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Institute for Tumor Biology
Professor Klaus Pantel
CTC-DTC Research: State of the Art & Perspectives
Pantel & Brakenhoff, Nat Rev Cancer, 2004; Pantel et al., Nat Rev Cancer, 2008
Primary Tumor Lymph-node metastasis
Hematogenous dissemination
Distant metastases
Lymphatic dissemination
Tumor Cell Dissemination Key Step in Tumor Progression
DTC
CTC
Izbicki/Pantel et al., NEJM, 1997 De Boer et al., NEJM, 2009
Detection of CTC in the peripheral blood
September 2013:
>400 registered clinical trials with CTC as biomarkers
> 13,000 publications in PubMed
Advantages over DTC detection:
•!Less invasive than BM sampling •!Pool of DTC from multiple distant sites
•! Metastasis biology
•! Novel CTC assays
!
!
!
ISMRC 2013, Paris: Scientific Sessions
Metastasis Biology
Primary tumor
Local relapse
Recirculation
Tumor-mass
dormancy
Metastasis
Escape
Tumor cell dormancy
DTC
Micrometastasis
Distant tissue (e.g. bone marrow)
Blood
Cancer micrometastases Klaus Pantel, Catherine Alix-Panabi• res and Sabine Riethdorf
NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 6 JUNE 2009 339
Tumor cell dissemination and cancer dormancy
(Uhr & Pantel, PNAS 2011)
Experimental findings: -! Reseeding of the primary tumor: Recirculation of breast cancer cells from the bone marrow to the primary site (J. Massague« s group, Kim et al, Cell 2009)
-! Escape from dormancy: VCAM1 promotes osteoclast differentiation & activation & attracts osteoclast progenitors (Y. Kang« s group, Lu/Pantel et al Cancer Cell 2011)
CTCs
DTCs
MNC
Ficoll gradient
Bone marrow
2 x 106 MNC per patient
Bone marrow aspirates taken from the upper iliac
crest
Immunocytochemistry: Cytokeratin staining with mAB A45-B/B3
Breast Cancer: 199/552 (36%) (Braun, Pantel et al. NEJM, 2000 & 2005)
Prostate Cancer: 86/193 (44.6%) (Koellermann/Pantel et al. JCO 2008) Nonmalignant disease: 2/191 (1%)
Detection of DTC in bone marrow
•! DTC detection correlates with metastatic AND locoregional relapse •! Most DTC are Ki67- and have CD44+/CD24- phenotype
•! DTC detection might be useful for stratification of bone-directed anti-cancer therapies (e.g., bisphosphonates, RANKL Abs)
•! Bisphosponate treatment reduces DTC counts and prevents metastatic & locoregional relapse
Cancer Dormancy: Research questions
•! Do all cancer patients have dormant tumor cells?
•! Can host factors induce or break dormancy? Stress? Inflammation?
•! Are there preferred reservoirs of dormant cells (e.g., bone marrow) ?
•! Does the immune system play a role in dormancy?
•! What is the effect of current therapies on dormant cells or
dormancy?
•! What signaling pathways or events reactivate dormant cells?
•! Do dormant cells have properties of cancer stem cells?
•! How does genetic background affect dormancy?
Uhr & Pantel PNAS 2011; Kang & Pantel, Cancer Cell 2013
Novel CTC Assays
CTC Enrichment Methods
Alix-Panabi• res & Pantel, Annu Rev Med, 2012 Pantel & Alix-Panabi• res, Trends Mol Med, 2010
Alix-Panabi• res & Pantel, Clin Chem, 2012
2013: > 50 different CTC
assays !
The technical
challenge: Finding one tumor cell in 106 Ð 108
normal blood cells
!"#$"%&'())(*'
''''
DNA
mRNA
intra-cytoplasmic proteins membrane proteins
secreted proteins by VIABLE cells
+,(-./0,'+&."1+'
nucleic acids
#002345*6457,08)69*'
mRNA
Tumor cell
CTC Identification Methods
Alix-Panabi• res et al., Clin Cancer Res, 2008
Cytokeratins as standard CTC markers BUT differential expression of individual CKs (Joosse/Pantel et al., Clin Cancer Res 2012)
Design of robust automated systems
for reproducible CTC detection
MagNestTM
Epithelial Cell Kit
Cristofanilli et al., NEJM, 2004 Riethdorf et al., CCR, 2007 & 2010
DeBono et al, CCR, 2008 Cohen et al, JCO, 2008 Krebs et al, JCO, 2012
CellSearchª System (FDA-cleared)
Enrichment of CTC with anti-EpCAM ferro fluids
CellSearchª System: Images of Tumor Cells
CK-PE
pos DAPI pos
CD45-APC neg
Tumor Cell
Cytoplasm Nucleus Cell Membrane Composite
= + -
Leukocyte nucleus
CD45+
Membrane Leukocyte Tumor Cell
Reaction chamber:
1. mRNA-capturing 2. c-DNA synthesis 3. PCR
Processing Fluid
sample inlet
dry reagents RT, TAQ, Primer, Reporter Alere Technologies GmbH Jena Page 1
Waste
Page 1
Automated multiplex q-RT-PCR: Lab-in-a-cartridge
Lysis chamber: binding of Biotin-Oligos to mRNA
Array: Signal monitoring of real time PCR
Challenge of CTC detection:
Epithelial-Mesenchymal Transition (EMT)
of carcinoma cells
(Bednarz-Knoll et al CMR 2012; Kang & Pantel, Cancer Cell 2013)
Tumor cell dissemination, plasticity and EMT
Dormancy > 10 years
Bednarz-Knoll, Alix-Panabi• res & Pantel Cancer & Met Rev 2012
Epithelial-Mesenchymal Plasticity of CTC
EpCAM, CK Vimentin
Expression profile of CTCs in breast cancer
Bednarz-Knoll, Alix-Panabieres & Pantel, 2011, Breast Cancer Res, 13: 282-293
EMT
(Mani/Weinberg, et al., Cell, 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 marker not downregulated during EMT, Cancer Res. Febr. 2013
Epithelial-Mesenchymal Transition in DTC line BC-M1
Bartkowiak et al. J Prot Res, 2009 Willipinski-Stapelfeldt et al. Clin Cancer Res, 2005
CTC in Clinical Studies
TNM 2010: CTC in new cM0(i+) Classification
Meta-Analysis of 49 studies comprising 6815 breast cancer patients
Zhang L, Riethdorf S!Pantel K, Clin Cancer Res, 2012.
0.89
0.690.64
0.510.47
0.79
0.56
0.40
0.29
0.19
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5
Time (years)
Prop
ortio
n Su
rviv
ing
.
CTC negCTC pos
0.860.78
0.620.58
0.34
0.78
0.51
0.29
0.15
0.060
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5
Time (years)
Prop
ortio
n Pr
ogre
ss F
ree
CTC negCTC pos
p<0.001
p<0.001
Progression-free survival
Overall survival
CTC detection: ICC & RT-PCR
ÒU rgent need for biomarkers to tailor systemic therapy in individual cancer patients, such as the
blood glucose test for directing insulin treatment of diabetesÓ
Alix-Panabi• res, Riethdorf, Pantel, Clin Cancer Res, 2008
Real-time monitoring of CTC (surrogate marker)
Abiraterone Phase III Study with CTC as secondary end point in patients with metastatic prostate cancer
Abiraterone 1000 mg daily Prednisone 5 mg BID
n = 797
Primary end point: •!OS (25% improvement;
HR 0.8) Secondary/tertiary end points: TTPP, rPFS, PSA response •!CTC enumeration
Efficacy end points (ITT)
Placebo daily
Prednisone 5 mg BID n = 398
R A N D O M I Z E D
2:1
•!1195 patients with progressive mCRPC
•!Failed 1 or 2
chemotherapy regimens
Planned Patients
HR, hazard ratio; ITT, intent to treat; H. Scher, ASCO 2011
Detection of CTC in early stage
cancer patients (low CTC counts):
Is the ability to release cancer cells into
the circulation relevant for the
development of distant metastases?
Prognostic impact of CTC in breast cancer patients without overt metastases
Rack, Janni et al, unpublished
Prognostic value of CTC in urinary bladder cancer
DFS HR: 4.6 CSS HR: 5.2
Molecular Characterization
of CTC
Alix-Panabi• res & Pantel, Clin Chem, 2013; Pantel & Alix-
Panabieres, Cancer Res. 2013
CTC as Liquid Biopsy for metastatic cells
Metastasis evolve many years after primary tumor resection and can harbor unique genomic alterations. Biopsy of metastases is an invasive and sometimes dangerous procedure. Can the molecular characterizaton of CTC reveal representative information on metastatic cells located at different sites ?
Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer
CTC without HER2 gene amplification
CTC with HER2 gene amplification
SK-BR-3
Red: CK ICC
Green: HER2 FISH
Red: Cen17 FISH
CTC without HER2 gene amplification
CTC with HER2 gene amplification
SK-BR-3
CTC without HER2 gene amplification
CTC with HER2 gene amplification
SK-BR-3
Red: CK ICC
Green: HER2 FISH
Red: Cen17 FISH
CB11 A0485 FISH
0
Composite CK DAPI CD45 HER2
A
B
1+
2+
3+
Composite CK DAPI CD45 HER2
MCF-7
BT20
T47D
SK-BR-3
BT474
C
CTC
MDA-MB-453
0
1+
1+
3+
3+
2+
CB11 A0485 FISH
0
Composite CK DAPI CD45 HER2
A
B
1+
2+
3+
Composite CK DAPI CD45 HER2
MCF-7
BT20
T47D
SK-BR-3
BT474
C
CTC
MDA-MB-453
0
1+
1+
3+
3+
2+
Riethdorf/Pantel et al., Clinical Cancer Res 2010 - Fehm/Pantel et al., Breast Cancer Res Treat 2010 Ignatiadis/Sotiriou et al PlosONE 2011 Ignatiadis/Pantel et al SABCS 2011
Discordance between HER2 status of
primary tumor and CTC
DETECT-III study: Anti-HER2 therapy (lapatinib) in metastatic breast cancer patients with HER2-negative primary tumors and HER2-positive CTC
Heterogeneity of ER status in CTCs of breast cancer patients with ER-positive primary tumors
ER+
ER -
ER CK DAPI CD45 Merge
ER-negative CTCs may survive endocrine therapy
Babayan, Joosse, Pantel et al., PLOS ONE 2013
CTC detection
CTC Capillary CTC
WGA +
-! Mutation analysis
-! CGH (conv./array)
-! NextGen Sequencing
Genomic Characterization of single CTC
CTC isolation
CRC patient #6 CRC patient #26
Distribution of mutations in primary tumor, metastases and CTC
Heitzer, Riethdorf, Pantel, Speicher et al, Cancer Res. 2013
Deep targeted sequencing revealed that 17 of 20 ãp rivate CTC mutationsÒ were also present in
subclones of the primary tumor and metastases
CTC and Other Circulating
Markers
Schwarzenbach/Hoon/Pantel, Nat Rev Cancer, 2011, Alix-Panabi• res/Schwarzenbach/Pantel, Annu Rev Med, 2012; Pantel & Alix-Panabieres, Cancer Res., 2013
Tumor-associated circulating cell-free nucleic
acids in blood
Micrometastasis
CTC
ctDNA: Diaz et al, Nature 2012 Heitzer et al, IJC 2013 microRNAs: Roth et al, BCR 2010 & PLosONE 2012
Diaz et al, Nature 2012 Heitzer et al, IJC 2013 Heitzer et al, IJC 2013 microRNAs: Roth et al, BCR 2010 & PLosONE 2012
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 Correlation CTC & Circulating microRNA: Breast Cancer: Madhavan, Pantel et al Clin Cancer Res 2012
BUT: ctDNA is released from apoptotic/necrotic cells Isolation of CTC allows in-depth molecular & functional characterization of viable cells including xenotransplantion into immunodeficient mice (Baccelli, Pantel et al, Nat. Biotech., 2013; Pantel et al., Nature Med., 2013 )
•! Estimation of the risk for metastatic relapse or metastatic progression
•! Stratification & real-time monitoring
! therapeutic targets and resistance mechanisms
! biology
Summary: Aims of Research on DTC & CTC
Center of Experimental Medicine
Institute of Tumor Biology - Klaus Pantel
•! Sabine Riethdorf/Christin Gasch •! Heidi Schwarzenbach •! Harriet Wikman/Michaela Wrage •! Katharina Effenberger •! Simon Joosse, Anna Babayan •! Kai Bartkowiak, Natalia Bednarz-Koll
Grant Support: DFG BMBF EU / ERC Dt. Krebshilfe Sander-Stiftung Roggenbuck-Stiftung
Micrometastasis Research Network at UCCH/UKE
Institut fŸ r Anatomie II
Institut fŸ r Klinische Chemie
Institut fŸ r Rechtsmedizin
Labor fŸ r Strahlenbiologie und -onkologie und -onkologie
Institut fŸ r Biochemie und
Molekularbiologie II. Medizinische
Klinik und Poliklinik
Klinik und Poliklinik fŸ r
Urologie
Klinik und Klinik und
Klinik und Poli- klinik fŸ r Mund-,
Kiefer- und Gesichts- chirurgie
Klinik und Poliklinik fŸ r
Neurochirurgie
I. Medizinische Klinik und Poliklinik
I. Medizinische
Klinik und Poliklinik fŸ r
Viszeralchirurgie
Klinik und Poliklinik fŸ r GynŠ kologie
Institut fŸ r Tumorbiologie
Institut fŸ r Tumorbiologie
University Medical Center Hamburg-Eppendorf (Germany)
German Cancer Research Center, (Germany)
Heinrich-Pette-Institut (Germany)
University of Graz (Austria)
Imperial College London (United Kingdom),
University of Utrecht (The Netherlands)
Free University of Amsterdam Medical Center (The Netherlands)
EU-Consortium-DISMAL
Start: November 2005 Coordinator: Klaus Pantel
SME 3 Agendia, (The Netherlands)
Lapeyronie Hospital, Montpellier, (France)
SME 2 TILL Photonics (Germany)
Leiden University Medical Center (The Netherlands),
Radium Hospital Oslo, (Norway)
Netherlands Cancer Institute (The Netherlands)
SME 1 Applied Imaging, (United Kingdom)
Catherine Alix-Panabieres, Montpellier: Prix cancer Gallet et Breton 2012
Klaus Pantel, Hamburg:
ERC Advanced Investigator Grant ãD ISSECTÒ (2011-2016)
ERA-NET TRANSCAN: CTC-SCAN Project (2013 Ð 2016)
2013
PAR
IS
CTC
2013 2013 2013
CTC
24-27, 2013