THE ROLE OF CIRCULATING

TUMOUR CELLS (CTCS) IN

CANCER MANAGEMENT

Klaus Pantel, MD, PhD

Chairman, Institute for Tumour Biology
AIMS OF RESEARCH ON CTCS
& CTDNA

‹ Screening & early detection of cancer

‹ Estimation of the risk for metastatic relapse or metastatic progression (prognostic

information)

‹ Stratification & real-time monitoring of therapies

‹ Identification of therapeutic targets and resistance mechanisms (biological

therapies)

‹ Understanding the biology of metastatic development

 TUMOUR CELL DISSEMINATION
AND CANCER DORMANCY

‹ Escape from dormancy (bone marrow): Primary

tumour
Local
‹ VCAM1 promotes osteoclast relapse

differentiation & activation & attracts

osteoclast progenitors (Lu/Pantel/Kang et al. Cancer Blood
CTCs
Cell 2011)
Distant tissue

‹ Tumour-induced osteoclast miRNA (e.g. bone

marrow)

changes as regulators and biomarkers of Recirculation DTCs DTC

osteolytic bone metastasis (Ell/Pantel/Kang et al., Tumour
Cancer Cell 2013) cell
dormancy
‹ Metabolic adaptation of DTCs is Tumour-mass

important for survival (LeBleu, Pantel, Kalluri et al.,

dormancy

Nature Cell Biol. 2014)

Micrometastasis

Escape

Metastasis

Reprinted by permission from Macmillan Publishers Ltd: Nat Rev Clin Oncol.Pantel K, et al., Nat Rev Clin Oncol.2009;6:339–51. Copyright 2009
 TUMOUR CELL
DISSEMINATION,
PLASTICITY AND EMT

Dormancy
> 10 years

Cancer Metastasis Rev, Plasticity of disseminating cancer cells in patients with epithelial malignancies, 2012; 31: 673–87. Bednarz-Knoll N, et al. © Springer
Science+Business Media, LLC 2012. With permission of Springer; Kang Y & Pantel K, Cancer Cell 2013;23:573–81; Joosse SA, et al.,
Individual cytokeratins can be downregulated and pan-cytokeratin antibody cocktail increases therefore the sensitivity of CTC assays. Clin. Cancer Res. 2012;18:993–1003;
Yokobor T, et al., Plastin-3 as new CTC marker not downregulated during EMT. Cancer Res 2013; 73:2059-–69
 CTC AS LIQUID BIOPSY FOR
METASTATIC CELLS
Metastasis evolve many years after ‹

primary tumour resection and can

harbor unique genomic alterations
Biopsy of metastases is an invasive ‹

and sometimes dangerous

procedure
Different metastatic sites show
The technical challenge: ‹

intra-patient heterogeneity
Finding one tumour cell in 106 – CTC
108 represent
normal blood cellscells
metastatic ‹

from different sites

Pantel K & Alix-Panabieres C, TMM 2010; Pantel & Alix-Panabieres, Cancer Res. 2013
Republished with permission of American Association for Clinical Chemistry, from Circulating Tumor Cells: Liquid Biopsy of Cancer, Alix-Panabières C, Pantel K, Clin
Chem, 2013; 59(1):110–118; permission conveyed through Copyright Clearance Center, Inc.
 CTC ENRICHMENT STRATEGIES
Biological properties
Protein expression
Positive selection
(a)
anti-E markers Ab
(E.g., EpCAM)
Physical properties
Label-free strategies
(e))
(c)
anti-M markers Ab
(E.g., N-Cadherin)

(d)
(f)
CTC +-
++ -DEP-
+
--
CTC
anti-E/M markers Ab
(E.g., Plastin 3)
+ DEP -
++ - -
+
Ex vivo
-CCellSearch
llllS
S h® system
t
- MagSweeper™ (g) Out
- EPHESIA CTC-chip CTC-iChip
- CTC-chip
- Velcro-like device
In vivo
Anti-CD45
-CCellCollector
llC ll t ®
- Photoacoustic nanodetector

Negative selection
(b)
anti-E markers Ab
Anti-CD45
CTCs

WBC WBC
W
C
CTC
RBC

Alix-Panabieres C & Pantel K,.Nature Rev.Cancer 2014;14:623–31

Images courtesy of Universitätsklinikum Hamburg Eppendorf
 APPROACHES FOR CTC
DETECTION
Immunocytological Molecular technologies Functional assays
technologies In vitro Cell Culture
anti-E/M markers Ab
anti
aan
nttii-m
marker
maark
rkeerr A
Abs
bss
(E.g., CK, Vimentin, E/N-Cadherin) - EPISPOT (E.g., CK19, HER2, EGFR, VEGF, PSA)

Viable
Fluo Ab2
CTC
CTC anti-tissue-specific
markers Ab LLiquid
iquid bead array
(E.g., PSA, Mammaglobin, MAGE) CTC mRNA
m
mRN A Ab1
Cell culture Immunospots

Secreted protein
anti-tumour associated
markers Ab
(E.g., HER2, EGFR) RT-qPCR
R
(single/multiple genes)) - Invasion assay
Functional
Technologies
g CTC

- Immunocytochemistry
- CellSearch® system
Fluo matrix
- Flow Cytometry
- DEParray® RNA-based
RNA-bbased TTechnologies
echnolo
Xenotransplantation models (CDx)

Viable CTC Metastases

days
with stem-cell properties
Alix-Panabieres C & Pantel K,.Nature Rev.Cancer 2014;14:623–31
Images courtesy of Universitätsklinikum Hamburg Eppendorf
CELLSEARCH™ SYSTEM
(FDA-CLEARED)
MagNestTM

7.5ml

Epithelial Cell Kit

Enrichment of CTC with

anti-EpCAM ferro fluids:
Captures tumour cells with very
low EpCAM expression
 CELLSEARCH™ SYSTEM:
IMAGES OF TUMOUR CELLS
Cytoplasm Nucleus Cell Membrane Composite

CK-PE DAPI CD45-APC Tumour cell

pos pos neg

+ - =

Leukocyte
Leukocyte nucleus CD45+ membrane
Tumour cell

Images courtesy of Universitätsklinikum Hamburg Eppendorf

 PROGNOSTIC VALUE OF CTC
COUNTS FOR SURVIVAL
In cancer patients with advanced disease

Breast Cancer Colorectal Cancer Prostate Cancer

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

Æ FDA approval
1) From N Engl J Med 2004, Cristofanilli M, et al., Circulating Tumor Cells, Disease Progression and Survival in metastatic breast
cancer, 351:781-791, Copyright © 2004, Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical
Society.
2) Reprinted with permission. © (2008) American Society of Clinical Oncology. All rights reserved. Cohen SJ, et al., J Clin Oncol 26(19), 2008:3213-3221
3) Reprinted from Clin Can Res, 2008, 14(19):6302-9, de Bono JS, et al., Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic
Castration-Resistant Prostate Cancer, with permission from AACR.
 CTCS BEFORE INITIATION OF
THERAPY IN 7.5 ML OF BLOOD –
FDA-APPROVAL

Reprinted from Clin Can Res, 2008;14(19):6302-9, de Bono JS, et al., Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic
Castration-Resistant Prostate Cancer, with permission from AACR.
PROGNOSTIC VALUE OF CTC
COUNTS FOR SURVIVAL
In cancer patients with advanced disease

Breast Cancer Colorectal Cancer Prostate Cancer

Cristofanilli, NEJM, 2004 Cohen, JCO, 2008 De Bono, Clin Can Res, 2008
 CONSORT DIAGRAM OF
CIRCULATING TUMOUR CELL (CTC)
COLLECTION IN S0421 STUDY

‹ Rising CTC at 3 weeks

associated with worse OS –
helpful for redirection and
optimization of therapy
Goldkorn A, et al., J Clin Oncol 2014;32 (11):1136-1142. Reprinted with permission. © (2014) American Society of Clinical Oncology. All rights reserved.
CLINICAL RELEVANCE
OF CTCS
Can changes in CTC counts
predict the efficacy of therapeutic
interventions (e.g., chemotherapy,
hormonal therapy)?
17 CENTRES PROVIDED DATA FOR
1944 ELIGIBLE PATIENTS
From 20 studies: Meta-analysis on raw data

2014
 CTCS VS. CONVENTIONAL
TUMOUR MARKERS
(Progression-free survival, p values) in metastatic breast cancer patients
receiving chemotherapy
Baseline 3-5 weeks 6-8 weeks
Model used
as reference CA15-3 BL + CEABL + CA15-3 BL + CEABL +
CTCBL CA15-3BL CEABL CTC3-5 CTC6-8
CA15-3 3-5 CEA 3-5 CA15-3 6-8 CEA 6-8
N patients 1193 914 593 436 357 289 279 215 170
CP 6 E-10 .10 .04

CP Few
.32 .12 5 E -05 .25 .35 9 E-05 .40
+CTCBL events
CP
+CTCBL .26 .41
+ CTC3-5
CP
Few
+CTCBL .36
+ CTC6-8 events

CTC=circulating tumour cells. CP=baseline clinicopathological model (appendix pp 3–5). CTCBL=CTC count at baseline.
CTC3–5=CTC count at 3–5 weeks. CTC6–8=CTC count at 6–8 weeks.
Bidard, Pierga, Michels, Pantel, et al., Lancet Oncology 2014, European Pooled Analysis of CTCs in metastatic BC (n=1944)
 IMPACT OF CTCS & LDH LEVEL
ON SURVIVAL
In prostate cancer patients treated with abiraterone
1.0

Probability of surviving 0.8

0.6

0.4

0.2

p < 0.001
0
0 3 6 9 12 15 18 21 24 27
Months from start of treatment
No. at risk
High risk 145 145 112 63 35 20 7 1 0 0
Intermediate risk 116 116 104 80 57 49 17 2 1 0
Low risk 450 450 439 405 364 329 238 110 14 1

The surrogate discriminates low-risk from high-risk patients

Reprinted with permission © 2015 American Society of Clinical Oncology. All rights reserved. Scher HI, et al., J Clin Oncol 2015; 33(12):1348-55
CTCS IN EARLY STAGE
CANCER PATIENTS
CHALLENGE
Very low number of CTCs
 PROGNOSTIC IMPACT OF CTC
IN BREAST CANCER PATIENTS
Without overt metastases
Multivariate analysis for DFS for different CTC cut-offs1
HR adjusted for treatment

Variable 0 vs ≥ 1 0, 1 vs ≥ 2 0–4 vs ≥ 5
CTCs in blood 1.878* 2.825* 4.035*
Pos/neg
Hormone receptor status 2.073* 2.020* 3.273
Pos/neg
Lymph node status 1.698* 1.664* 1.574*
Pos/neg
Grading 2.961* 3.182* 3.245
G1 vs G2–3
Tumour size 1.629* 1.655* 2.573*
T1 vsT2–4

*p<0.05

1. San Antonio Breast Cancer Symposium; December 8–12, 2010;

Rack B, Janni W, Pantel K, et al., J Natl Cancer Inst 2014;106:pii: dju066.
 PROGNOSTIC VALUE OF CTC IN
URINARY BLADDER CANCER
Survival outcomes: Independent prognostic factor
Median follow-up: 18 months

DFS HR: 4.6 CSS HR: 5.2

Reprinted from Eur Urol 61(4), Rink M, et al., Prognostic Role and HER2 Expression of Circulating Tumor Cells in Peripheral Blood of Patients Prior to Radical
Cystectomy: A Prospective Study, :810–7, copyright 2012 with permission of European Association of Urology.
 ERA-NET TRANSCAN:
CTC-SCAN PROJECT
High-risk Prostate Cancer (stage M0)
Partners: Germany, France, Greece, Poland, Austria

Coordinator: Klaus Pantel, Hamburg

 NEW APPROACH:
IN VIVO CAPTURE OF CTC

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

1. Image courtesy of GILUPI Nanomedizin GmbH. Available at: http://www.gilupi.com/

2. Saucedo-Zeni N, et al., A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and
structured medical wireInt J Oncol. 2012;1:1241–1250.This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported
License.
Gorges TM, et al., Clin Cancer Res 2016;22:2197-206
 TNM 2010: CTC IN NEW CM0(I+)
CLASSIFICATION

Distant Metastases (M)

M0 No clinical or radiographic evidence of
distant metastases
cM0(i+) No clinical or radiographic evidence of
distant metastases, but deposits of
molecularly or microscopically detected
tumour cells in circulating blood, bone
marrow, or other nonregional nodal tissue
that are no larger than 0.2 mm in a patient
without symptoms or signs of metastases
M1 Distant detectable metastases as
determined by classic clinical and
radiographic means and/or histologically
proven larger than 0.2 mm

AJCC 7th Ed Cancer Staging Manual

 HAEMATOGENEOUS SPREAD OF
PRIMARY BRAIN TUMOURS:
Detection of CTCs in glioma patients (~20%)

GFAP EGFR
stain amplification
Translational relevance

Glioma patients with CTCs may not be used

Single cell as transplant donors
isolation CTCs may serve as liquid biopsy

Single Cell
CGH

From Müller C, et al., The role of circulating tumor cells (CTCs) in cancer management. Sci transl Med; 2014, 6:247ra101. Reprinted with permission from AAAS.
MOLECULAR
CHARACTERISATION
OF CTC
Therapeutic targets
Resistance mechanisms
 DETECTION OF THERAPEUTIC
TARGETS ON CTC:
HER2 oncogene in breast cancer

Discordance
DETECT-III study: Anti-HER2 therapy (lapatinib) in metastatic between
breast cancer HER2
patients
status of primary
with HER2-negative primary tumours and HER2-positive CTC tumour
and CTC

Reprinted from Clinical Cancer Res. Copyright 2010, 16(9): 2634-2645, Riethdorf S, et al., Detection and HER2 Expression of Circulating Tumor Cells: Prospective
Monitoring in Breast Cancer Patients Treated in the Neoadjuvant GeparQuattro Trial, with permission from AACR.
Fehm T, et al., Breast Cancer Res Treat 2010;124(2):403-12. Ignatiadis M, et al., PlosONE, 2011; 6(1).e15624 - Ignatiadis/Pantel, et al., SABCS, 2011
 HETEROGENEITY OF ER STATUS IN
CTCS OF BREAST CANCER PATIENTS
With ER-positive primary tumours

ER CK DAPI CD45 Merge

E
R
+

E
R
-

ER-negative CTCs may survive endocrine therapy

Babayan A, Hannemann J, Spötter J, Müller V, Pantel K, Joosse SA (2013). Heterogeneity of Estrogen Receptor Expression in Circulating Tumor Cells from Metastatic
Breast Cancer Patients. PLoS ONE 8(9): e75038. © 2013 Babayan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
License.
 GENOMIC CHARACTERISATION
OF SINGLE CTC
CTC detection

CTC isolation

WGA +
‹ Mutation analysis
‹ CGH (conv./array)
‹ NextGen Sequencing

CTC Capillary CTC

Images courtesy of Prof Klaus Pantel and from Riethdorf S, et al., Clin Cancer Res 2007;13:920–8
 GENOMIC PROFILES (CNVS)
OF ER+ AND ER- CTCS
In breast cancer patients determined by NGS

ER- ER+

Presented at ISMRC 2013; Babayan A, Pantel K, et al., PlosONE 2013;8:e75038.

Images courtesy of Prof Klaus Pantel.
 AR-V7 IN CTCS OF METASTATIC
PROSTATE CANCER PATIENTS
Association with resistance to enzalutamide and arbiraterone

From Antonarakis ES, et al., AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028–38. Copyright © 2014,
Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.
 AR-V7 IN CTCS OF METASTATIC
PROSTATE CANCER PATIENTS
Association with resistance to enzalutamide and arbiraterone

From Antonarakis ES, et al., AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028–38. Copyright © 2014,
Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.
 DISTRIBUTION OF MUTATIONS IN
PRIMARY TUMOUR, METASTASES AND CTC
Colon cancer patient #6 Colon cancer patient #26

Deep targeted sequencing revealed that 17 of 20

“private CTC mutations䇿 were also present
in subclones of the primary tumour and metastases

Reprinted from Cancer Research. Copyright 2013, 73(10): 2965-75, Heitzer E, et al., Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH
and Next-Generation Sequencing, with permission from AACR.
FUNCTIONAL STUDIES
ON CTCS
FUNCTIONAL ANALYSES OF CTCS
IN XENOGRAFT ASSAYS AND CELL LINES
 NUCLEID ACIDS (DNA, RNA) AS
BLOOD-BASED BIOMARKERS
In cancer patients
Release by dying cells Active secretion by viable cells

Tumour cell
Tumour cell
Tumour cell

cell-free RNA

reee DNA
cell-free A

Exosomes

Schwarzenbach H, Pantel K, et al., Nature Rev. Cancer 2011; 426-437; Nature Rev. Clin. Oncol. 2014; Pantel K, et al., Nature Med. 2013; Speicher MR & Pantel K, Nature Biotech. 2014
 COMPARISON OF PLASMA DNA
CONCENTRATIONS IN PATIENTS
With localised M0 (n=69) and M1 (n=12)

Reprinted from Clinical Cancer Research. Copyright 2009, 15(3), 1032-1038, Schwarzenbach H, Cell-free Tumor DNA in Blood Plasma As a Marker for
Circulating Tumor Cells in Prostate Cancer, with permission from AACR.
 TMPRSS-ERG-ASSOCIATED 3 MB
DELETION ON CHROMOSOME 21
And mapping of the breakpoint on ctDNA in prostate cancer

Heitzer E, et al., Genome Medicine 2013;5:30. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 2.0)
(http://creativecommons.org/licenses/by/2.0)
 2013

How can the analysis of DNA fragments released from apoptotic/necrotic

cells reveal important information on resistant tumour cells surviving
therapy?
(Schwarzenbach, Hoon, Pantel, Nat. Rev. Cancer 2011; Pantel et al., Nature Med., 2013; Speicher & Pantel, Nat. Biotech. 2014)

Reprinted from Cancer Research. Copyright 2013, 73(21), 6384-6388, Pantel K, Alix-Panabières C, Real-time Liquid Biopsy in Cancer Patients: Fact or Fiction?,
with permission from AACR
 COMPARATIVE ANALYSIS OF CTCS
AND CTDNA IN BREAST CANCER
1. Progressive disease with increasing liver metastases and ascites – no chemoT

ctDNA levels may not always reflect disease progression

2. Excessive numbers of CTCs (~50.000/7.5 ml) in three blood samples; each with multiple homogeneous
in cancer patients
copy number changes and mutations in CTCs

CTCs analyses are not restricted to dying cancer cells and provide
CTC complementary information

3. However, very low concentration of ctDNA fragments at each measurement

ctDNA

Heidary M, et al., Breast Cancer Research 2014;16:421. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0) (http://creativecommons.org/licenses/by/4.0)
EFFECT OF CYTOTOXIC THERAPY
(E.G., CHEMOTHERAPY) ON CTCS
AND CTDNA
Before therapy Therapy sensitive tumour cells: undergo
nd release
apoptosis and eas DN
DNA

Tumour Î ctDNA

Therapy resistant tumour cells:

do not undergo apoptosis and can disseminate
After therapy through the blood

Î no ctDNA
Î CTCs available for DNA analyses

CTC Top: Tumour consists of heterogeneous clones that are sensitive

Tumo
mouuur
Tumour or resistant to cytotoxic therapies.
Bottom: Cytotoxic therapies kill sensitive tumour cells, leading
CTC
to the release of ctDNA from these dying cells into the
circulation, while CTCs are derived from resistant clones.
Reprinted by permission from Macmillan Publishers Ltd: Nature Medicine. Wan L, et al., Tumor metastasis: moving new biological insights into the clinic.Nat Med
2013:19:1450-1464. Copyright 2013.
 2016

CTCs and ctDNA provide

complementary information
as liquid biopsy

Alix-Panabières C, Pantel K. Cancer Discov 2016 ;6:479-91.

CENTER OF EXPERIMENTAL
MEDICINE INSTITUTE OF TUMOUR
BIOLOGY - KLAUS PANTEL

ERC Advanced Investigator Grant “DISSECT䇿 (2011-2016)

EU/IMI Consortium „CANCER-ID“ (2015-2019)
‹ Sabine Riethdorf/Christin Gasch ‹ Grant Support:
‹ Heidi Schwarzenbach DFG, BMBF, EU / ERC, EU/IMI Cancer-ID
‹ Harriet Wikman/Michaela Wrage Dt. Krebshilfe, Sander-Stiftung, Roggenbuck-Stiftung
‹ Katharina Effenberger
‹ Juliane Hannemann/Simon Joosse
‹ Kai Bartkowiak, Natalia Bednarz-Koll
THANK YOU!