0.969

r

1 Immunoassay, DPC Immulite; 2 Immunoassay, Bayer ADVIA Centaur; 3 Radioimmunoassay (Diagnostic Systems L.);4 ColorMetric (Bayer). Samples run in negative-ion mode; 5 Radioimmunoassay, DPC Coat-A-Count;

6 Radioimmunoassay (ICN Pharmaceuticals); 7 Extracted radioimmunoassay (DPC)

Guo et al, Arch Pathol Lab Med 2004; 128: 469-475

Relative differences between Testosterone concentrations measured with DPC RIA and LC-MS/MS

-100

-80

-60

-40

-20

0

20

40

60

80

100

0 2 4 6 8

Mean testosterone [µg/L]

Rela

tive D

iffe

rence %

male

female

Streit et al (unpublished)

men

women

Practicability and costs associated with tandem-MS or immunoassays

LC/MS-MS Immunoassay

Ease of use difficult easy

Expertise high low

Turnaround time intermediate short

Investment cost high moderate/low

Direct cost low high

Technician time intermediate short

Selectivity high intermediate/low

Availability outside working hours

restricted unrestricted

Automation low/intermediate high

Effort for test development

low/intermediate high

Adatpted from Armstrong, Oellerich, Applied Pharmacokinetics and Pharmacodynamics4th ed Lippincott Williams and Wilkins, 2006:30-39

Diagnostic molecular pathology

Categories of

proteomics

Potential

applications

Expression

proteomics

Structural

proteomicsFunctional

proteomics

Genome

project

Proteome

profiling

Stucture of

Protein complexes

Subcellular

proteomics

Biomarker

discovery

Protein-protein

interactions

Mechanisms or

functions of protein

compartmentalization/

translocation

Genomic dataProteomic data

Prove the existence of genes

Elucidate the mechanisms of diseases,

aging and protein functions

Facilitate therapy

Benefits

Integration

Proteomics and genomics integration

Adapted from Lau et al, Acta Bioch Bioph Sin 2003;35:965-975

Hortin, G. AACC 2007

The Challenge of Molecular Diversity

Genome

(25,000 Genes)

Transcriptome

(> 100,000 mRNAs)

Proteome

(> 1,000,000 ? Proteins)

Peptidome

(> 10,000,000 ? Peptides)

Polymorphisms,

Translocations

Duplications

mRNA expression

mRNA sequences

Protein concentration

Protein structure

Protein localization

TranscriptionSplicing

Proteolysis

TranslationProtein Processing

Peptide concentration

Peptide structure

Peptide localization

Molecular pathology - Genomics

• Genome-wide approaches

- gene-expression profiles

- markers from association studies

• Circulating nucleic acids (CNA)

• MicroRNAs (miRNAs)(repression of target mRNAs, impact on protein output)

Single-gene, candidate-pathway-gene and genome-wide

pharmacogenomic approaches

Cheok et al, Nature Reviews / Cancer 2006;6:117-128

Gene-expression profiles and drug sensitivity in leukemia

Cheok et al, Nature Reviews / Cancer 2006;6:117-128

Genome-wide association study

• SLCO1B1 influx transporter variants and statin-

induced myopathy

• Genomewide association study using ~ 300,000

markers in 85 subjects with myopathy and 90 controls,

all of whom were taking 80mg simvastatin daily

The SEARCH Collaborative Group, N Engl J Med 2008; 359

Trend in the association between myopathy and each SNP


Risk of myopathy associated with simvastatin


SLCO1B1 rs4149056 genotype

Conclusions – SLCO1B1 variants

- SLCO1B1 encodes OATP1B1 regulating the hepatic uptake of statins

- SLCO1B1 polymorphism affects the pharmacokinetics of active simvastatin acid

- SLCO1B1 c.521C variant allele:

• enhances risk of systemic adverse effects

• reduced uptake of simvastatin acid by OATP1B1 into the liver could reduce its cholesterol –lowering efficacy

- Clinical usefulness of SLCO1B1 variants for guiding therapy so far not verified

Circulating nucleic acids (CNA)

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing

of DNA in maternal plasma.

Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W, Leung TY, Foo CH, Xie B, Tsui NB, Lun FM, Zee BC, Lau TK, Cantor CR, Lo YM.

Proc Natl Acad Sci USA 2008; 105: 20458-63

Conclusion:

14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. This new approach is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Pharmacogenetic testing in clinical practice

� Impact on patient management

in the areas of

− oncology

− psychiatry

− infectious diseases

− cardiology

Oncology pharmacogenomics

Therapeutic resistance

CYP2D6 and tamoxifen:

DNA matters in breast cancer

Janelle M. Hoskins1,3, Lisa A. Carey2 & Howard L.McLeod

Nature Reviews Cancer 2009;9:576-586

Conclusion:

Genotype-guided tamoxifen administration may be useful to optimize treatment of breast cancer. Large clinical trials are needed before this change is advocated.

Treatment of endocrine – responsive breast cancer

Estrogen receptor inhibitor Tamoxifen

Aromatase inhibitors (e.g. Anastrozol, Letrozol, Exemestan)

Therapeutic uses

Breast cancer

- pre-, postmenopausal

- adjuvant therapy after primary treatment

- metastases

Breast cancer

- postmenopausal

- adjuvant therapy after primary treatment

- metastases

Costs

21 € (100 tablets) 573 € (100 tablets)

Polymorphisms

CYP2D6 CYP19 (?)

Inhibition by co-medications (e.g. fluoxetine)

Partial metabolic pathway of tamoxifen andits interaction with estrogen receptors

Hoskins et al, Nature 2009;9:576-586

Frequencies of CYP2D6 allelic variants

Adapted from Brockmöller et al., Pharmacogenomics 2000; 1:125-151;

Raimundo et al, Clin Pharmacol Ther 2004; 76:128Wennerholm et al, Pharmacogenetics 2001; 11:417-27

Allele Function Caucasians % Asians % Africans %

*MxN ultra rapid 1.8 1 2

*3 inactive 2 0

*4 inactive 21 1 4

*5 inactive 2 4.1 4

*6 inactive 1

*7 inactive 0.1

*9 reduced 2

*10 reduced 1.5 50 6

*17 reduced 0 34

*29 reduced 20

*41 reduced 8.4

AmpliChip CYP2D6 and CYP2C19 microarray

more than 15,000 probes CYP2D6 29 SNPs

CYP2C19 *1, *2, *3

labeled target DNA

oligonucleotide probe

***

*

2.340

285

370

650

CYP2D6 extensive metabolizer CYP2D6 poor metabolizer

(full activity) (no acitivity)

von Ahsen et al, J Lab Med 2009;33:293-301

Estrogen receptor targeted activity

by Tamoxifen and metabolites

660

290

213

600

4-hydroxy-Tamoxifen

Endoxifen

N-desmethyl-Tamoxifen

Tamoxifen

CYP2D6 stratified disease - free survival under tamoxifen treatment

Schroth et al, JAMA. 2009;302:1429

German/USA breast cancer cohort, >95% ER+, TAM treated, n=1325

CYP2D6 EM homo

unstratified cohort

CYP2D6 EM het, IM

CYP2D6 PMDis

ease

–fr

ee

su

rv

iva

l

Association between CYP2D6 inhibition and breast cancer recurrence under tamoxifen treatment

CYP2D6

no

t re

lev

an

t r

elev

an

t

TL Lash et al, Lancet Oncol 2009:10:825-33

Wegmann P. et al,

Breast Cancer Res 2005

Okishiro M. et al,

Cancer 2009

Nowell SA. et al,

Breast Cancer Res Treat 2005

Wegmann P. et al,


Goetz MP. et al,


Newman WG .et al,

Clin Cancer Res 2008

Schroth W. et al,

J Clin Oncol 2007

Ramon YC. et al,

Breast Cancer Res Treat 2009

Xu Y. et al,

Ann Oncol 2008

Kiyotani K. et al ,

Cancer Scl 2008

Tamoxifen: proposed dosage adjustments

CYP2D6 predicted phenotype

PM IM EM

Aromatase inhibitors

$ 350/ mo

(Tamoxifen 100 mg/d ? )

Tamoxifen

40 mg/d

$ 16/ mo

Tamoxifen

20 mg/d

$ 8/ mo

Weck, K. AACC 2010

Drugs for which revised labeling includes pharmacogenetic information

DrugRelevant area of medicine

Year FDA

approved revised

labeling

Approximate rate of ADRs or resistance

Gene or allele

Other drugs with

Pharmacogenetic

Relationship to gene

or allele

6-Mercaptopurine Oncology 2003 1-10% ADRs TPMTAzathioprine, 6-Thioguanine

Irinotecan Oncology 2004 30-40% ADRs UGT1A1Tropotecan, Nilotinib,

Protease Inhibitors

Tamoxifen Oncology Pending 30% resistant CYP2D6 Codeine

5-Fluorouracil Oncology Pending 20% ADRs DYPD Capecitabine

McMillin, G.A., Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Burtis C.A,

Ashwood E.R, Bruns D.E eds. 5th ed., Elsevier Philadelphia 2010; in press

Drugs for which revised labeling includes pharmacogenetic information

DrugRelevant area of medicine

Year FDA

approved revised

labeling

Approximate rate of ADRs or resistance

Gene or allele

Other drugs with

Pharmacogenetic

Relationship to gene or allele

Atomoxetine Psychiatry 2004 5-10% ADRs CYP2D6Antidepressants,

Amphetamine

CarbamazepinePsychiatry,

Neurology2007 10% ADRs HLA-B*1502

Pjenytoin,

Lamotrigine

AbacavirInfectious

Desease2007 5-8% ADRs HLA-B*5701

IsoniazidInfectious

DeseasePending NAT

Rifampin

Hydralazine

Warfarin Cardiology 2007 5-40% ADRs CYP2C9, VKORC1

Clopidogrel Cardiology 2009 30% resistant CYP2C19

Voriconazole, Omeprazole, Diazepam, Nelfinavir, Antidepressants, Tamoxifen

McMillin, G.A., Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Burtis C.A,

Ashwood E.R, Bruns D.E eds. 5th ed., Elsevier Philadelphia 2010; in press

Molecular pathology - Proteomics

Proteome analysis is a powerful tool

to investigate:

- biomarkers of disease

- antigens of pathogens

- drug target proteins

- posttranslational modifications

Dynamic Range of Plasma Proteins

From: Anderson and Anderson Mol. Cell. Prot. (2003)

Proteome of conidial surface associated proteins

of Aspergillus fumigatus reflecting potential

vaccine candidates and allergens

Abdul R. Asif 1, Michael Oellerich 1, Victor W. Amstrong 1,

Birgit Riemenschneider 2, Michel Monod 3 and Utz Reichard 2*

J Proteome Res 2006; 5:954-962

Conclusion:

Conidial surface proteins are of interest because of their possibly crucial role in triggering certain Aspergillus-related allergic diseases. Genomic information on conidia compared well with our proteomic findings.

Asif et al J. Proteome Res. 2006;5:954-962 (online version)

Novel allergens of Aspergillus fumigatus

Consistently IgE reactive proteins with individual ABPA

patients’ sera = 30 Proteins

• Secreted = 12 Proteins (7 novel)

• Cytosolic/Hyphal = 18 Proteins (12 novel)

8 novel consistently IgE reactive proteins1. Improvement of existing crude extract based ABPA diagnostic testing

2. Desensibilization strategies

Patent No. 1278/DEL/2010

Indo-German BMBF Project (IND 06/040)

Bharat et al, J Proteome Res (submitted)

Marketed small-molecule drug targets by biochemical class

Hopkins et al, Nature Reviews 2002, 1:727-730

GPCRs 30%

Ion channels 7%

Transporters 4%

Nuclear hormone

receptors 4%

Enzymes 47%

DNA 1%

Integrins 1%

Miscellaneous 2%

Others receptors 4%

GPCR, G-protein coupled receptor

Exploitable drug targets of the

human genome: ~ 600 – 1.500

Increase of

cytosolic Ca2+ DAG

PKCCa2+ dependent enzymes

NFAT NF-κκκκB AP-1

GEF (on Ras)

Erk, JNK, p38

Oellerich T et al, Mol Cell Proteomics 2009;8:1738-50

Functional Proteomics - B cell receptor (BCR) mediated signaling -

Increase of

cytosolic Ca2+ DAG

PKCCa2+ dependent enzymes

NFAT NF-κκκκB AP-1

GEF (on Ras)

Erk, JNK, p38

Oellerich T et al, Mol Cell Proteomics 2009;8:1738-50

Functional Proteomics - B cell receptor (BCR) mediated signaling -

Mutation at adaptor protein SLP-65

Biomarkers in cancer

• Biomarkers may play a greater role in the future for cancer staging, grading and selection of therapy

• Addition of individual biomarkers in the assessment of histological grade could increase the utility of grading for predicting response to therapy

• Biomarker candidates must undergo careful clinical validation

• Few markers have so far been integrated into clinical practice (e.g. HER2/neu)

Adapted from Ludwig et al, Nature reviews / Cancer 2005; 5:845-856

‘‘Water, water, every where,

Nor any drop to drink‘‘S. Coleridge, Rime of the Ancient Mariner, 1798

Molecular pathology – outlook

• Proteomics will provide new sensitive tools for: -

- early detection of neoplasms

- assessment of tumor malignancy

- discovery of novel drug target proteins

- biomarkers of diseases

• Molecular diagnostics including cytogenetics will be helpful to

further improve difficult diagnostic and management pathways

• To translate the promise into reality from bench to bedside is

the real challenge and a task for the pathologist

Proteomics - NIH Funded Grants

Rifai, N.; 2010

Future of Pathology

Future of Pathology

„The future has many names:

for the weak it is the unattainable

for the timid it is the unknown

for the courageous it is the chance“

Victor Hugo (1802 – 1885)

Pathology / Laboratory medicine – future challenges

What could be done?

→ enhance the role of pathologists as physicians and leaders in medicine

→ implement scientific innovations into diagnostics

→ ensure multidisciplinary cooperation to develop best practices for new and old technologies

→ improve harmonisation in education

→ preserve independent university departments with fundedchairs for pathology and laboratory medicine to providehigh quality education, practice and research

→ build stronger bridges between our professional societies

Role of professional societies

Promotion of laboratory medicine in

Germany by DGKL

• Foundation for pathobiochemistry and molecular diagnostics

(scholarships, grants, university chairs)

• CME

(education, training in laboratory medicine)

• Official journals (CCLM, J Lab Med)

• Reference Institute for Bioanalytics (RfB)

(quality control schemes)

• Member of DAkkS (accreditation organisation)

2010

Proteomics:

Advances in ProteinAnalysis for the Clinical Laboratory

Glen Hortin, Leigh Anderson and Steve Carr

Rifai, N.; 2010

Leading scientific journals in laboratory medicine

Rifai, N.; 2010

Leading scientific journals in laboratory medicine

World Association of Societies of Pathology and Laboratory Medicine

(WASPaLM)

45 Societies in 34 Countries

World Association of Societies of Pathology and Laboratory Medicine

(WASPaLM)

45 Societies in 34 Countries

Creation of global opportunities for

cooperation in education, research,

practice and commerce

Thank you

Documents

6c_03