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