developments in liquid biopsy in oncology · center of experimental medicine, department of tumor...

Simon Joosse (s.joosse@uke.de)

University Medical Center Hamburg-Eppendorf

Center of Experimental Medicine, Department of Tumor Biology

Developments in liquid biopsy in

oncology

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Simon Joosse (s.joosse@uke.de)

Joosse et al. (2014) EMBO Mol Med

Metastatic cascade – Tumor cell dissemination

CTCs = Circulating Tumor Cells

DTCs = Disseminated Tumor Cells

EMT = epithelial-mesenchymal transition

MET = mesenchymal-epithelial transition

DTC

Extravasation

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Simon Joosse (s.joosse@uke.de)

Circulating tumor markers as liquid biopsy

Elazezy & Joosse (2018) Comput Struct Biotechnol J

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Simon Joosse (s.joosse@uke.de)

Alix-Panabières & Pantel (2016) Cancer Discovery

Aims of liquid biopsy

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Simon Joosse (s.joosse@uke.de)

Circulating tumor cell (CTC) enrichment

Joosse et al. (2014) EMBO Mol Med

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Simon Joosse (s.joosse@uke.de)

CellSave Preservative Tube Enrichment of EpCAM positive cells and staining for Keratin and CD45

Cytoplasm Nucleus Cell membrane Composite

CD45+ membrane

Keratin-PEpositive

DAPI

positive

CD45-APC

negative Tumor cell

Leukocyte Tumor cells

CTC enrichment - CellSearch System (FDA-cleared)

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Simon Joosse (s.joosse@uke.de)

CTC enrichment - in vivo capture

Saucedo-Zeni et al. (2012) Int J Oncol

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Simon Joosse (s.joosse@uke.de)

Gorges et al. (2016) Clin Cancer Res

in vivo capture of CTCs in lung cancer patients

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Simon Joosse (s.joosse@uke.de)

Gorges et al. (2016) Clin Cancer Res

in vivo capture of CTCs in lung cancer patients

corr=0.36, p=0.003

p<0.0001

p=0.0013 p=0.127

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Simon Joosse (s.joosse@uke.de)

Gorges et al. (2016) Clin Cancer Res

in vivo capture of CTCs in lung cancer patients

- Improved capture of CTCs in lung cancer

- Molecular characterization

- Predictive value

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Simon Joosse (s.joosse@uke.de)

Alix-Panabières & Pantel (2016) Cancer Discovery

Screening and early

detection of cancer

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Simon Joosse (s.joosse@uke.de)

ctDNA for early detection

Elazezy & Joosse (2018) Comput Struct Biotechnol J

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Simon Joosse (s.joosse@uke.de)

Fernandez-Cuesta et al. (2016) EBioMedicine

Krimmel et al. (2016) PNAS

Challenges of early detection of cancer

TP53 mutation detection in cfDNA of ovarian

cancer patients and control subjects

TP53 mutation detection in cfDNA of

lung cancer patients and control subjects

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Simon Joosse (s.joosse@uke.de)

Challenges of early detection of cancer

Detection of leukemia mutations in non-cancer subjects

Genovese et al. (2014) NEJM

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Simon Joosse (s.joosse@uke.de)

Alix-Panabières & Pantel (2016) Cancer Discovery

Risk for metastatic

relapse (prognosis)

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Simon Joosse (s.joosse@uke.de)

Bone-marrow aspirates

from upper iliac crests

Breast cancer (M0): 199/552 (36%)Braun et al. NEJM 2000

Bone-marrow

Ficoll gradient

Mononuclear cells

Keratin staining Tumor cell screening

adapted from: Pantel & Brakenhoff (2004) Nat Rev Cancer

Prostate cancer (pM0N0): 86/193 (44.6%)Köllermann et al. JCO 2008

Nonmalignant

disease: 2/191 (1%)

Prognostic value of Disseminated Tumor Cells (DTCs)

DTCs correlate with relapse

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Simon Joosse (s.joosse@uke.de)

Prognosis of liquid biopsy in advanced stage cancer - CTCs

De Bono et al. (2008) Clin Cancer Res

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Simon Joosse (s.joosse@uke.de)

Prognosis of liquid biopsy in early stage cancer - CTCs

Breast cancer

Bladder cancer

Head & neck cancer

Testicular germ cell cancer

Colorectal cancer

EpCAM+/K+ CTCs are associated

with unfavorable prognosis in

early stage cancer

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Simon Joosse (s.joosse@uke.de)

Prognosis of liquid biopsy in early stage breast cancer - ctDNA

Neoadjuvant

chemotherapy SurgeryPost-

surgeryStandard follow-up

Baseline

Post-surgery

Mutation tracking

Garcia-Murillas et al. (2015) Sci Transl Med

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Simon Joosse (s.joosse@uke.de)

Alix-Panabières & Pantel (2016) Cancer Discovery

Therapeutic targets

and resistance

mechanismsDo not d

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Simon Joosse (s.joosse@uke.de)

Identification of therapeutic targets and resistance mechanisms- ER in breast cancer

Johnston Dowsett (2003) Nat Rev Cancer

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Simon Joosse (s.joosse@uke.de)

Babayan et al. (2013) PLoS One

ER K DAPI CD45 Merge

Ove

rall

su

rviv

al

p = 0.033

HR: 7.38

CTC+

CTC-

Therapy relevant markers

expressed in CTCs

Identification of therapeutic targets and resistance mechanisms- ER in breast cancer

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Simon Joosse (s.joosse@uke.de)

Identification of therapeutic targets and resistance mechanisms- PD1-PDL1

Pardoll et al. (2012) Nat Rev Cancer

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Simon Joosse (s.joosse@uke.de)

Identification of therapeutic targets and resistance mechanisms- PD1-PDL1

Mazel et al. (2015) Mol Onc

PD-L1 expression found on CTCs

in 11/16 patients

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Simon Joosse (s.joosse@uke.de)

CTC without HER2 gene amplification

CTC with HER2 gene amplification

SK-BR-3

Red: CK

ICCGreen: HER2

FISH

Red: Cen17

FISH

CTC without HER2 gene amplification

CTC with HER2 gene amplification

SK-BR-3

CTC without HER2 gene amplification

CTC with HER2 gene amplification

SK-BR-3

Red: CK

ICCGreen: HER2

FISH

Red: Cen17

FISH

Riethdorf et al. (2010) Clin Cancer Res

DETECT-III study:

Anti-HER2 therapy in metastatic

breast cancer patients with

HER2-neg primary tumors but

HER2-pos CTC

Identification of therapeutic targets and resistance mechanisms- HER2 in breast cancer

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Simon Joosse (s.joosse@uke.de)

Cayrefourcq et al. (2015) Cancer Res

AACR Award for most cited article

in Cancer Research 2015

Characterization through cell line

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Simon Joosse (s.joosse@uke.de)

• Different substrates can be used as liquid biopsy

(e.g., CTCs, cfDNA, exosomes)

• Liquid biopsy can be used

• To stratify cancer patients

• To monitor therapy response

• For early detection

• For characterization of cancer heterogeneity

• Challenging in symptom-free subjects

• Liquid biopsy can be used for personalized treatment

Conclusion / Take home message (1)

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Simon Joosse (s.joosse@uke.de)

Understanding the biology of

metastasis

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Simon Joosse (s.joosse@uke.de)

Cancer heterogeneity and liquid biopsy

Alix-Panabières & Pantel (2010) Clin Cancer Res

CTCs = Circulating Tumor Cells

DTCs = Disseminated Tumor Cells

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Simon Joosse (s.joosse@uke.de)

Circulating tumor markers as liquid biopsy

Joosse & Pantel (2015) Cancer Cell

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Simon Joosse (s.joosse@uke.de)

Tumor DNA

Reference DNA

Chromosome

Cop

y n

um

be

r va

lue

Joosse et al. (2007) BMC Cancer

Array CGH Next Generation Sequencing

DNA fragments

Molecular characterization of single tumor cells

Babayan et al. (2016) Oncotarget

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Simon Joosse (s.joosse@uke.de)

NG

S

Single SKBR3 cell #1

Chromosome 8 Chromosome 17

aC

GH

NG

S

SKBR3 pool (reference)

NG

S

Chromosome 8 Chromosome 17

Single SKBR3 cell #2

Single cell vs genomic

r2 = 0.91 Babayan et al. (2016) Oncotarget

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Simon Joosse (s.joosse@uke.de)

Joosse et al. (2014) EMBO Mol Med

Metastatic cascade – Tumor cell dissemination

CTCs = Circulating Tumor Cells

DTCs = Disseminated Tumor Cells

EMT = epithelial-mesenchymal transition

MET = mesenchymal-epithelial transition

DTC

Extravasation

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Simon Joosse (s.joosse@uke.de)

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 (%)

Pantel & Brakenhof (2004) Nat Cancer Rev

Bone marrow is a reservoir for DTCs

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Simon Joosse (s.joosse@uke.de)

Croucher et al. (2016) Nat Rev Cancer

Bone metastasis

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Simon Joosse (s.joosse@uke.de)

Mohme et al. (2017) Sci Rep

CTC release through cement augmentation of vertebral metastases

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Simon Joosse (s.joosse@uke.de)

Mohme et al. (2017) Sci Rep

CTC release through cement augmentation of vertebral metastases

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Simon Joosse (s.joosse@uke.de)

Mohme et al. (2017) Sci Rep

CTC release through cement augmentation of vertebral metastases

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Simon Joosse (s.joosse@uke.de)

Joosse and Pantel (2013) Cancer Research

What drives tumor dissemination?

EMT = epithelial-mesenchymal transition

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Simon Joosse (s.joosse@uke.de)

vs.

Classification of early stage breast tumors

according to DTC status

Expression profiling on micro-dissected

tumor tissue

Bioinformatic analysis

and validation

of identified genes

DTC- DTC+

RAI2→

ER+ breast tumors

Drivers of tumor cell dissemination

Werner et al. (2015) Cancer Discovery

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Simon Joosse (s.joosse@uke.de)

Epithelial-mesenchymal transition: tumor cell dissemination

Esposito and Kang (2015) Cancer Discovery

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Simon Joosse (s.joosse@uke.de)

Joosse and Pantel (2013) Cancer Research

What enables cell survival?

EMT = epithelial-mesenchymal transition

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Simon Joosse (s.joosse@uke.de)

Giuliano et al. (2018) Cancer Res

Intravasation of tumor cells

Single cell dissemination Cell cluster dissemination

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Simon Joosse (s.joosse@uke.de)

Aceto et al. (2014) Cell

CTC Clusters have higher metastatic potential compared to single CTCs

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Simon Joosse (s.joosse@uke.de)

Aceto et al. (2014) Cell

CTC Clusters have higher metastatic potential compared to single CTCs

Prostate cancer patientsBreast cancer patients

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Simon Joosse (s.joosse@uke.de)

Joosse and Pantel (2013) Cancer Research

What plays a role in extravasation of CTCs?

EMT = epithelial-mesenchymal transition

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Simon Joosse (s.joosse@uke.de)

Extravasation of tumor cells

Follain et al. (2018) Dev Cell

Caudal Plexus

Head Intravascular injection

of tumor cells (2.103)

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Simon Joosse (s.joosse@uke.de)

Follain et al. (2018) Dev Cell

Extravasation of tumor cells

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Simon Joosse (s.joosse@uke.de)

Joosse and Pantel (2013) Cancer Research

What determines the metastatic location?

EMT = epithelial-mesenchymal transition

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Simon Joosse (s.joosse@uke.de)

Extracellular vesicles (EVs) carry a tumor’s instructions

Elazezy & Joosse (2018) Comput Struct Biotechnol J

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Simon Joosse (s.joosse@uke.de)

Exosomes determine organotropic metastasis

Hoshino et al. (2015) Nature

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Simon Joosse (s.joosse@uke.de)

Genomic progression in colorectal cancer metastasis

Joosse et al. (2018) Clin Chem

Chromosomal

aberration

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Simon Joosse (s.joosse@uke.de)

• Liquid biopsy may be used to study cancer biology:

• Intravasation

• CTC survival

• Extravasation

• Outgrowth of metastasis

• Tumor heterogeneity

Conclusion / Take home message (2)

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