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

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 Liquid Biopsy Biopsy for metastatic for for metastatic for metastatic cells metastatic cells 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 on metastatic cells located at different sites ? Alix Alix- -Panabières & Pantel, Panabières & Pantel, Clin Clin Chem Chem , , 2012 2012

Detection Detection of of CTC in CTC in the the peripheral peripheral blood blood September 2013: September 2013: > 400 registered > 400 registered clinical 400 400 i t i t d d clinical trials li i li i l t i l l t i l trials with with CTC ith ith CTC CTC CTC as as biomarkers biomarkers biomarkers biomarkers

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 one tumor tumor cell cell 10 6 – 10 10 8 in in 10 normal normal blood blood Alix- Alix -Panabières & Pantel, Panabières & Pantel, Clin Chem Clin Chem , , 2012 2012 cells cells 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

New approach: In vivo capture of CTC (1.5 L blood) Proof-of-principle data in breast, lung and prostate cancer

CTC Identification Methods CTC Identification Methods Real Real ‐ time RT time RT ‐ PCR PCR Cytokeratins as standard CTC markers Cytokeratins as standard CTC markers BUT differential expression of individual CKs (Joosse/Pantel et al., Clin Cancer Res 2012) nucleic acids nucleic acids nucleic acids nucleic acids mRNA DNA DNA intra intra- -cytoplasmic proteins cytoplasmic proteins > 13,000 publications on CTC in membrane proteins membrane proteins PubMed 2013 PubMed 2013 mRNA Immunocytochemistry Immunocytochemistry Tumor cell Tumor cell secreted secreted proteins by proteins by VIABLE cells VIABLE cells EPISPOT assay EPISPOT assay Alix Alix- -Panabières Panabières et al., et al., Clin Cancer Res, 2008 Clin Cancer Res, 2008

Design Design of of robust robust automated automated systems systems for for reproducible reproducible CTC CTC detection detection

CellSearch™ System CellSearch™ System (FDA (FDA- -cleared) cleared) MagNest TM TM MagNest Epithelial Cell Kit Enrichment of CTC with Enrichment of CTC with anti anti- -EpCAM ferro fluids EpCAM ferro fluids p Cristofanilli et al., NEJM, 2004 Cristofanilli et al., NEJM, 2004 Riethdorf et al., CCR, 2007 & 2010 Riethdorf et al., CCR, 2007 & 2010 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

CellSearch™ System CellSearch ™ System: Images of Tumor Cells : Images of Tumor Cells Cytoplasm Nucleus Cell Membrane Composite Cytoplasm Nucleus Cell Membrane Composite DAPI DAPI CD45- CD45 -APC APC Tumor Cell Tumor Cell CK- CK -PE PE pos pos pos pos p neg neg g - + = Leukocyte Leukocyte CD45 + + Leukocyte Leukocyte CD45 Tumor Cell Tumor Cell nucleus nucleus Membrane Membrane

Prognostic value of CTC counts for survival Prognostic value of CTC counts for survival i i in cancer patients with advanced disease in cancer patients with advanced disease ti ti t t ith ith d d d di d di Breast Cancer Breast Cancer Colorectal Cancer Colorectal Cancer Prostate Cancer Prostate Cancer Cohen, JCO, 2008 Cohen, JCO, 2008 Cohen, JCO, 2008 Cohen, JCO, 2008 De Bono, Clin Can Res, 2008 De Bono, Clin Can Res, 2008 De Bono, Clin Can Res, 2008 De Bono, Clin Can Res, 2008 Christofanilli, NEJM, 2004 Christofanilli, NEJM, 2004 Christofanilli NEJM 2004 Christofanilli NEJM 2004  FDA  FDA FDA clearance FDA clearance clearance clearance

Detection Detection of of 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 development of distant metastases? l t f di t t t t ?

Prognostic impact of CTC in breast cancer patients without overt metastases Rack, Janni et al, unpublished

Meta-Analysis of 49 studies comprising 6815 breast cancer patients p Progression-free survival CTC detection: ICC & RT CTC detection: ICC & RT- 1 1 CTC neg 0.86 PCR 0.78 CTC pos 0.8 0.78 ess Free 0.62 0.58 0.6 0.6 Proportion Progre 0.51 0.34 0.4 Overall survival 0.29 p<0.001 0.2 0.15 0.15 1 0.06 0.89 0 CTC neg 0 1 2 3 4 5 0.8 0.79 CTC pos 0.69 Time (years) 0.64 . ortion Surviving 0.6 0.51 0.56 0.47 0.4 0.40 0.29 Propo 0.2 0.19 p<0.001 0 0 1 2 3 4 5 Ti Time (years) ( ) Zhang L, Riethdorf S…Pantel K, Clin Cancer Res , 2012.

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

Challenge Challenge Challenge of Challenge of of CTC of CTC CTC detection CTC detection detection: detection: Epithelial Epithelial-Mesenchymal Epithelial-Mesenchymal Epithelial Mesenchymal Transition (EMT) Mesenchymal Transition (EMT) Transition (EMT) Transition (EMT) of of carcinoma carcinoma cells cells

Tumor cell dissemination, plasticity and EMT ( Bednarz-Knoll et al CMR 2012; Kang & Pantel, Cancer Cell 2013) Dormancy > 10 years 10

Epithelial-Mesenchymal Plasticity of CTC EpCAM CK EpCAM, CK Vimentin Vimentin Bednarz-Knoll, Alix-Panabières & Pantel Cancer & Met Rev 2012

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

M l M l Molecular Molecular Characterization l l Characterization of Ch Ch t t i i ti ti of CTC f CTC CTC CTC f for for therapeutic th therapeutic targets th ti ti t t targets t t („real ( ( („real-time liquid l l ti time liquid biopsy ti li li id bi id bi biopsy“) “)

Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer HER2 oncogene in breast cancer CTC without HER2 gene amplification CTC without HER2 gene amplification CTC without HER2 gene amplification A A A A Composite Composite CK DAPI CD45 HER2 CK DAPI CD45 HER2 DETECT DETECT-III study: Anti DETECT III study: Anti DETECT III study: Anti HER2 therapy (lapatinib) in metastatic breast cancer III study: Anti-HER2 therapy (lapatinib) in metastatic breast cancer HER2 therapy (lapatinib) in metastatic breast cancer HER2 therapy (lapatinib) in metastatic breast cancer Red: CK Red: CK Red: CK Red: CK Red: Cen17 Red: Cen17 Red: Cen17 Red: Cen17 Green: HER2 Green: HER2 ICC ICC FISH FISH FISH FISH 0 0 patients with HER2- patients with HER2 -negative primary tumors and HER2 negative primary tumors and HER2- -positive CTC positive CTC 1+ 1+ CTC CTC CTC with HER2 gene amplification CTC with HER2 gene amplification CTC with HER2 gene amplification CTC with HER2 gene amplification CTC with HER2 gene amplification CTC with HER2 gene amplification 2+ 2+ 2+ 2+ 3+ 3+ C C B B Composite Composite CK DAPI CD45 HER2 CK DAPI CD45 HER2 CB11 CB11 A0485 FISH A0485 FISH MCF-7 MCF-7 0 0 SK-BR-3 SK-BR-3 SK-BR-3 BT20 BT20 1+ 1+ T47D T47D 1+ 1+ Discordance between HER2 Discordance between HER2 MDA- MDA- status of primary tumor and status of primary tumor and 2+ 2+ MB-453 MB-453 CTC CTC CTC CTC SK-BR-3 SK-BR-3 3+ 3+ BT474 BT474 3+ 3+ 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

Heterogeneity of ER status in CTCs of breast cancer patients with ER-positive primary tumors Babayan, Joosse, Pantel et al., PLOS ONE 2013 Babayan, Joosse, Pantel et al., PLOS ONE 2013 ER CK DAPI CD45 Merge ER CK DAPI CD45 Merge E R R + E R R - ER-negative CTCs may survive endocrine therapy

Genomic Characterization of single CTC Genomic Characterization of single CTC CTC detection CTC isolation WGA + - Mutation analysis - Mutation analysis - CGH (conv./array) - NextGen Sequencing NextGen Sequencing CTC Capillary CTC

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