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Future of Pathology and Laboratory Medicine Michael Oellerich Department of Clinical Chemistry Georg-August-University Gรถttingen Germany www.clinchem.med.uni-goettingen.de


German universities in the 17th – 18th century

GĂśttingen 1737


Universities founded: Gรถttingen 1737 Princeton 1746 Columbia 1754

George II, King of England and Elector of Hanover


Friedrich Gustav Jacob Henle Anatomist and Pathologist 1809 - 1885

Robert Koch Physician and Bacteriologist 1843 - 1910

Nobel Prize 1905 Physiology and Medicine


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Raslavicus P. (unpublished)


Evolution of laboratory testing in the USA 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with economic effects on independent lab revenue, and resurgence of hospital laboratory work (2005 - --) Raslavicus P. (unpublished)


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

Clinical Laboratory 2010

Clinical Laboratory 1935


Clinical Chemistry at UMG Teaching

Department of Clinical Chemistry

Core laboratory (routine and stat service)

External hospitals

UMG laboratories

Research

Clinical trials

Proteomics

MPI Biophysical chemistry


Pathology (in future)

Transfusion Medicine

Nephrology/ Rheumatology

Medical Microbiology / Virology

Hematology / Oncology

Clinical Chemistry

Multidisciplinary diagnostic network at UMG

UMG – laboratories incl. TLA-platform Centre for Bioanalytics


Laboratory Medicine is currently undergoing a rapid concentration process in Germany Lab 1 Lab 2

Partnerships

Lab 1

Lab 3

Lab 3

Core Lab with satellite labs

Core Lab

Lab 1

Lab 2 Lab 3 Lab 2 Bobrowski 2008


Market shares of leading private laboratory chains in Europe


Expenses for laboratory tests in Germany Expenses: ~6.1 bn â‚Ź per year private insurance compulsory insurance (out-pts) compulsory insurance (hosp. pts)

BĂśrmann, Asklepios, Medilys 2010


Market shares of laboratory chains in Germay

Bรถrmann, Asklepios, Medilys 2010


Total Laboratory Automation (TLA)

Consolidation

Information management Preanalytics

Analytics

Postanalytics

• Sorting • Centrifugation • Distribution

• Plasma • Blood • CSF

• Validation • Interpretation • Transmission

Integration Trend is toward increased use of automation, particularly modular and task-targeted solutions, by medical laboratories across the globe. R. Michel info@DarkDaily.com 2010 Trillium-Report 2008


Integrated diagnostics

Information management Prevention Early diagnosis

Diagnostics

Therapy

In vitro

In vivo

(Laboratory medicine, Pathology)

(Radiology, Ultrasonography)

Multidisciplinary networks useful to best promote personalized medicine Trillium-Report 2008


Laboratory Medicine Physicians - Chemical Pathologists in Germany Practising 942

Private laboratories

Hospitals

Other

313

113

516

Bobrowski 2008


Hospitals with Physicians for Laboratory Medicine / Microbiology in Germany Total number of hospitals

2197

- with division for laboratory medicine 493

(22.4%)

- with laboratory medicine physicians 160

( 7.3%)

-> Tendency to merge hospital laboratories Bobrowski 2008


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 Bundes채rztekammer 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; info@DarkDaily.com


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


Pathology – future challenges 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


Pathology – future challenges 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


Pathology – need for transformation

„The circumstances in our environment, the pace of innovation, the extraordinary flood of new technologies – these factors are driving our 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)


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

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


Application of LC-MS/MS in endocrinology

Steroid Profiles using Liquid Chromatography–Tandem Mass Spectrometry with Atmospheric Pressure Photoionization Source Tiedong Guo, MS; Michael Chan, PhD; Steven J. Soldin, PhD

Conclusion: The improved specificity and simultaneous quantification features afforded by this method represent distinct advantages over current immunoassays. Arch Pathol Lab Med 2004; 128:469-475


Correlation between Tandem Mass Spectrometry and Immunoassays Steroid DHEAS1 Cortisol2 Androstenedione3 Estriol4 Progesterone5 DHEA3 11-Deoxycortisol6 Testosterone6 17α-Hydroxyprogesterone7 Estradiol1 1

Equations y= 1.15x + 43.18 y= 1.036x + 18.28 y= 1.051x + 0.769 y= 1.132x + 0.079 y= 1.236x – 0.502 y= 1.973x + 2.063 y= 0.795x + 1.176 y= 0.919x – 0.064 y= 1.587x + 0.123 y= 1.436 + 0.252

n 50 50 50 13 50 27 15 50 46 43

r 0.971 0.983 0.905 0.959 0.988 0.886 0.908 0.971 0.988 0.969

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 Relative Difference %

60 40 20

men male

0

women female

-20 -40 -60 -80 -100 0

2

4

6

8

Mean tes tos terone [Âľg/L]

Streit et al (unpublished)


Practicability and costs associated with tandem-MS or immunoassays Ease of use Expertise Turnaround time Investment cost Direct cost Technician time Selectivity Availability outside working hours Automation Effort for test development

LC/MS-MS

Immunoassay

difficult high intermediate high low intermediate high

easy low short moderate/low high short intermediate/low

restricted

unrestricted

low/intermediate

high

low/intermediate

high

Adatpted from Armstrong, Oellerich, Applied Pharmacokinetics and Pharmacodynamics 4th ed Lippincott Williams and Wilkins, 2006:30-39


Diagnostic molecular pathology Proteomics and genomics integration Categories of proteomics

Potential applications

Expression proteomics

Structural proteomics

Functional proteomics

Proteome profiling

Stucture of Protein complexes

Subcellular proteomics

Biomarker discovery

Protein-protein interactions

Genome project

Mechanisms or functions of protein compartmentalization/ translocation

Genomic data

Proteomic data Integration Prove the existence of genes Benefits

Elucidate the mechanisms of diseases, aging and protein functions

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


The Challenge of Molecular Diversity Genome (25,000 Genes)

Polymorphisms, Translocations Duplications

Transcription Splicing

Transcriptome (> 100,000 mRNAs)

mRNA expression mRNA sequences

Translation Protein Processing

Proteome (> 1,000,000 ? Proteins)

Protein concentration Protein structure Protein localization

Proteolysis

Peptidome (> 10,000,000 ? Peptides)

Peptide concentration Peptide structure Peptide localization Hortin, G. AACC 2007


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 statininduced 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

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


Risk of myopathy associated with simvastatin

SLCO1B1 rs4149056 genotype

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


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.

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


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

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

Nature Reviews Cancer 2009;9:576-586


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 Costs 21 € (100 tablets) Polymorphisms CYP2D6 Inhibition by co-medications (e.g. fluoxetine)

Breast cancer - postmenopausal - adjuvant therapy after primary treatment - metastases 573 € (100 tablets) CYP19 (?)


Partial metabolic pathway of tamoxifen and its interaction with estrogen receptors

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


Frequencies of CYP2D6 allelic variants Allele *MxN *3 *4 *5 *6 *7 *9 *10 *17 *29 *41

Function Caucasians % Asians % Africans % ultra rapid 1.8 1 2 inactive 2 0 inactive 21 1 4 inactive 2 4.1 4 inactive 1 inactive 0.1 reduced 2 reduced 1.5 50 6 reduced 0 34 reduced 20 reduced 8.4

Adapted from Brockmรถller et al., Pharmacogenomics 2000; 1:125-151; Raimundo et al, Clin Pharmacol Ther 2004; 76:128 Wennerholm et al, Pharmacogenetics 2001; 11:417-27


AmpliChip CYP2D6 and CYP2C19 microarray more than 15,000 probes CYP2D6 29 SNPs CYP2C19 *1, *2, *3

labeled target DNA oligonucleotide probe

**

* *


Estrogen receptor targeted activity by Tamoxifen and metabolites CYP2D6 extensive metabolizer (full activity)

4-hydroxy-Tamoxifen Endoxifen N-desmethyl-Tamoxifen Tamoxifen

370 650

CYP2D6 poor metabolizer (no acitivity)

285

290 660

213

2.340

600

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


CYP2D6 stratified disease - free survival under tamoxifen treatment

Disease– free survival

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

CYP2D6 EM homo unstratified cohort CYP2D6 EM het, IM CYP2D6 PM

Schroth et al, JAMA. 2009;302:1429


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, Breast Cancer Res 2007 Goetz MP. et al, Breast Cancer Res 2007 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

CYP2D6 not relevant relevant

Association between CYP2D6 inhibition and breast cancer recurrence under tamoxifen treatment

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


Tamoxifen: proposed dosage adjustments CYP2D6 predicted phenotype PM

IM

EM

Aromatase inhibitors

Tamoxifen 40 mg/d

Tamoxifen 20 mg/d

$ 350/ mo

$ 16/ mo

$ 8/ mo

(Tamoxifen 100 mg/d ? ) Weck, K. AACC 2010


Drugs for which revised labeling includes pharmacogenetic information

Drug

Relevant area of medicine

Year FDA approved revised labeling

6-Mercaptopurine

Oncology

2003

Approximate rate Gene or of ADRs or allele resistance

1-10% ADRs

Other drugs with Pharmacogenetic Relationship to gene or allele

TPMT

Azathioprine, 6-Thioguanine

Irinotecan

Oncology

2004

30-40% ADRs

UGT1A1

Tropotecan, 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 Drug

Relevant area of medicine

Year FDA approved Approximate rate of ADRs or revised resistance labeling

Gene or allele

Atomoxetine

Psychiatry

2004

5-10% ADRs

CYP2D6

2007

10% ADRs

HLA-B*1502

2007

5-8% ADRs

HLA-B*5701

Carbamazepine Abacavir Isoniazid Warfarin

Clopidogrel

Psychiatry, Neurology Infectious Desease Infectious Desease Cardiology

Cardiology

Pending 2007

2009

NAT 5-40% ADRs

30% resistant

Other drugs with Pharmacogenetic Relationship to gene or allele Antidepressants, Amphetamine Pjenytoin, Lamotrigine

Rifampin Hydralazine

CYP2C9, VKORC1

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* 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. J Proteome Res 2006; 5:954-962


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 proteins 1. Improvement of existing crude extract based ABPA diagnostic testing 2. Desensibilization strategies Patent No. 1278/DEL/2010 Bharat et al, J Proteome Res (submitted)

Indo-German BMBF Project (IND 06/040)


Marketed small-molecule drug targets by biochemical class Exploitable drug targets of the human genome: ~ 600 – 1.500

Enzymes 47%

GPCRs 30%

DNA 1% Integrins 1%

Ion channels 7%

Miscellaneous 2%

Transporters 4%

Others receptors 4%

Nuclear hormone receptors 4%

GPCR, G-protein coupled receptor Hopkins et al, Nature Reviews 2002, 1:727-730


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

Increase of cytosolic Ca2+ Ca2+ dependent enzymes NFAT

DAG

GEF (on Ras)

PKC

Erk, JNK, p38

NF-κ κB

AP-1

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

Increase of cytosolic Ca2+ Ca2+ dependent enzymes NFAT

DAG

GEF (on Ras)

PKC

Erk, JNK, p38

NF-κ κB

AP-1

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


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 funded chairs for pathology and laboratory medicine to provide high 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)


Leading scientific journals in laboratory medicine

2010 Proteomics: Advances in Protein Analysis for the Clinical Laboratory Glen Hortin, Leigh Anderson and Steve Carr

Rifai, N.; 2010


Leading scientific journals in laboratory medicine

Rifai, N.; 2010


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) Creation of global opportunities for cooperation in education, research, practice and commerce

45 Societies in 34 Countries


Thank you

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