Klaus PantelInstitut für Tumorbiologie

TUMOR DORMANCY: RELEVANCE IN BREAST CANCER

Primary tumor

Local relapse

Recirculation

Tumor-mass

dormancy

Metastasis

Escape

Tumor cell dormancy

DTC

Micrometastasis

Distant tissue(e.g. bone marrow)

Blood

NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 6 JUNE 2009 339

Tumor cell dissemination and cancer dormancy

VCAM1 promotes osteoclast differentiation & activation & attracts osteoclast progenitors (Lu/Pantel/Kang et al Cancer Cell 2011)Tumor-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)RAI2 as new metastasis-suppressor (Werner, Wikman, Pantel et al, Cancer Discovery 2015)Exosome-mediated homing of metastatic cells to specific distant sites (Hoshino, Pantel, Bissell, Peinado, Lyden et al., Nature, 2015)

CTCs

DTCs

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?

Kang & Pantel, Cancer Cell 2013

MNC

Ficollgradient

Bonemarrow

2 x 106 MNCper 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%)

DTC in bone marrow of cancer patients

MNC

Ficollgradient

Bonemarrow

2 x 106 MNCper 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%)

DTC in bone marrow of cancer patients

• DTC detection correlates with metastatic AND locoregional relapse• Most DTC are Ki67- and have CD44+/CD24- stem cell phenotype

• DTCs can occupy the hematopoietic stem cell niches

Breast Cancer

Prostate cancer*

Lung cancer (NSCLC)

Gastric cancer

Esophageal cancer

Colorectal cancer

Pancreatic cancer

Head and neck cancer

20-40

20-30

40-60

35-60

30-40

20-30

10-20

20-30

Tumor type Detection rate (%)

BM is a homing organ and putative reservoirfor DTC derived from various primary sites

vs.

Classification of early stage breast tumors according to DTC status

Expression profiling on micro-dissected tumor tissue

Bioinformatic analysisand validation

of identified genes

Identification of DTC-associated genes

BM- BM+

Luminal A/B, HR+ Luminal A/B, HR+

RAI2→

Werner, Wikman, Pantel et al, Cancer Discovery, 2015

S

RAI2

GATA3FOXOA1GRHL2

CTBP2

CTBP2GATA3FOXOA1GRHL2

VimentinZEB1SNAI1 SNAI2

CDH1

Hematogenous dissemination

All-trans retinoic acid

RAI2 loss

Editorial: Esposito & Kang, Cancer Discovery, 2015

RAI2 as potential new metastasis-suppressor gene

Werner, Wikman, Pantel et al, Cancer Discovery, 2015

Detection of CTC in the peripheral blood

August 2015: > 16,000 reports in PubMed

Advantages over DTC detection:• Less invasive than BM sampling

• Pool of DTC from multiple distant sites

2010

• 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 Liquid Biopsy

Alix-Panabières & Pantel, Clin Chem, 2013; Pantel & Alix-

Panabieres, Cancer Res. 2013

CTC as Liquid Biopsyfor 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.

Intra-patient heterogeneity of metastases at different sites

CTC/ctDNA might reveal representative informationon metastatic cells located atdifferent sites

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

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

anti-E markers Ab

Anti-CD45

Alix-Panabieres & Pantel, Nature Rev. Cancer 2014

New approach: In vivo capture of CTC

Lung cancer CTCs: Gorges, Pantel et al., CCR 2015

anti-tumor associated markers Ab

anti-E/M markers Ab

anti-tissue-specific markers Ab

CTC

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

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

(E.g., HER2, EGFR)

Immunocytological technologies

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

Technologies

Functional assaysMolecular technologies

Liquid bead array

RNA-based Technologies

CTC mRNA

Xenotransplantation models (CDx)

Viable CTC with stem-cell properties

Cell culture

anti-marker Abs(E.g., CK19, HER2, EGFR, VEGF, PSA)- EPISPOT

Viable CTC

- Invasion assay

In vitro Cell Culture

Fluo matrix

Functional CTC

Metastases

RT-qPCR (single/multiple genes)

Secreted protein

days

ImmunospotsAb1

Fluo Ab2

Alix-Panabieres & Pantel, Nature Rev. Cancer 2014

Approaches for CTC detection

Bednarz-Knoll, Alix-Panabières & Pantel Cancer & Met Rev 2012

Epithelial-Mesenchymal Plasticity of CTCs

EpCAM, CK Vimentin

Intermediate E/M Phenotypes:Potential MICs!

CTCs in early stage cancer patients:

Detection of MRD for risk assessment and

improved staging

MagNestTM

Epithelial Cell Kit

CellSearch™ System (FDA-cleared)

Enrichment of CTC with anti-EpCAM ferro fluids:

Captures tumor cells with very low EpCAM

expression

7.5ml

CellSearch™ System: Images of Tumor Cells

CK-PEpos

DAPIpos

CD45-APCneg

Tumor Cell

Cytoplasm Nucleus Cell Membrane Composite

=+ -

Leukocyte nucleus

CD45+

Membrane

LeukocyteTumor Cell

Prognostic impact of CTC in breast cancer patients without overt metastases

Rack, Janni, Pantel et al, JNCI 2014

TNM 2010: CTC in new cM0(i+) Classification

Monitoring of CTCs:

Can early changes in CTC counts predict

the efficacy of therapeutic interventions (e.g., chemotherapy, hormonal therapy)?

2014

17 centres provided data for 1944 eligible patients from 20 studiesMeta-analysis on raw data.

CTCs vs. conventional tumor markers (PFS, p values) in metastatic

breast cancer patients receiving chemotherapy

Model used as referen-ce

(

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 6-8

CEABL + 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

Bidard, Pierga, Michels, Pantel et al, Lancet Oncology 2014, European Pooled

Analysis of CTCs in metastatic BC (n=1944)

Characterization of CTCs at the

DNA, RNA and protein level:

- Therapeutic targets

- Resistance mechanisms

Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer

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

Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer

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

Genomic profiles (CNAs) of ER+ and ER-CTCs in breast cancer patients

determined by NGS

ER+ER-

The blockade of immune checkpoints in cancer immunotherapyDrew M. Pardoll

Nature Reviews Cancer 12, 252-264 (April 2012)

PD1-PDL1 mediated immune blockade as cancer target

PD-L1 expression on CTCs in breast cancer

Mazel, Pantel, Alix-Panabieres et al, Mol. Oncol. 2015(Editorial by R. David in Lancet Oncol. 2015)

PD-L1 expression on CTCs in breast cancer

Mazel, Pantel, Alix-Panabieres et al, Mol. Oncol. 2015(Editorial by R. David in Lancet Oncol. 2015)

PD-L1 is frequently expressed on CTCs (> 60% of patients) in metastatic

breast cancer patients

Functional studies on

CTCs

DTC cell lines as models for functional analyses

Stem cell markersUnfolded protein response

Bartkowiak et al., J Prot Res 2010 & Cancer Res. 2015Grabinski/Jücker et al., Cell Signal. 2011Bartkowiak et al., Cancer Res. 2015

DTC adapt to hypoxic conditions in bone marrow

Potential Metastasis-initiating Cells: EPCAMlow, CD44+, CD47+ and cMET+

2013

How can the analysis of DNA fragments released from apoptotic/necrotic cells reveal important information on

resistant tumor cells surviving therapy? (Schwarzenbach, Hoon, Pantel, Nat. Rev. Cancer 2011; Pantel et al., Nature

Med., 2013; Speicher & Pantel, Nat. Biotech. 2014)

2013

Comparative analysis of CTCs and ctDNA in breast cancer

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

Heidary, Speicher, Pantel et al, Breast Cancer Res. 2014

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

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

ctDNA

Comparative analysis of CTCs and ctDNA in breast cancer

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

Heidary, Speicher, Pantel et al, Breast Cancer Res. 2014

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

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.

cell-free DNA

Tumor cell

Tumor cell

Tumor cell

Exosomes (Proteins, RNA, DNA?)

cell-free RNA

Release by dying cells

Nucleid Acids (DNA, microRNA) as blood-based biomarkers in cancer patients

Active secretion by viable cells

Schwarzenbach, Pantel et al., Nature Rev. Cancer 2011; Nature Rev. Clin. Oncol. 2014; Pantel et al., Nature Med. 2013; Speicher & Pantel, Nature Biotech. 2014

Tumor-educated platelets as new blood biomarkers(Best et al., Cancer Cell 2015; Editorial from Joosse/Pantel)

CANCER-ID EU Konsortium 2015-2020

Non-EFPIA/non-SME

Non-profitorganizations

SMEs

ClinicalsitesEFPIA

Academicinstitutions

32 partners:• 6 EFPIA companies (lead Bayer, co-

lead Menarini)• 17 academic/clinical sites• 6 SMEs• 2 non-profit organizations• 1 non-SME/non-EFPIA

Prof. Klaus Pantel

Prof. LeonTerstappen

Page 43 CANCER-ID evaluation hearing Brussels- Oct 7th 2014

Scientific Managment: Klaus Pantel, UKE (Leon Terstappen)Coordination: Thomas Schlange, BAYER (Barbara Baggiani)

CANCER-ID EU Konsortium 2015-2020

Non-EFPIA/non-SME

Non-profitorganizations

SMEs

ClinicalsitesEFPIA

Academicinstitutions

32 partners:• 6 EFPIA companies (lead Bayer, co-

lead Menarini)• 17 academic/clinical sites• 6 SMEs• 2 non-profit organizations• 1 non-SME/non-EFPIA

Prof. Klaus Pantel

Prof. LeonTerstappen

Page 44 CANCER-ID evaluation hearing Brussels- Oct 7th 2014

Scientific Managment: Klaus Pantel, UKE (Leon Terstappen)Coordination: Thomas Schlange, BAYER (Barbara Baggiani)

Blood-based Diagnostic in Lung and Breast Cancer (CTCs, ctDNA & cfmiRNA)33 EU Partners (Academic institutions &

EFPIA companies)

Center of Experimental Medicine

Institute of Tumor Biology - Klaus Pantel

• Sabine Riethdorf/Tobias Gorges• Heidi Schwarzenbach, B. Steinbach• Harriet Wikman/Stefan Werner• Simon Joosse, Anna Babayan• Kai Bartkowiak, Natalia Bednarz-Koll• A. Andreas, C. Coith, J. Kropidlowski, M. Stoupiec

Grant Support:

DFGBMBFEU / ERCDt. KrebshilfeSander-Stiftung Roggenbuck-Stiftung

Center of Experimental Medicine

Institute of Tumor Biology - Klaus Pantel

• Sabine Riethdorf/Tobias Gorges• Heidi Schwarzenbach, B. Steinbach• Harriet Wikman/Stefan Werner• Simon Joosse, Anna Babayan• Kai Bartkowiak, Natalia Bednarz-Koll• A. Andreas, C. Coith, J. Kropidlowski, M. Stoupiec

Grant Support:

DFGBMBFEU / ERCDt. KrebshilfeSander-Stiftung Roggenbuck-Stiftung

Klaus Pantel, Hamburg: ERC Advanced Investigator Grant

„DISSECT“ (2011-2016)

Micrometastasis Research Network at UCCH/UKE

Institut fürAnatomie II

Institut fürKlinischeChemie

Institut fürRechtsmedizin

Labor fürStrahlenbiologie und -onkologie

Institut fürBiochemie und

MolekularbiologieII. Medizinische

Klinik undPoliklinik

Klinik undPoliklinik für

Urologie

Klinik und Poli-klinik für Mund-,

Kiefer- und Gesichts-chirurgie

Klinik undPoliklinik für

Neurochirurgie

I. MedizinischeKlinik undPoliklinik

Klinik undPoliklinik für

Viszeralchirurgie

Klinik undPoliklinik für Gynäkologie

Institut fürTumorbiologie

Institut fürTumorbiologie