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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