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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