THE ROLE OF CIRCULATING TUMOUR CELLS (CTCS) IN CANCER MANAGEMENT

Klaus Pantel, MD, PhD

Chairman, Institute for Tumour Biology

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

AIMS OF RESEARCH ON CTCS & CTDNA

Primarytumour

Local relapse

Recirculation

Tumour-mass dormancy

Metastasis

Escape

Tumourcell

dormancy

DTC

Micrometastasis

Distant tissue(e.g. bone marrow)

Blood CTCs

DTCs

Escape from dormancy (bone marrow):VCAM1 promotes osteoclast differentiation & activation & attracts osteoclast progenitors (Lu/Pantel/Kang et al. Cancer Cell 2011)

Tumour-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis (Ell/Pantel/Kang et al., Cancer Cell 2013)

Metabolic adaptation of DTCs is important for survival (LeBleu, Pantel, Kalluri et al., Nature Cell Biol. 2014)

TUMOUR CELL DISSEMINATION AND CANCER DORMANCY

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

Dormancy > 10 years

TUMOUR CELL DISSEMINATION, PLASTICITY AND EMT

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

Metastasis evolve many years after primary tumour resection and can harbor unique genomic alterationsBiopsy of metastases is an invasive and sometimes dangerous procedureDifferent metastatic sites show intra-patient heterogeneityCTC represent metastatic cells from different sites

The technical challenge: Finding one tumour cell in 106 – 108 normal blood cells

CTC AS LIQUID BIOPSY FOR METASTATIC CELLS

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, ClinChem, 2013; 59(1):110–118; permission conveyed through Copyright Clearance Center, Inc.

Biological propertiesProtein expression

anti-M markers Abanti-E markers Ab

anti-E/M markers Ab

- CellSearch® system - MagSweeper™- EPHESIA CTC-chip- CTC-chip- Velcro-like device

Ex vivo

- CellCollector®

- Photoacoustic nanodetector

In vivo

CC llllSS hh®® t

C llC ll t ®

Physical propertiesLabel-free strategies

CTC

CTC

(a)

(E.g., EpCAM)

(E.g., Plastin 3)

(E.g., N-Cadherin)

(e)

(d)

(c)

++++

++

+

+

- -

-- ----

++++++++

-----

+++++++++

DEP

+++++++++ + -- ---

----++++++++++++ ---------DEP

(f)

Anti-CD45

WBC

Negative selection(b)

CTCs

Positive selection

WBC

CTC-iChip(g) Out

)

RBCCTC

WBC

RBC

WWCTCC

anti-E markers Ab

Anti-CD45

CTC ENRICHMENT STRATEGIES

Alix-Panabieres C & Pantel K,.Nature Rev.Cancer 2014;14:623–31Images courtesy of Universitätsklinikum Hamburg Eppendorf

anti-tumour associatedmarkers Ab

anti-E/M markers Ab

anti-tissue-specificmarkers Ab

CTC

(E.g., CK, Vimentin, E/N-Cadherin)

(E.g., PSA, Mammaglobin, MAGE)

(E.g., HER2, EGFR)

Immunocytologicaltechnologies

- Immunocytochemistry- CellSearch® system - Flow Cytometry- DEParray®

Technologiesg

Functional assaysMolecular technologies

Liquid bead array

RNA-based Technologies

CTC mRNA

Xenotransplantation models (CDx)

Viable CTC with stem-cell properties

Cell culture

antiananananaananaannaanaan -nnnnttttttttttt marker Abstititiitittitt -------mamamaamamaaaaaaaaarkrkrkrkrkrrkrkkrkrrkkkerererererererererereeere AAAAAAAAAAAAAAAbsbsbsbsbsbsbssbsssbssbsmmmmmmmmmmmmmmmmmmm(E.g., CK19, HER2, EGFR, VEGF, PSA)- EPISPOT

Cell culture

Viable CTC

- Invasion assay

In vitro Cell Culture

Fluo matrix

FunctionalCTC

Metastases

RRNNAA-bbaasseedd TTeecchhnnolo

LLiiquid bead arrayA

RT-qPCR(single/multiple genes)

mRNm A

R)

Secreted protein

days

ImmunospotsAb1

Fluo Ab2

APPROACHES FOR CTC DETECTION

Alix-Panabieres C & Pantel K,.Nature Rev.Cancer 2014;14:623–31Images courtesy of Universitätsklinikum Hamburg Eppendorf

MagNestTM

Epithelial Cell Kit

Enrichment of CTC with anti-EpCAM ferro fluids:

Captures tumour cells with very low EpCAM expression

7.5ml

CELLSEARCH™ SYSTEM (FDA-CLEARED)

CK-PEpos

DAPIpos

CD45-APCneg

Tumour cell

Cytoplasm Nucleus Cell Membrane Composite

=+ -

Leukocyte nucleus CD45+ membrane LeukocyteTumour cell

CELLSEARCH™ SYSTEM: IMAGES OF TUMOUR CELLS

Images courtesy of Universitätsklinikum Hamburg Eppendorf

PROGNOSTIC VALUE OF CTC COUNTS FOR SURVIVALIn cancer patients with advanced disease

Breast CancerChristofanilli, NEJM, 2004

Prostate CancerDe Bono, Clin Can Res, 2008

Colorectal CancerCohen, JCO, 2008

FDA approval1) 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), 2008:3213-32213) 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 SURVIVALIn cancer patients with advanced disease

Breast CancerCristofanilli, NEJM, 2004

Prostate CancerDe Bono, Clin Can Res, 2008

Colorectal CancerCohen, JCO, 2008

Rising CTC at 3 weeks associated with worse OS –helpful for redirection and optimization of therapy

CONSORT DIAGRAM OF CIRCULATING TUMOUR CELL (CTC) COLLECTION IN S0421 STUDY

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 CTCSCan changes in CTC counts predict the efficacy of therapeutic interventions (e.g., chemotherapy, hormonal therapy)?

2014

17 CENTRES PROVIDED DATA FOR 1944 ELIGIBLE PATIENTSFrom 20 studies: Meta-analysis on raw data

Model usedas reference

Baseline 3-5 weeks 6-8 weeks

CTCBL CA15-3BL CEABL CTC3-5 CA15-3 BL + CA15-3 3-5

CEABL + CEA 3-5 CTC6-8 CA15-3 BL +

CA15-3 6-8CEABL + CEA 6-8

N patients 1193 914 593 436 357 289 279 215 170

CP 6 E-10 .10 .04

CP+CTCBL .32 .12 5 E -05 .25 .35 9 E-05 .40 Few

eventsCP+CTCBL+ CTC3-5

.26 .41

CP+CTCBL+ CTC6-8

.36 Few events

CTCS VS. CONVENTIONAL TUMOUR MARKERS(Progression-free survival, p values) in metastatic breast cancer patients receiving chemotherapy

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 SURVIVALIn prostate cancer patients treated with abiraterone

Prob

abilit

y of s

urviv

ing

242115930

0

0.2

0.4

0.6

0.8

1.0

Months from start of treatmentNo. at risk

6 12 18 27

High riskIntermediate risk

Low risk

145116450

145116450

112104439

6380

405

3557

364

2049

329

717

238

12

110

01

14

001

p < 0.001

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 PATIENTSCHALLENGEVery low number of CTCs

PROGNOSTIC IMPACT OF CTC IN BREAST CANCER PATIENTSWithout overt metastases

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.

Variable

HR adjusted for treatment

0 vs ≥ 1 0, 1 vs ≥ 2 0–4 vs ≥ 5

CTCs in bloodPos/neg

1.878* 2.825* 4.035*

Hormone receptor statusPos/neg

2.073* 2.020* 3.273

Lymph node statusPos/neg

1.698* 1.664* 1.574*

GradingG1 vs G2–3

2.961* 3.182* 3.245

Tumour sizeT1 vsT2–4

1.629* 1.655* 2.573*

*p<0.05

Multivariate analysis for DFS for different CTC cut-offs1

PROGNOSTIC VALUE OF CTC IN URINARY BLADDER CANCERSurvival 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 PROJECTHigh-risk Prostate Cancer (stage M0) Partners: Germany, France, Greece, Poland, Austria

Coordinator: Klaus Pantel, Hamburg

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

NEW APPROACH: IN VIVO CAPTURE OF CTC

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 UnportedLicense.Gorges TM, et al., Clin Cancer Res 2016;22:2197-206

1

2

TNM 2010: CTC IN NEW CM0(I+) CLASSIFICATION

Distant Metastases (M)M0 No clinical or radiographic evidence of

distant metastasescM0(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

GFAP stain

EGFR amplification

Single cell isolation

Single CellCGH

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

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.

Translational relevance

Glioma patients with CTCs may not be used as transplant donors

CTCs may serve as liquid biopsy

MOLECULAR CHARACTERISATION OF CTCTherapeutic targets Resistance mechanisms

Discordance between HER2 status of primary tumour

and CTC

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

DETECTION OF THERAPEUTIC TARGETS ON CTC: HER2 oncogene in breast cancer

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

With ER-positive primary tumours

ER+

ER-

ER CK DAPI CD45 Merge

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.

HETEROGENEITY OF ER STATUS IN CTCS OF BREAST CANCER PATIENTS

CTC detection

CTC Capillary CTC

WGA + Mutation analysisCGH (conv./array)NextGen Sequencing

GENOMIC CHARACTERISATION OF SINGLE CTC

CTC isolation

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- CTCSIn 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 PATIENTSAssociation 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.

Colon cancer patient #6 Colon cancer patient #26

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

in subclones of the primary tumour and metastases

DISTRIBUTION OF MUTATIONS IN PRIMARY TUMOUR, METASTASES AND CTC

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

cell-free DNA

Tumour cell

Tumour cell

reee DNAA

Tumour cell

Exosomes

cell-free RNA

Release by dying cells Active secretion by viable cells

NUCLEID ACIDS (DNA, RNA) AS BLOOD-BASED BIOMARKERSIn cancer patients

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 PATIENTSWith 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 21And 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)

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)

2013

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

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

CTC

1. Progressive disease with increasing liver metastases and ascites – no chemoT

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

ctDNA

ctDNA levels may not always reflect disease progression in cancer patients

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

COMPARATIVE ANALYSIS OF CTCS AND CTDNA IN BREAST CANCER

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)

Therapy sensitive tumour cells: undergo apoptosis and release DNA

ctDNA

Therapy resistant tumour cells: do not undergo apoptosis and can disseminate through the blood

no ctDNACTCs available for DNA analyses

nd release DNeas

After therapy

CTC

CTC

Before therapy

Tumour

TumourururTumomouu

EFFECT OF CYTOTOXIC THERAPY (E.G., CHEMOTHERAPY) ON CTCS AND CTDNA

Top: Tumour consists of heterogeneous clones that are sensitive or resistant to cytotoxic therapies.Bottom: Cytotoxic therapies kill sensitive tumour cells, leading 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 providecomplementary informationas liquid biopsy

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

CENTER OF EXPERIMENTAL MEDICINE INSTITUTE OF TUMOUR BIOLOGY - KLAUS PANTEL

Sabine Riethdorf/Christin GaschHeidi SchwarzenbachHarriet Wikman/Michaela WrageKatharina EffenbergerJuliane Hannemann/Simon JoosseKai Bartkowiak, Natalia Bednarz-Koll

Grant Support: DFG, BMBF, EU / ERC, EU/IMI Cancer-IDDt. Krebshilfe, Sander-Stiftung, Roggenbuck-Stiftung

ERC Advanced Investigator Grant “DISSECT (2011-2016)EU/IMI Consortium „CANCER-ID“ (2015-2019)

THANK YOU!