Klaus Pantel. Institut für Tumorbiologie TUMOR DORMANCY: RELEVANCE IN BREAST CANCER

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1 Klaus Pantel Institut für Tumorbiologie TUMOR DORMANCY: RELEVANCE IN BREAST CANCER

2 Tumor cell dissemination and cancer dormancy Primary tumor Local relapse 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) Blood Distant tissue (e.g. bone marrow) Recirculation Tumormass dormancy Micrometastasis Metastasis DTCs Escape CTCs DTC Tumor cell dormancy NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 6 JUNE

3 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

4 DTC in bone marrow of cancer patients Bone marrow Ficoll gradient 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%) Bone marrow aspirates taken from the upper iliac crest MNC Immunocytochemistry: Cytokeratin staining with mab A45-B/B3 2 x 10 6 MNC per patient

5 DTC in bone marrow of cancer patients Bone marrow aspirates taken from the upper iliac crest Bone marrow Ficoll gradient 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 detection correlates with metastatic AND locoregional relapse Most DTC are Ki67- and MNChave CD44+/CD24- stem cell phenotype DTCs can occupy the hematopoietic stem cell niches Immunocytochemistry: Cytokeratin staining with mab A45-B/B3 2 x 10 6 MNC per patient

6 BM is a homing organ and putative reservoir for DTC derived from various primary sites Tumor type Detection rate (%) Breast Cancer Prostate cancer* Lung cancer (NSCLC) Gastric cancer Esophageal cancer Colorectal cancer Pancreatic cancer Head and neck cancer

7 Identification of DTC-associated genes Luminal A/B, HR+ BM- Luminal A/B, HR+ BM+ Classification of early stage breast tumors according to DTC status vs. Expression profiling on micro-dissected tumor tissue RAI2 Bioinformatic analysis and validation of identified genes Werner, Wikman, Pantel et al, Cancer Discovery, 2015

8 Editorial: Esposito & Kang, Cancer Discovery, 2015 RAI2 All-trans retinoic acid CTBP2 RAI2 as potential new metastasis-suppressor gene RAI2 loss S GATA3 FOXOA1 GRHL2 CTBP2 GATA3 FOXOA1 GRHL2 Vimentin ZEB1 SNAI1 SNAI2 CDH1 Hematogenous dissemination Werner, Wikman, Pantel et al, Cancer Discovery, 2015

9 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

10 2010

11 Aims of Liquid Biopsy 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

12 CTC as Liquid Biopsy for 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 information on metastatic cells located at different sites Alix-Panabières & Pantel, Clin Chem, 2013; Pantel & Alix- Panabieres, Cancer Res. 2013

13 Biological properties Protein expression Positive Selection (a) anti-e markers Ab (E.g., EpCAM) CTC anti-e/m markers Ab (E.g., Plastin 3) Ex vivo anti-m markers Ab (E.g., N-Cadherin) CTC (c) - CellSearch system - MagSweeper - EPHESIA CTC-chip - CTC-chip - Velcro-like device In vivo - CellCollector - Photoacoustic nanodetector Physical properties Label-free strategies (d) (e) (f) DEP DEP CTC-iChip (g) Out Anti-CD45 Negative Selection (b) Anti-CD45 CTCs anti-e markers Ab WBC Alix-Panabieres & Pantel, Nature Rev. Cancer 2014 CTC WBC RBC

14 New approach: In vivo capture of CTC Lung cancer CTCs: Gorges, Pantel et al., CCR 2015

15 Approaches for CTC detection Immunocytological technologies Molecular technologies Functional assays In vitro Cell Culture anti-e/m markers Ab (E.g., CK, Vimentin, E/N-Cadherin) - EPISPOT anti-marker Abs (E.g., CK19, HER2, EGFR, VEGF, PSA) CTC anti-tumor associated markers Ab (E.g., HER2, EGFR) anti-tissue-specific markers Ab (E.g., PSA, Mammaglobin, MAGE) CTC mrna RT-qPCR (single/multiple genes) Liquid bead array Viable CTC Cell culture - Invasion assay Fluo Ab2 Ab1 Secreted protein Immunospots Technologies Functional CTC - Immunocytochemistry - CellSearch system - Flow Cytometry - DEParray RNA-based Technologies Fluo matrix Xenotransplantation models (CDx) Alix-Panabieres & Pantel, Nature Rev. Cancer 2014 Viable CTC with stem-cell properties days Metastase s

16 Epithelial-Mesenchymal Plasticity of CTCs Intermediate E/M Phenotypes: Potential MICs! EpCAM, CK Vimentin Bednarz-Knoll, Alix-Panabières & Pantel Cancer & Met Rev 2012

17 CTCs in early stage cancer patients: Detection of MRD for risk assessment and improved staging

18 CellSearch System (FDA-cleared) MagNest TM 7.5ml Epithelial Cell Kit Enrichment of CTC with anti-epcam ferro fluids: Captures tumor cells with very low EpCAM expression

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

20 Prognostic impact of CTC in breast cancer patients without overt metastases Rack, Janni, Pantel et al, JNCI 2014

21 TNM 2010: CTC in new cm0(i+) Classification

22 Monitoring of CTCs: Can early changes in CTC counts predict the efficacy of therapeutic interventions (e.g., chemotherapy, hormonal therapy)?

23 centres provided data for 1944 eligible patients from 20 studies Meta-analysis on raw data.

24 CTCs vs. conventional tumor markers (PFS, p values) in metastatic breast cancer patients receiving chemotherapy CTCBL ( baseline 3-5 weeks 6-8 weeks Model used as reference CA15-3BL CEABL CTC3-5 CA15-3 BL + CA CEABL + CEA 3-5 CTC6-8 CA15-3 BL + CA CEABL + CEA 6-8 N patients CP 6 E CP +CTCBL CP +CTCBL + CTC3-5 CP +CTCBL + CTC E E Few events Few events Bidard, Pierga, Michels, Pantel et al, Lancet Oncology 2014, European Pooled Analysis of CTCs in metastatic BC (n=1944)

25 Characterization of CTCs at the DNA, RNA and protein level: - Therapeutic targets - Resistance mechanisms

26 Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer Discordance between HER2 status of primary tumor and CTC Riethdorf/Pantel et al., Clinical Cancer Res Fehm/Pantel et al., Breast Cancer Res Treat Ignatiadis/Sotiriou et al, PlosONE, Ignatiadis/Pantel et al, SABCS, 2011

27 Detection of therapeutic targets on CTC: HER2 oncogene in breast cancer DETECT-III study: Anti-HER2 therapy (lapatinib) in metastatic breast cancer patients with HER2-negative primary tumors and HER2-positive CTC Discordance between HER2 status of primary tumor and CTC Riethdorf/Pantel et al., Clinical Cancer Res Fehm/Pantel et al., Breast Cancer Res Treat Ignatiadis/Sotiriou et al, PlosONE, Ignatiadis/Pantel et al, SABCS, 2011

28 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 E R + E R - ER-negative CTCs may survive endocrine therapy

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

30 Genomic profiles (CNAs) of ER+ and ER- CTCs in breast cancer patients determined by NGS ER- ER+

31 PD1-PDL1 mediated immune blockade as cancer target The blockade of immune checkpoints in cancer immunotherapy Drew M. Pardoll Nature Reviews Cancer 12, (April 2012)

32 PD-L1 expression on CTCs in breast cancer Mazel, Pantel, Alix-Panabieres et al, Mol. Oncol (Editorial by R. David in Lancet Oncol. 2015)

33 PD-L1 expression on CTCs in breast cancer PD-L1 is frequently expressed on CTCs (> 60% of patients) in metastatic breast cancer patients Mazel, Pantel, Alix-Panabieres et al, Mol. Oncol (Editorial by R. David in Lancet Oncol. 2015)

34 Functional studies on CTCs

35 DTC cell lines as models for functional analyses DTC adapt to hypoxic conditions in bone marrow Unfolded protein response Stem cell markers Bartkowiak et al., J Prot Res 2010 & Cancer Res Grabinski/Jücker et al., Cell Signal Bartkowiak et al., Cancer Res. 2015

36 Potential Metastasis-initiating Cells: EPCAM low, CD44 +, CD47 + and cmet +

37 2013

38 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)

39 Comparative analysis of CTCs and ctdna in breast cancer 1. Progressive disease with increasing liver metastases and ascites no chemot 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 Heidary, Speicher, Pantel et al, Breast Cancer Res. 2014

40 Comparative analysis of CTCs and ctdna in breast cancer 1. Progressive disease with increasing liver metastases and ascites no chemot ctdna levels may not always reflect disease progression in 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 CTCs CTCanalyses are not restricted to dying cancer cells and provide complementary information. 3. However, very low concentration of ctdna fragments at each measurement ctdna cancer patients. Heidary, Speicher, Pantel et al, Breast Cancer Res. 2014

41 Nucleid Acids (DNA, microrna) as blood-based biomarkers in cancer patients Release by dying cells Tumor cell Tumor cell Active secretion by viable cells Tumor cell cell-free RNA cell-free DNA Exosomes (Proteins, RNA, DNA?) Schwarzenbach, Pantel et al., Nature Rev. Cancer 2011; Nature Rev. Clin. Oncol. 2014; Pantel et al., Nature Med. 2013; Speicher & Pantel, Nature Biotech. 2014

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

43 CANCER-ID EU Konsortium Scientific Managment: Klaus Pantel, UKE (Leon Terstappen) Coordination: Thomas Schlange, BAYER (Barbara Baggiani) EFPIA Academic institutions Prof. Klaus Pantel Clinical sites 32 partners: 6 EFPIA companies (lead Bayer, colead Menarini) 17 academic/clinical sites 6 SMEs 2 non-profit organizations 1 non-sme/non-efpia Prof. Leon Terstappen SMEs Non-profit organizations Non-EFPIA/ non-sme Page 43 CANCER-ID evaluation hearing Brussels- Oct 7th 2014

44 EFPIA Academic institutions CANCER-ID EU Konsortium Scientific Managment: Klaus Pantel, UKE (Leon Terstappen) Coordination: Thomas Schlange, BAYER (Barbara Baggiani) Prof. Klaus Pantel Clinical sites 32 partners: 6 EFPIA companies (lead Bayer, colead Menarini) 17 academic/clinical sites 6 SMEs 2 non-profit organizations 1 non-sme/non-efpia Non-profit organizations Blood-based Diagnostic in Lung and Breast Cancer (CTCs, ctdna & cfmirna) Prof. Leon Terstappen 33 EU Partners (Academic SMEs institutions & EFPIA companies) Non-EFPIA/ non-sme Page 44 CANCER-ID evaluation hearing Brussels- Oct 7th 2014

45 Center of Experimental Medicine Institute of Tumor Biology - Klaus Pantel Grant Support: 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 DFG BMBF EU / ERC Dt. Krebshilfe Sander-Stiftung Roggenbuck-Stiftung

46 Center of Experimental Medicine Institute of Tumor Biology - Klaus Pantel Sabine Riethdorf/Tobias Gorges Heidi Schwarzenbach, B. Steinbach Harriet Wikman/Stefan Werner Klaus Pantel, Hamburg: Simon Joosse, Anna Babayan Kai Bartkowiak, Natalia Bednarz-Koll A. Andreas, C. Coith, J. Kropidlowski, M. Stoupiec Grant Support: DFG BMBF ERC Advanced Investigator EU Grant / ERC Dt. Krebshilfe DISSECT ( ) Sander-Stiftung Roggenbuck-Stiftung

47 Micrometastasis Research Network at UCCH/UKE Institut für Klinische Chemie Institut für Anatomie II Klinik und Poliklinik für Gynäkologie Klinik und Poliklinik für Viszeralchirurgie I. Medizinische Klinik und Poliklinik Institut für Rechtsmedizin Institut für Tumorbiologie Klinik und Poliklinik für Neurochirurgie Labor für Strahlenbiologie und -onkologie Institut für Biochemie und Molekularbiologie II. Medizinische Klinik und Poliklinik Klinik und Poliklinik für Mund-, Kiefer- und Gesichtschirurgie Klinik und Poliklinik für Urologie

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