InstituteInstitute forfor TumorTumor BiologyBiologyInstitute Institute forfor Tumor Tumor BiologyBiology

Klaus PantelKlaus Pantel

“Liquid Biopsy”: Current Status and Future PerspectivePerspective

CTCCTC asas LiquidLiquid BiopsyBiopsyforfor metastaticmetastatic cellscellsforfor metastaticmetastatic cellscells

Metastasis evolve many years y yafter primary tumor resection and can harbor unique genomic alterations.

Biopsy of metastases is an invasive and sometimesinvasive and sometimes dangerous procedure.

Can the molecularCan the molecular characterizaton of CTCreveal representativeinformationon metastatic cells located atdifferent sites ?

AlixAlix--Panabières & Pantel, Panabières & Pantel, Clin Clin ChemChem, , 20122012

DetectionDetection ofof CTC in CTC in thethe peripheralperipheral bloodblood

September 2013: September 2013:

400 i t d400 i t d li i lli i l t i lt i l ithith CTCCTC> 400 registered > 400 registered clinicalclinical trialstrials withwith CTC CTC asas

biomarkersbiomarkersbiomarkersbiomarkers

CTC Enrichment MethodsCTC Enrichment Methods Enr chm nt M tho s Enr chm nt M tho s

2013: > 50 different CTC

!assays !

The The technicaltechnicalchallengechallenge: :

FindingFinding oneonetumortumor cellcellin in 101066 –– 101088

normal normal bloodbloodAlixAlix--Panabières & Pantel, Panabières & Pantel, Annu Rev MedAnnu Rev Med, 2012, 2012Pantel & AlixPantel & Alix--Panabières, Panabières, Trends Mol MedTrends Mol Med, 2010, 2010

AlixAlix--Panabières & Pantel, Panabières & Pantel, Clin ChemClin Chem, , 20122012cellscells

New approach: In vivo capture of CTC (1.5 L blood)

Proof-of-principle data in breast, lung and prostate cancer

CTC Identification MethodsCTC Identification MethodsRealReal‐‐time RTtime RT‐‐PCRPCR

Cytokeratins as standard CTC markers

nucleic acidsnucleic acids

Cytokeratins as standard CTC markersBUT differential expression of individual CKs(Joosse/Pantel et al., Clin Cancer Res 2012)

DNA

nucleic acidsnucleic acids

mRNA

DNAintraintra--cytoplasmic proteinscytoplasmic proteins

membrane proteinsmembrane proteins> 13,000 publications on CTC in PubMed 2013

mRNA

ImmunocytochemistryImmunocytochemistry

PubMed 2013

secreted secreted

Tumor cellTumor cell

EPISPOT assayEPISPOT assay

proteins by proteins by VIABLE cellsVIABLE cells

AlixAlix--Panabières Panabières et al., et al., Clin Cancer Res, 2008Clin Cancer Res, 2008

Design Design ofof robust robust automatedautomated systemssystems

forfor reproduciblereproducible CTC CTC detectiondetection

CellSearch™ System CellSearch™ System (FDA(FDA--cleared)cleared)

MagNestMagNestTMTM

Epithelial Cell Kit

Enrichment of CTC with Enrichment of CTC with antianti--EpCAM ferro fluidsEpCAM ferro fluids

Cristofanilli et al., NEJM, 2004Cristofanilli et al., NEJM, 2004Riethdorf et al., CCR, 2007 & 2010Riethdorf et al., CCR, 2007 & 2010

pp

DeBono et al, CCR, 2008DeBono et al, CCR, 2008Cohen et al, JCO, 2008Cohen et al, JCO, 2008Krebs et al, JCO, 2012Krebs et al, JCO, 2012

CellSearchCellSearch™ System™ System: Images of Tumor Cells: Images of Tumor Cells

Cytoplasm Nucleus Cell Membrane CompositeCytoplasm Nucleus Cell Membrane Composite

CKCK--PEPEpospos

DAPIDAPIpospos

CD45CD45--APCAPCnegneg

Tumor CellTumor Cellpp gg

=+ -

Leukocyte Leukocyte nucleusnucleus

CD45CD45+ +

MembraneMembrane

LeukocyteLeukocyteTumor CellTumor Cell

Prognostic value of CTC counts for survival Prognostic value of CTC counts for survival i ti t ith d d dii ti t ith d d diin cancer patients with advanced diseasein cancer patients with advanced disease

Breast CancerBreast CancerChristofanilli NEJM 2004Christofanilli NEJM 2004

Prostate CancerProstate CancerDe Bono, Clin Can Res, 2008De Bono, Clin Can Res, 2008

Colorectal CancerColorectal CancerCohen, JCO, 2008Cohen, JCO, 2008Christofanilli, NEJM, 2004Christofanilli, NEJM, 2004 De Bono, Clin Can Res, 2008De Bono, Clin Can Res, 2008Cohen, JCO, 2008Cohen, JCO, 2008

FDAFDA clearanceclearance FDA FDA clearanceclearance

DetectionDetection ofof CTC in CTC in earlyearly stagestage

cancercancer patientspatients ((lowlow CTC CTC countscounts):):

Is the ability to release cancer cells into

the circulation relevant for the

d l t f di t t t t ?development of distant metastases?

Prognostic impact of CTC in breast cancer patients without overt metastases

Rack, Janni et al, unpublished

Meta-Analysis of 49 studies comprising 6815 breast cancer patientsp

1

Progression-free survivalCTC detection: ICC & RT-

0.860.78

0.620.58

0.78

0.6

0.8

1

ess F

ree

CTC negCTC pos

CTC detection: ICC & RTPCR

0.340.51

0.29

0.150.2

0.4

0.6

Prop

ortio

n Pr

ogre

p<0.001 Overall survival0.89

0.690.64

0.790.8

1

.

CTC negCTC pos

0.15

0.060

0 1 2 3 4 5

Time (years)

0.510.470.56

0.40

0.29

0.4

0.6

ortio

n Su

rviv

ing

0.19

0

0.2

0 1 2 3 4 5

Ti ( )

Prop

o

p<0.001

Zhang L, Riethdorf S…Pantel K, Clin Cancer Res, 2012.

Time (years)

TNM 2010: CTC in new cM0(i+) Classification

ChallengeChallenge ofof CTCCTC detectiondetection::Challenge Challenge ofof CTC CTC detectiondetection::

EpithelialEpithelial--MesenchymalMesenchymal Transition (EMT)Transition (EMT)EpithelialEpithelial--MesenchymalMesenchymal Transition (EMT) Transition (EMT)

ofof carcinomacarcinoma cellscells

(Bednarz-Knoll et al CMR 2012; Kang &

Tumor cell dissemination, plasticity and EMT

Pantel, Cancer Cell 2013)

Dormancy 10> 10 years

Epithelial-Mesenchymal Plasticity of CTC

EpCAM CK VimentinEpCAM, CK Vimentin

Bednarz-Knoll, Alix-Panabières & Pantel Cancer & Met Rev 2012

Expression profile of CTCs in breast cancer

EMTEMT

(Mani/Weinberg, (Mani/Weinberg, et alet al., ., CellCell, 2008;) , 2008;)

Yu et al, Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science, Febr. 2013

Yokobori Mimori Pantel Mori et al Plastin 3 as new CTC markerYokobori, Mimori, Pantel, Mori et al. Plastin-3 as new CTC markernot downregulated during EMT, Cancer Res. Febr. 2013

Bednarz-Knoll, Alix-Panabieres & Pantel, 2011, Breast Cancer Res, 13: 282-293

M l lM l l Ch t i tiCh t i ti ff CTCCTCMolecularMolecular CharacterizationCharacterization ofof CTC CTC

ff th tith ti t tt tfor for therapeutictherapeutic targetstargets( l( l ti li idti li id bibi ““))(„real(„real--time liquid time liquid biopsybiopsy““))

Detection of therapeutic targets on CTC: HER2 oncogene in breast cancerHER2 oncogene in breast cancer

CTC without HER2 gene amplification

Red: CK Red: Cen17

CTC without HER2 gene amplificationCTC without HER2 gene amplification

Red: CK Red: Cen17A Composite CK DAPI CD45 HER2A Composite CK DAPI CD45 HER2DETECTDETECT III study: AntiIII study: Anti HER2 therapy (lapatinib) in metastatic breast cancerHER2 therapy (lapatinib) in metastatic breast cancer

CTC with HER2 gene amplification

Red: CK ICC

Green: HER2 FISH

Red: Cen17 FISH

CTC with HER2 gene amplificationCTC with HER2 gene amplification

Red: CK ICC

Green: HER2 FISH

Red: Cen17 FISH

0

A

1+

2+

CTC

0

A

1+

2+

CTC

DETECTDETECT--III study: AntiIII study: Anti--HER2 therapy (lapatinib) in metastatic breast cancer HER2 therapy (lapatinib) in metastatic breast cancer

patients with HER2patients with HER2--negative primary tumors and HER2negative primary tumors and HER2--positive CTCpositive CTCCTC with HER2 gene amplificationCTC with HER2 gene amplificationCTC with HER2 gene amplification

CB11 A0485 FISHComposite CK DAPI CD45 HER2B

2+

3+

C CB11 A0485 FISHComposite CK DAPI CD45 HER2B

2+

3+

CSK-BR-3SK-BR-3SK-BR-3

MCF-7

BT20

0

1+

MCF-7

BT20

0

1+

T47D

MDA-MB-453

1+

2+

T47D

MDA-MB-453

1+

2+

Discordance between HER2 Discordance between HER2 status of primary tumor and status of primary tumor and CTCCTC

SK-BR-3

BT474 3+

3+SK-BR-3

BT474 3+

3+

CTCCTC

Riethdorf/Pantel et al., Clinical Cancer Res 2010 - Fehm/Pantel et al., Breast Cancer Res Treat 2010Ignatiadis/Sotiriou et al, PlosONE, 2011 - Ignatiadis/Pantel et al, SABCS, 2011

Heterogeneity of ER status in CTCs of breast cancer patients with ER-positive primary tumors

Babayan, Joosse, Pantel et al., PLOS ONE 2013

ER CK DAPI CD45 Merge

Babayan, Joosse, Pantel et al., PLOS ONE 2013

ER

ER CK DAPI CD45 Merge

R+

ERR-

ER-negative CTCs may survive endocrine therapy

Genomic Characterization of single CTCGenomic Characterization of single CTC

CTC detection

CTC isolation

WGA +

- Mutation analysis- Mutation analysis

- CGH (conv./array)

NextGen Sequencing

CTC Capillary CTC

- NextGen Sequencing

Detection of mutations in genes relevant for resistance of targeted therapies (eg EGFR inhibition)targeted therapies (eg, EGFR inhibition)

Gasch, Pantel, Riethdorf et al., Clin. Chem. 2012

Distribution of mutations in primary tumor, metastases and CTC

CRC patient #6 CRC patient #26

Deep targeted sequencing revealed that 17 of 20 „private CTC mutations“ were also present in„private CTC mutations were also present in

subclones of the primary tumor and metastases

Heitzer, Riethdorf, Pantel, Speicher et al, Cancer Res. 2013

TumorTumor--associated associated circulating cellcirculating cell--free nucleic free nucleic

CTCgg

acids in bloodacids in blood

MicrometastasisKRAS Mutations:KRAS Mutations:

C l ti CTC & Ci l ti T DNA Diaz et al, Nature 2012Heitzer et al, IJC 2013microRNAs:microRNAs:

Correlation CTC & Circulating Tumor DNA:Prostate Cancer: Schwarzenbach, Alix-Panabieres, Pantel et al., Clin Cancer Res 2009; Breast cancer: Dawson et al, NEJM, 2013; Colon Cancer: Heitzer, Pantel et al, Int J Cancer 2013 Roth et al, BCR 2010 &

PLosONE 2012Int J Cancer, 2013Correlation CTC & Circulating microRNA:Breast Cancer: Madhavan, Pantel et al Clin Cancer Res 2012

BUT: ctDNA is released from apoptotic/necrotic cellsIsolation of CTC allows in-depth molecular & functional characterization ofviable cells including xenotransplantion into immunodeficient mice (Baccelli, Pantel et al, Nat. Biotech., 2013; Pantel et al., Nature Med., 2013 )

Schwarzenbach/Schwarzenbach/HoonHoon/Pantel, /Pantel, Nat Nat RevRev Cancer, Cancer, 2011, Alix2011, Alix--PanabièresPanabières/Schwarzenbach/Pantel, /Schwarzenbach/Pantel, AnnuAnnu RevRevMedMed, 2012; Pantel & Alix, 2012; Pantel & Alix--Panabieres, Cancer Panabieres, Cancer ResRes., 2013 ., 2013

AimsAims ofof Research on Research on CirculatingCirculating Tumor CellsTumor Cellsgg

•• EstimationEstimation ofof thethe riskrisk for for metastaticmetastatic relapserelapse orormetastaticmetastatic progressionprogression ((prognosticprognostic informationinformation))

•• StratificationStratification & & realreal--timetime monitoringmonitoring ofof therapiestherapies

•• IdentificationIdentification ofof therapeutictherapeutic targetstargets andand resistanceresistancepp ggmechanismsmechanisms ((biologicalbiological therapiestherapies))

•• Understanding Understanding thethe biologybiology ofof metastaticmetastatic developmentdevelopment

early disseminationearly disseminationMetastasis Models

early stage cancerearly stage cancerDTCsDTCs

early stage cancerearly stage cancergenetic genetic

progressionprogression

Dormancy

advanced stage canceradvanced stage cancer

overt metastasisovert metastasisDTCsDTCs

overt metastasisovert metastasisno metastasisno metastasis PantelPantel et al., et al., NatureNature RevRev CancerCancer 20082008

Cancer Cancer DormancyDormancy: : Research Research questionsquestions

DD llll ti tti t hh d td t tt llll ??•• DoDo allall cancercancer patientspatients havehave dormantdormant tumortumor cellscells??

•• CanCan hosthost factorsfactors induceinduce oror breakbreak dormancydormancy?? Stress?Stress? InflammationInflammation??

•• AreAre therethere preferredpreferred reservoirsreservoirs ofof dormantdormant cellscells (e(e..gg..,, bonebone marrowmarrow)) ??

•• DoesDoes thethe immuneimmune systemsystem playplay aa rolerole inin dormancydormancy??•• DoesDoes thethe immuneimmune systemsystem playplay aa rolerole inin dormancydormancy??

•• WhatWhat isis thethe effecteffect ofof currentcurrent therapiestherapies onon dormantdormant cellscells oror

dormancydormancy??

•• WhatWhat signalingsignaling pathwayspathways oror eventsevents reactivatereactivate dormantdormant cellscells??g gg g p yp y

•• DoDo dormantdormant cellscells havehave propertiesproperties ofof cancercancer stemstem cellscells??

•• HowHow doesdoes geneticgenetic backgroundbackground affectaffect dormancydormancy??

Uhr & Pantel PNAS 2011; Kang & Pantel, Cancer Uhr & Pantel PNAS 2011; Kang & Pantel, Cancer CellCell 20132013

Metastasis Metastasis BiologyBiologygygy

Center of Experimental MedicineCenter of Experimental Medicine

Institute of Tumor Biology Institute of Tumor Biology -- Klaus PantelKlaus Pantelgygy

•• Sabine Riethdorf/Christin GaschSabine Riethdorf/Christin GaschGrant Support:Grant Support:

•• Heidi SchwarzenbachHeidi Schwarzenbach•• Harriet Wikman/Michaela Harriet Wikman/Michaela WrageWrage•• Katharina EffenbergerKatharina Effenberger

DFGDFGBMBFBMBFEU / ERCEU / ERC•• Katharina EffenbergerKatharina Effenberger

•• Simon Joosse, Anna Simon Joosse, Anna Babayan•• Kai Bartkowiak, Natalia BednarzKai Bartkowiak, Natalia Bednarz--KollKoll

Dt. KrebshilfeDt. KrebshilfeSanderSander--Stiftung Stiftung RoggenbuckRoggenbuck--StiftungStiftungRoggenbuckRoggenbuck StiftungStiftung

Micrometastasis Research Network at UCCH/UKEMicrometastasis Research Network at UCCH/UKE

Institut fürAnatomie II Viszeralchirurgie

Klinik undPoliklinik für

Viszeralchirurgie

Klinik undPoliklinik für Gynäkologie

Institut fürKlinischeChemie

I. MedizinischeKlinik undPoliklinik

Institut fürRechtsmedizin

Klinik undPoliklinik für

Neurochirurgie

Institut fürTumorbiologie

Institut fürTumorbiologie

Labor fürStrahlenbiologie und -onkologie

Klinik und Poli-Klinik und Poli-klinik für Mund-,

Kiefer- und Gesichts-chirurgieg

Molekularbiologie

Institut fürBiochemie und

MolekularbiologieII. Medizinische

Klinik undPoliklinik

Klinik undPoliklinik für

Urologie

chirurgie

Poliklinik

EUEU--ConsortiumConsortium--DISMALDISMALUniversity Medical Center Hamburg-Eppendorf (Germany)

Free University of Amsterdam Medical Center (The Netherlands)

Start: November 2005 Coordinator: Klaus Pantel

Imperial College London (United Kingdom),

Radium Hospital Oslo, (Norway)

German Cancer Research Center, (Germany)

SME 1 Applied Imaging, (United Kingdom)

ERC Advanced Investigator Grant DISSECT“ (2011-2016)

University of Graz (Austria)

University of Utrecht (The Netherlands)

„DISSECT (2011-2016)

ERA NET TRANSCAN CTC SCANHeinrich-Pette-Institut (Germany)

Netherlands Cancer Institute (The Netherlands)

ERA-NET TRANSCAN: CTC-SCAN Project (2013 – 2016)

Lapeyronie Hospital, Montpellier, (France)

SME 2 TILL Photonics (Germany)

( )

SME 3 Agendia, (The Netherlands)

Leiden University Medical Center (The Netherlands),

24-27, 2013