Klaus Pantel Klaus Pantel Liquid Biopsy: Current Status and Future - - PowerPoint PPT Presentation

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Institute Institute for Institute Institute for for Tumor for Tumor Tumor Biology Tumor Biology Biology Biology Klaus Pantel Klaus Pantel Liquid Biopsy: Current Status and Future Perspective Perspective CTC CTC as as Liquid

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Institute Institute for for Tumor Tumor Biology Biology Institute Institute for for Tumor Tumor Biology Biology

Klaus Pantel Klaus Pantel “Liquid Biopsy”: Current Status and Future Perspective Perspective

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CTC CTC as as Liquid Liquid Biopsy Biopsy for for metastatic metastatic cells cells for for metastatic metastatic cells cells

Metastasis evolve many years y y after primary tumor resection and can harbor unique genomic alterations. Biopsy of metastases is an invasive and sometimes invasive and sometimes dangerous procedure. Can the molecular Can the molecular characterizaton of CTC reveal representative information

  • n metastatic cells located at

different sites ?

Alix Alix-

  • Panabières & Pantel,

Panabières & Pantel, Clin Clin Chem Chem, , 2012 2012

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Detection Detection of

  • f CTC in

CTC in the the peripheral peripheral blood blood

September 2013: September 2013: 400 i t d 400 i t d li i l li i l t i l t i l ith ith CTC CTC > 400 registered > 400 registered clinical clinical trials trials with with CTC CTC as as biomarkers biomarkers biomarkers biomarkers

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CTC Enrichment Methods CTC Enrichment Methods Enr chm nt M tho s Enr chm nt M tho s

2013: > 50 different CTC ! assays ! The The technical technical challenge challenge: : Finding Finding one

  • ne

tumor tumor cell cell in in 10 106 – 10 108 normal normal blood blood

Alix Alix-

  • Panabières & Pantel,

Panabières & Pantel, Annu Rev Med Annu Rev Med, 2012 , 2012 Pantel & Alix Pantel & Alix-

  • Panabières,

Panabières, Trends Mol Med Trends Mol Med, 2010 , 2010 Alix Alix-

  • Panabières & Pantel,

Panabières & Pantel, Clin Chem Clin Chem, , 2012 2012

cells cells

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New approach: In vivo capture of CTC (1.5 L blood)

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

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CTC Identification Methods CTC Identification Methods

Real Real‐time RT time RT‐PCR PCR

Cytokeratins as standard CTC markers

nucleic acids nucleic acids

Cytokeratins as standard CTC markers BUT differential expression of individual CKs

(Joosse/Pantel et al., Clin Cancer Res 2012)

DNA

nucleic acids nucleic acids

mRNA DNA

intra intra-

  • cytoplasmic proteins

cytoplasmic proteins membrane proteins membrane proteins

> 13,000 publications on CTC in PubMed 2013

mRNA

Immunocytochemistry Immunocytochemistry

PubMed 2013

secreted secreted

Tumor cell Tumor cell

EPISPOT assay EPISPOT assay

proteins by proteins by VIABLE cells VIABLE cells

Alix Alix-

  • Panabières

Panabières et al., et al., Clin Cancer Res, 2008 Clin Cancer Res, 2008

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Design Design of

  • f robust

robust automated automated systems systems for for reproducible reproducible CTC CTC detection detection

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CellSearch™ System CellSearch™ System (FDA

(FDA-

  • cleared)

cleared)

MagNest MagNestTM

TM

Epithelial Cell Kit

Enrichment of CTC with Enrichment of CTC with anti anti-

  • EpCAM ferro fluids

EpCAM ferro fluids

Cristofanilli et al., NEJM, 2004 Cristofanilli et al., NEJM, 2004 Riethdorf et al., CCR, 2007 & 2010 Riethdorf et al., CCR, 2007 & 2010

p

DeBono et al, CCR, 2008 DeBono et al, CCR, 2008 Cohen et al, JCO, 2008 Cohen et al, JCO, 2008 Krebs et al, JCO, 2012 Krebs et al, JCO, 2012

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CellSearch CellSearch™ System ™ System: Images of Tumor Cells : Images of Tumor Cells

Cytoplasm Nucleus Cell Membrane Composite Cytoplasm Nucleus Cell Membrane Composite

CK CK-

  • PE

PE pos pos DAPI DAPI pos pos CD45 CD45-

  • APC

APC neg neg Tumor Cell Tumor Cell p g

= +

  • Leukocyte

Leukocyte nucleus nucleus CD45 CD45+

+

Membrane Membrane Leukocyte Leukocyte Tumor Cell Tumor Cell

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Prognostic value of CTC counts for survival Prognostic value of CTC counts for survival i ti t ith d d di i ti t ith d d di in cancer patients with advanced disease in cancer patients with advanced disease

Breast Cancer Breast Cancer

Christofanilli NEJM 2004 Christofanilli NEJM 2004

Prostate Cancer Prostate Cancer

De Bono, Clin Can Res, 2008 De Bono, Clin Can Res, 2008

Colorectal Cancer Colorectal Cancer

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

 FDA FDA clearance clearance  FDA FDA clearance clearance

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Detection Detection of

  • f CTC in

CTC in early early stage stage cancer cancer patients patients ( (low low CTC CTC counts counts): ):

Is the ability to release cancer cells into the circulation relevant for the d l t f di t t t t ? development of distant metastases?

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Prognostic impact of CTC in breast cancer patients without overt metastases

Rack, Janni et al, unpublished

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Meta-Analysis of 49 studies comprising 6815 breast cancer patients p

1

Progression-free survival CTC detection: ICC & RT-

0.86 0.78 0.62 0.58

0.78

0.6 0.8 1 ess Free CTC neg CTC pos

CTC detection: ICC & RT PCR

0.34

0.51 0.29 0.15

0.2 0.4 0.6 Proportion Progre

p<0.001

Overall survival

0.89 0.69 0.64 0.79 0.8 1 . CTC neg CTC pos

0.15 0.06

1 2 3 4 5 Time (years) 0.51 0.47 0.56 0.40 0.29 0.4 0.6

  • rtion Surviving

0.19 0.2 1 2 3 4 5

Ti ( )

Propo

p<0.001

Zhang L, Riethdorf S…Pantel K, Clin Cancer Res, 2012.

Time (years)

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

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Challenge Challenge of

  • f CTC

CTC detection detection: Challenge Challenge of

  • f CTC

CTC detection detection: Epithelial Epithelial-Mesenchymal Mesenchymal Transition (EMT) Transition (EMT) Epithelial Epithelial-Mesenchymal Mesenchymal Transition (EMT) Transition (EMT)

  • f
  • f carcinoma

carcinoma cells cells

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(Bednarz-Knoll et al CMR 2012; Kang &

Tumor cell dissemination, plasticity and EMT

Pantel, Cancer Cell 2013)

Dormancy 10 > 10 years

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Epithelial-Mesenchymal Plasticity of CTC EpCAM CK Vimentin EpCAM, CK Vimentin

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

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Expression profile of CTCs in breast cancer EMT EMT

(Mani/Weinberg, (Mani/Weinberg, et al et al., ., Cell Cell, 2008;) , 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 Yokobori, Mimori, Pantel, Mori et al. Plastin-3 as new CTC marker not downregulated during EMT, Cancer Res. Febr. 2013

Bednarz-Knoll, Alix-Panabieres & Pantel, 2011, Breast Cancer Res, 13: 282-293

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M l l M l l Ch t i ti Ch t i ti f CTC CTC Molecular Molecular Characterization Characterization of

  • f CTC

CTC f th ti th ti t t t t for for therapeutic therapeutic targets targets

( l ( l ti li id ti li id bi bi “) („real („real-time liquid time liquid biopsy biopsy“)

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

CTC without HER2 gene amplification

Red: CK Red: Cen17

CTC without HER2 gene amplification CTC without HER2 gene amplification

Red: CK Red: Cen17

A

Composite CK DAPI CD45 HER2

A

Composite CK DAPI CD45 HER2

DETECT DETECT III study: Anti III study: Anti HER2 therapy (lapatinib) in metastatic breast cancer HER2 therapy (lapatinib) in metastatic breast cancer

CTC with HER2 gene amplification

Red: CK ICC Green: HER2 FISH Red: Cen17 FISH

CTC with HER2 gene amplification CTC with HER2 gene amplification

Red: CK ICC Green: HER2 FISH Red: Cen17 FISH

A

1+ 2+ CTC

A

1+ 2+ CTC

DETECT DETECT-III study: Anti III study: Anti-HER2 therapy (lapatinib) in metastatic breast cancer HER2 therapy (lapatinib) in metastatic breast cancer patients with HER2 patients with HER2-

  • negative primary tumors and HER2

negative primary tumors and HER2-

  • positive CTC

positive CTC

CTC with HER2 gene amplification CTC with HER2 gene amplification CTC with HER2 gene amplification

CB11 A0485 FISH Composite CK DAPI CD45 HER2

B

2+ 3+

C

CB11 A0485 FISH Composite CK DAPI CD45 HER2

B

2+ 3+

C

SK-BR-3 SK-BR-3 SK-BR-3

MCF-7 BT20 1+ MCF-7 BT20 1+ T47D MDA- MB-453 1+ 2+ T47D MDA- MB-453 1+ 2+

Discordance between HER2 Discordance between HER2 status of primary tumor and status of primary tumor and CTC CTC

SK-BR-3 BT474 3+ 3+ SK-BR-3 BT474 3+ 3+

CTC CTC

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

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Heterogeneity of ER status in CTCs of breast cancer patients with ER-positive primary tumors

Babayan, Joosse, Pantel et al., PLOS ONE 2013

ER CK DAPI CD45 Merge

Babayan, Joosse, Pantel et al., PLOS ONE 2013

E R

ER CK DAPI CD45 Merge

R + E R R

  • ER-negative CTCs may survive endocrine therapy
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Genomic Characterization of single CTC Genomic Characterization of single CTC

CTC detection CTC isolation WGA +

  • Mutation analysis
  • Mutation analysis
  • CGH (conv./array)

NextGen Sequencing CTC

Capillary

CTC

  • NextGen Sequencing
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Detection of mutations in genes relevant for resistance of targeted therapies (eg EGFR inhibition) targeted therapies (eg, EGFR inhibition)

Gasch, Pantel, Riethdorf et al., Clin. Chem. 2012

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Distribution of mutations in primary tumor, metastases and CTC

CRC patient #6 CRC patient #26

Deep targeted sequencing revealed that 17 of 20 „private CTC mutations“ were also present in „private CTC mutations were also present in subclones of the primary tumor and metastases

Heitzer, Riethdorf, Pantel, Speicher et al, Cancer Res. 2013

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Tumor Tumor-

  • associated

associated circulating cell circulating cell-

  • free nucleic

free nucleic

CTC

g acids in blood acids in blood

Micrometastasis

KRAS Mutations: KRAS Mutations:

C l ti CTC & Ci l ti T DNA

Diaz et al, Nature 2012 Heitzer et al, IJC 2013

microRNAs: microRNAs:

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

Roth et al, BCR 2010 & PLosONE 2012

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 )

Schwarzenbach/ Schwarzenbach/Hoon Hoon/Pantel, /Pantel, Nat Nat Rev Rev Cancer, Cancer, 2011, Alix 2011, Alix-

  • Panabières

Panabières/Schwarzenbach/Pantel, /Schwarzenbach/Pantel, Annu Annu Rev Rev Med Med, 2012; Pantel & Alix , 2012; Pantel & Alix-

  • Panabieres, Cancer

Panabieres, Cancer Res Res., 2013 ., 2013

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Aims Aims of

  • f Research on

Research on Circulating Circulating Tumor Cells Tumor Cells g

  • Estimation

Estimation of

  • f the

the risk risk for for metastatic metastatic relapse relapse or

  • r

metastatic metastatic progression progression (prognostic prognostic information information)

  • Stratification

Stratification & & real real-

  • time

time monitoring monitoring of

  • f therapies

therapies

  • Identification

Identification of

  • f therapeutic

therapeutic targets targets and and resistance resistance p g mechanisms mechanisms (biological biological therapies therapies)

  • Understanding

Understanding the the biology biology of

  • f metastatic

metastatic development development

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early dissemination early dissemination

Metastasis Models

early stage cancer early stage cancer DTCs DTCs early stage cancer early stage cancer genetic genetic progression progression  Dormancy advanced stage cancer advanced stage cancer

  • vert metastasis
  • vert metastasis

DTCs DTCs

  • vert metastasis
  • vert metastasis

no metastasis no metastasis

Pantel Pantel et al., et al., Nature Nature Rev Rev Cancer Cancer 2008 2008

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Cancer Cancer Dormancy Dormancy: : Research Research questions questions

D ll ll ti t ti t h d t d t t ll ll ?

  • Do

Do all all cancer cancer patients patients have have dormant dormant tumor tumor cells cells?

  • Can

Can host host factors factors induce induce or

  • r break

break dormancy dormancy? ? Stress? Stress? Inflammation Inflammation? ?

  • Are

Are there there preferred preferred reservoirs reservoirs of

  • f dormant

dormant cells cells (e

(e. .g g. ., , bone bone marrow marrow) ) ?

  • Does

Does the the immune immune system system play play a role role in in dormancy dormancy?

  • Does

Does the the immune immune system system play play a role role in in dormancy dormancy?

  • What

What is is the the effect effect

  • f
  • f

current current therapies therapies

  • n
  • n

dormant dormant cells cells or

  • r

dormancy dormancy? ?

  • What

What signaling signaling pathways pathways or

  • r events

events reactivate reactivate dormant dormant cells cells? ? g g g g p y p y

  • Do

Do dormant dormant cells cells have have properties properties of

  • f cancer

cancer stem stem cells cells? ?

  • How

How does does genetic genetic background background affect affect dormancy dormancy? ?

Uhr & Pantel PNAS 2011; Kang & Pantel, Cancer Uhr & Pantel PNAS 2011; Kang & Pantel, Cancer Cell Cell 2013 2013

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Metastasis Metastasis Biology Biology gy gy

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Center of Experimental Medicine Center of Experimental Medicine Institute of Tumor Biology Institute of Tumor Biology -

  • Klaus Pantel

Klaus Pantel gy gy

  • Sabine Riethdorf/Christin Gasch

Sabine Riethdorf/Christin Gasch

Grant Support: Grant Support:

  • Heidi Schwarzenbach

Heidi Schwarzenbach

  • Harriet Wikman/Michaela

Harriet Wikman/Michaela Wrage Wrage

  • Katharina Effenberger

Katharina Effenberger

DFG DFG BMBF BMBF EU / ERC EU / ERC

  • Katharina Effenberger

Katharina Effenberger

  • Simon Joosse, Anna

Simon Joosse, Anna Babayan

  • Kai Bartkowiak, Natalia Bednarz

Kai Bartkowiak, Natalia Bednarz-

  • Koll

Koll

  • Dt. Krebshilfe
  • Dt. Krebshilfe

Sander Sander-

  • Stiftung

Stiftung Roggenbuck Roggenbuck-Stiftung Stiftung Roggenbuck Roggenbuck Stiftung Stiftung

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SLIDE 31

Micrometastasis Research Network at UCCH/UKE Micrometastasis Research Network at UCCH/UKE

Institut für Anatomie II Viszeralchirurgie Klinik und Poliklinik für Viszeralchirurgie Klinik und Poliklinik für Gynäkologie Institut für Klinische Chemie

  • I. Medizinische

Klinik und Poliklinik Institut für Rechtsmedizin Klinik und Poliklinik für Neurochirurgie Institut für Tumorbiologie Institut für Tumorbiologie Labor für Strahlenbiologie und -onkologie

Klinik und Poli- Klinik und Poli- klinik für Mund-, Kiefer- und Gesichts- chirurgie

g Molekularbiologie Institut für Biochemie und Molekularbiologie

  • II. Medizinische

Klinik und Poliklinik Klinik und Poliklinik für Urologie

chirurgie

Poliklinik

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SLIDE 32

EU EU-

  • Consortium

Consortium-

  • DISMAL

DISMAL

University Medical Center Hamburg-Eppendorf (Germany) Free University of Amsterdam Medical Center (The Netherlands)

Start: November 2005 Coordinator: Klaus Pantel

Imperial College London (United Kingdom), Radium Hospital Oslo, (Norway) German Cancer Research Center, (Germany) SME 1 Applied Imaging, (United Kingdom) ERC Advanced Investigator Grant

DISSECT“ (2011-2016)

University

  • f Graz (Austria)

University of Utrecht (The Netherlands)

„DISSECT (2011-2016) ERA NET TRANSCAN CTC SCAN

Heinrich-Pette- Institut (Germany) Netherlands Cancer Institute (The Netherlands)

ERA-NET TRANSCAN: CTC-SCAN Project (2013 – 2016)

Lapeyronie Hospital, Montpellier, (France) SME 2 TILL Photonics (Germany) ( ) SME 3 Agendia, (The Netherlands) Leiden University Medical Center (The Netherlands),

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24-27, 2013