theranostik – perspektiven für die personalisierte medizin ... · economic impact of companion...

1

Theranostik – Perspektiven für die personalisierte Medizin in der

Infektionsdiagnostik

Till BachmannUniversity of Edinburgh

Workshop Infektionsdiagnostik25. Januar 2012Immanuel Klinik Rüdersdorf

Division of Pathway Medicine @ Edinburgh BioQuarter

•• University of EdinburghUniversity of Edinburgh

•• College of Medicine and College of Medicine and Veterinary MedicineVeterinary MedicineVeterinary MedicineVeterinary Medicine

•• 13 Principal Investigators13 Principal Investigators•• 70 staff and students70 staff and students


•• Pathway Biology of Pathway Biology of Infection and ImmunityInfection and Immunity

•• Biochip Biochip MedicineMedicine•• Stratified MedicineStratified Medicine•• Global HealthGlobal Health

2

Talk Outline

• Personalised Medicine

• Companion Diagnostics

• Infectious Diseases & Antibiotic Resistance

• New Technology Solutions for ID Diagnostics


• New Technology Solutions for ID Diagnostics

Not Every Drug Works with Every Patient


Clinical application of pharmacogenetics. Spear BB, Heath-Chiozzi M, Huff J. Trends Mol Med. 2001 May;7(5):201-4.

3

Personalised Medicines

Right Medicine

Right Patient

Therapeutic Need

Right Patient

Right Disease

Right TimeInnovative Medicines


Right Dose

Right Response

Right Price

Patient Targeted Therapies

Keys issues withmedicines today

are …..

*5th – 12th September 2005

Efficacy (does the patient

get better?)

SafetySafety (is the drug safe for the patient?)

4

Industry’s View

“For every single Pharma product …the biomarker research and the development of potentially companion diagnostics is a

“For every single Pharma product …the biomarker research and the development of potentially companion diagnostics is acompanion diagnostics is a standard part of the development process” (Severin Schwan, Roche CEO, June 2007)

companion diagnostics is a standard part of the development process” (Severin Schwan, Roche CEO, June 2007)


7

http://www.currentpartnering.com

Technology Path to PGx

Bio-k

Drugs

Assaysmarker


Personalised Medicine

5

Personalised Medicine - The Beginnings

• Herceptin/Trastuzumab (Genentech/Roche)– Antibody against Human Epidermal growth

f t R t 2 (HER2 ( bB2 HER2/ ))

• HercepTest (DakoCytomation)• Pathway (Ventana/Roche)

– Identify which patients overexpress Her2

• PathVysion (Vysis/Abbott)Id tif hi h ti t h lti l i f

factor Receptor 2 (HER2 (erbB2, HER2/neu))


– Identify which patients have multiple copies of Her2 gene

http://www.herceptin.comhttp://www.dako.comhttp://www.ventanamed.comhttp://www.abbottmolecular.com/PathVysionHER2DNAProbeKit_5138.aspx

Drugs in the Personalized Medicine Cabinet 2011

Workshop Infektionsdiagnostik25. Januar 2012Immanuel Klinik Rüdersdorf The Case for Personalized Medicine, 3rd Edition, 2011, The Personalized Medicine Coalition (PMC)

6

Companies in Companion Diagnostics

ELISABead Array

Microarray Sequencing

y

Invader

In VivoVirus assay

RT-PCRMethylation


Nature Biotechnology 26, 509 - 517 (2008)

FISH

IHC

miRNA

23 Personalised Medicine M&A Deals 2011

Workshop Infektionsdiagnostik25. Januar 2012Immanuel Klinik RüdersdorfThe Burrill Report October 2011 http://www.burrillreport.com/article-the_october_2011_issue_of_the_burrill_report.html

7

Economic Impact of Companion TestsDiscounted Cash Flow - No Dx, NPV = $892 (15% dcf)

500

1000

1500

2000

2500

ue

& C

ost

s

No CDx

-1000

-500

0

500

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020

Year

Rev

en

Revenue Costs

Discounted Cash Flow - Companion Dx, NPV = $2,694 (10% dcf)

2000

2500

ed M

edic

ines

Ltd

dmed

icin

es.c

o.uk

/

No CDx


-1000

-500

0

500

1000

1500

2000

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020

Rev

enu

e &

Co

sts

Year

Revenue Additional Revenue Costs Dx test

Edd

ie B

lair,

Inte

grat

e

http

://w

ww

.inte

grat

ed

CDx

Preclinical Phase I Phase 2 Phase 3 Filing Post-Approval

Companion Diagnostics Development Scenario 1 – CDx to be developed

Rx Development

BM Assay Development

C

Rx & Dx data filed for approval Diagnostic launched at

ti

Companion diagnostic identified and NOT available


Commercial IVD

Cross-over

Eg. Herceptin

same time as drug

Chris Chamberlain

8

Reimbursement is a Key Driver

• Health Technology Assessment (cf UK NICE)*Hughes NRDD 8: 261 (2009)

To implement this risk-sharing agreement, clinicians are required to measure the levels of serum M protein (SMP; a specific biomarker for tumour load) after a maximum of four cycles of treatment. If the patient has a reduction in SMP of 50% or more, indicating a complete or partial response, treatment will continue and the NHS will pay. If not, the JnJ must rebate the full cost.Hughes NRDD 6: 945 (2007)


Andrew Dillon, Chief Executive of NICE commented ….”the NHS will only pay for the drug when it has been proven to work…”

Hughes NRDD 6: 945 (2007)

Eddie Blair, Integrated Medicines Ltd 2010


Health Care

Provider

DxDeveloper

PM Package

9

Companies Entering the Infectious Disease CDx Market I


annualreport.roche.com

Companies Entering the Infectious Disease CDx Market II


18www.biocartis.com

10

Companies Entering the Infectious Disease CDx Market III


www.genomeweb.com

Companies Entering the Infectious Disease CDx Market IV


www.ivdtechnology.com

11

Not Every Drug Works with Every PatientThe Problem of Antibiotic Resistance

• Growing antibiotic resistance threatens the effectiveness of antibiotics

• Serious infections in humans• Overuse and misuse of antibiotics leads to

Bug

• Overuse and misuse of antibiotics leads to resistance

• 2.5m extra hospital days• 380,000 extra infections, 25,000 extra

deaths (4 indications)• 1.5b € per year extra costs in EU• beta-lactams most used antibiotics• Some resistance rates have more than

doubled in the past five years


doubled in the past five years• Few antibacterial agents with new

mechanisms of action under development (gram-)

ECDC, EMEA, Technical report: The bacterial challenge: time to react, 2009

Antibiotic Resistance Develops Quickly


Targeting virulence: a new paradigm for antimicrobial therapy, Anne E Clatworthy, Emily Pierson & Deborah T Hung, Nature Chemical Biology 3, 541 - 548 (2007)

12

Antibiotic Prescribing and Consumption


• Antibiotic prescribing and consumption varies between European countries• Primary care accounts for about 80 to 90% of all antibiotic prescriptions, mainly

for respiratory tract infections

European Center for Disease Prevention and Control, http://www.ecdc.europa.eu

Numbers of unique beta-lactamase enzymes identified since the introduction

of the first beta-lactam antibiotics


Davies, J. Et Al. 2010. Microbiol. Mol. Biol. Rev. 74(3):417-433

13

Worldwide distribution of different classes of CTX-M beta-lactamases


Davies, J. et al. 2010. Microbiol. Mol. Biol. Rev. 74(3):417-433

Do beta-lactamase variants matter therapeutically? I

“As demonstrated in this study, the KPC ariants ha e different s sceptibilities and


Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4. Wolter DJ, Kurpiel PM, Woodford N, Palepou MF, Goering RV, Hanson ND.Antimicrob Agents Chemother. 2009 Feb;53(2):557-62

variants have different susceptibilities and hydrolytic properties. This may contribute to the difficulty that clinical laboratories have encountered in identifying KPC-producing organisms. “

14

Unmet Need in Clinical Microbiology

Pathogen Drug Resistance

Therapy


Rapid Tests to Enable Prospective Therapy


Diagnostic DNA Microarrays

15

Microbial Antibiotic

SpeciesSNP

DPM Microbial DNA Microarray Portfolio

Microbial Antibiotic

ResistancePathogens

ESBL (SHV, TEM, CTX-M)

OXA, AmpC

KPC

gyrA/parC

Sepsis

Fungi

Protozoa

GastroenteritisHCV


E.coli Pathogenicity

fimH ECOR

Environment

Cyanobacteria

Spotting• Microgrid II• Epoxy slides• Spotted capture probes• 20 - 25 nt

Genotyping DNA Microarray Assay

Sample DNAextraction

Amplification and Labeling

Imaging &Identification

Hybridization


• Crude sample

• Pure cultures

extraction• AE resin

and Labeling• PCR• Cy3-dCTP• DNase

Identification

• ScanarrayExpress

• 1 h

16

Serin β-lactamases Metallo β-lactamases

Antibiotic Resistance: Extended Spectrum Beta Lactamases

Carbapenemases

Class A*Group 2**

Class C*Group 1**

Class D*Group 2d**

Class B*Group3**

Cephalosporinaseschromosomal, plasmid encodedall β-Lactams except Carbapenemsno inhibition with clavulanic acid

e.g. AmpC

Carbapenemaseschromosomal, plasmid encodedall β-Lactams except Monobactamsno inhibition with clavulanic acid

e.g. IMP, NDM-1

Cloxacillin (Oxacillin) hydrolyzing enzymeschromosomal, plasmid encoded

e.g. OXA

plasmid encodedinhibition with clavulanic acid2be broad spectrum β-Lactams 2br inhibitor resistant

e.g. TEM, SHV, CTX-M, KPC

point mutations (SNPs) can change resistance phenotype


Molekulare und biochemische Grundlagen der Beta-Lactam-Resistenz durch Beta-Lactamasen. Irith Wiegand. Chemotherapie Journal, 12. Jahrgang, Heft 6/2003

Extended Spectrum Beta-Lactamase (ESBL)

Inhibitor Resistant (IR)

mutant beta-lactamases differ in 1 to 5 amino acid positions

* older Classification according to Ambler (1980)** Classification according to Bush, Jacoby and Medeiros (BJM)

SNP Detection Microarray Principle

allele-specific hybridisation


spotting controlneg. hyb. controlpos. hyb. controlprocess controlperfect matchTEM1mismatch TEM1

17

ESBL Microarray

TEM SHV CTX-M

CTX-M groups

CTX M M1

Specifications

3 chip modules 14 subarrays

process control (PC)

spotting control (SC)

positive control (PHC)

negative control (NHC)

CTX-M group M1

CTX-M group M8/25

CTX-M group M2PMs TEM-1, SHV-1 and CTX-M15

14 subarrays 155 probe sets 2060 spots


CTX-M group M9

Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. Leinberger DM, Grimm V, Rubtsova M, Weile J, Schröppel K, Wichelhaus TA, Knabbe C, Schmid RD, Bachmann TT. J Clin Microbiol. 2010 Feb;48(2):460-71.

ESBL Microarray Testing of Clinical Isolates

SHV-12 SHV type beta lactamase13 x

ESBLs

SHV-5SHV-12 + CTX-M15

CTX-M15 CTX-M type beta lactamase42 x


CTX-M15 + CTX-M14bCTX-M14

CTX-M9

CTX-M3

no ESBL variant no TEM, SHV or CTX-M ESBL6 x

Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. Leinberger DM, Grimm V, Rubtsova M, Weile J, Schröppel K, Wichelhaus TA, Knabbe C, Schmid RD, Bachmann TT. J Clin Microbiol. 2010 Feb;48(2):460-71.

18

Klebsiella pneumoniae carbapenemase (KPC) microarray

• Confers resistance to Carbapenems• Carbapenems classed as “drug of last

resort”• First reported in North Carolina inFirst reported in North Carolina in

2001 by Yigit et al. • Found in New York, Israel, China,

South America, ...Perfect Match(KPC-3)


Peter H, Berggrav K, Thomas P, Pfeifer Y, Witte W, Dobrindt U, Bachmann TT, Rapid and Sensitive Detection of Carbapenem Resistance Directly from Urine Samples using DNA Microarrays. submitted

KPC Reference Strains for Assay Development


urine dilutions 1x109 – 1 CFU / ml

QIAprep Spin Miniprep Kit (Qiagen)

Urine Bacterial DNA Isolation Kit (Norgen)


19

KPC Microarray Results I

Array 1 (K. pneumoniae, VA 367, KPC-3)

Array readout results:



Identification Directly from Spiked Urine Samples

Qiagen K. pneumoniae (KPC-3)

LOD4000 CFU / ml urine

Norgen


LOD360 CFU / ml urine


20

Limit of Detection (LOD)

Analysis directly from spiked urine samples



Improving the Resolution of SNP Detection

C TA G

hybridisation

C TA G C TA G

hybridisation digestiondigestion


Schulze H, Barl T, Vase H, Baier S, Thomas P, Giraud G, Crain J, Bachmann TT, Enzymatic on-chip enhancement of genotyping DNA microarrays. submitted

21

Nuclease Treated KPC Arrays with Clinical Isolates

1.21.2

Nuclease treatedBuffer control

0

0.2

0.4

0.6

0.8

1

SNP 147 SNP 308 SNP 716 SNP 814

SNP Position

A

G

C

T

0

0.2

0.4

0.6

0.8

1

SNP 147 SNP 308 SNP 716 SNP 814

SNP Position

A

G

C

T


PCR amplified KPC-3 positive sample labeled with Cy3 was hybridized to the KPC array and treated with 1:10 dilution of nuclease (left) or a control (1x Taq PCR buffer) (right) at 42ºC for 30 min. Normalized signal intensity data.

CEL nuclease substantially enhanced SNP discrimination of a KPC microarray tested with PCR products derived from clinical isolates.

Schulze H, Barl T, Vase H, Baier S, Thomas P, Giraud G, Crain J, Bachmann TT, Enzymatic on-chip enhancement of genotyping DNA microarrays. submitted


Point of Care Testing

22

• Integration Of Nucleic Acid, Protein And Small Molecule Detection On A Rapid, Point-of-care Multi-parameter Platform

• 4 year $15M funding provided by ITI Scotland and

Biosensing Platform Project

• 4 year, $15M funding provided by ITI Scotland and the European Regional Development Fund

• Demonstrator cartridge & instrument• Microfluidics core capability • Prototype system designed for flexible development• Short time-to-result achieved

Total TTR of 10 – 15minSample preparation <2minPlasma to cDNA ~6minqPCR <7minqPCR <7min

• Small molecule (antibiotic) detection• Multiplex and Multiparameter assays• Novel detection modalities• Partners

ITI-Scottish Enterprise, GlasgowLab901, Edinburgh, ScotlandDivision of Pathway Medicine, EdinburghAxis-Shield Diagnostics, Dundee

Chronic Wound CareProgramme

• Development of a an easy-to-use, portable medical device that can be readily applied to diagnose and treat chronic wounds in a clinical environment and in the community.

• University of Edinburgh, Zisys Ltd. research provider

• Aim: Molecular MRSA detection from clinical specimen without PCR

23

Electrochemical Impedance Spectroscopy (EIS)

• Label free detection

• Small AC potential is applied to an electrochemical cell and the

3’

Fe(CN)6-3/-4

Fe(CN)6-3/-4

current response measured

• Interrogate different frequencies ranging from 0.1 -100,000 Hz

5’

Probe

Target 10 nM


Target 10 nM

Target 50 nM0.00

E - Excitation potential I - current response

Detection of MRSA using EIS

3

4 0 nM mecA + 3 S.D

Rat

io

4

5

6

7

R2 = 0.9920

e R

atio

0.01 0.1 1 100

1

2

Sig

na

l In

cre

ase

mecA PCR product / nM

0 10 20 30 40 50 60 700

1

2

3

4

Sig

nal I

ncr

ease

MRSA mecA PCR product / nM

• PCR Product • PCR Product


OO

O

O

NH2

• PCR Product• PNA Probes

• PCR Product• PNA Probes + Spacer

Corrigan D., et al. Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates. Biosensors Bioelectronics in revision

24

Enzymatic Signal Amplification to Increase EIS Signal

1000 PMMM

[Fe3(CN)6]3- + e-[Fe3(CN)6]4-[Fe3(CN)6]3- + e-[Fe3(CN)6]4-

0 5 10 15 20 25 301

10

100

MM

Am

pli

fica

tio

n f

acto

r

Localised precipitation

[ 3( )6][ 3( )6][Fe3(CN)6] e[Fe3(CN)6]


0 5 10 15 20 25 30

t [min]

• SNP discrimination• Localised precipitation• Detection even before 5 min feasible

Alkaline phosphatase enzymatic signal amplification for fast, sensitive impedimetric DNA detection. Kaatz M,Schulze H, Ciani I, Lisdat F, Mount AR, Bachmann TT. Analyst. 2012 Jan 7;137(1):59-63.

PM

MM

Towards NDM-1 Point of Care Testing using EIS

3

0

0.5

1

1.5

2

2.5

NDM-1 noncomplementary Target

del

ta R

ct

Control

Test


Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Kumarasamy KK et al. Lancet Infect Dis. 2010 Sep;10(9):597-602.

p y g

Sample

• 15 min incubation• Synthetic target

25

Sample Manipulation to Improve Assay Performance

25 nm• Dielectrophoresis (DEP)• Trapping of bacterial RNA by DEP in order to

AC field

achieve faster and more sensitive detection


16S rRNA (1541 bases) 23S rRNA (2904 bases)

Dielectrophoretic manipulation of ribosomal RNA. Giraud G, Pethig R, Schulze H, Henihan G, Terry JG, Menachery A, Ciani I, Corrigan D, Campbell CJ, Mount AR, Ghazal P, Walton AJ, Crain J, Bachmann TT.Biomicrofluidics. 2011 Jun;5(2):24116

Frequency Dependency of rRNA Dielectrophoresis

Spherical rRNA Stretched rRNA0.01 MHz0.1 MHz

1 MHz3 MHz

5 MHz7 MHz

10 MHz30 MHz50 MHz



26

Manipulation of rRNA in Real Time


DEP at 3 MHz 4V -------------------------- 1 minute --------------------- DEP at 50 MHz 4V


Conclusion and Outlook

• Personalised Medicine is becoming a viable approachin healthcare.

• IVD and Pharma partnering is rising• IVD and Pharma partnering is rising.

• Omics research feeds into constant stream of novel biomarkers.

• Rx demand for Dx creates opportunities for biosensing developments.

• Antibiotic resistance creates an unmet need for therapies


Image http://ocjewishexperience.wordpress.com

of infectious disease.

• New technologies offer advanced possibilities for infectious disease diagnostics.

27

Acknowledgments

Grace HenihanIlenia CianiMarie CraigonCatriona AndersonMiriam Kaatz

Julie LubyDiana Sisu

Stuart EmberMizan Khondoker

Davide PapaleChaker Tlili

Alison HardieAnne HolmesHolger SchulzeHarald PeterAlan RossDamion CorriganJon Terry

Chaker TliliNate Wittenberg

Gerard GiraudJohn Henry

John Beattie

Eddie Blair


Peter Ghazal, Ron Pethig, Jason Crain, Anthony Walton, Colin Campbell, Andy Mount,Ken Pierce, Rohit Mistry, Carmelo Volpe, Larry Wangh, Jay Lewington

Kate Templeton, Paddy Gibb, Matthew Young,Fred Lisdat, and all former funders, collaboration partners and colleagues

Eddie Blair

theranostik – perspektiven für die personalisierte medizin ... · economic impact of companion...

Documents