“Liquid biopsies as prognostic and predictive biomarkers; ready for the clinic?” AR-V7 expression in CTCs from patients with castration- resistant prostate cancer & Feasibility studies of circulation tumor DNA analysis John W. M. Martens Dept of Medical Oncology, Erasmus MC Cancer Institute Rotterdam Donderdag 16 april 2015 Conflict of Interest � Funding/collaboration received from: � Veridex (BC/CRC CTC studies) � Sanofi (CRPC CTC-ARv7 studies) � Thermo-Fisher (early access to oncomine panels) � Cytotrack & Olink (collaborator in EU-project CAREMORE) � Philips research (Molecular diagnostics BC) � Therawis (Prognostic markers) � Patent applications: Our dept. owns a patent on the ARv7 in CTCs 1
Outline of the presentation � State-of-the-art of genomics research (focus BC) � Introduction about liquid biopsies (CTCs, ctDNA, EVs) � Application of CTC count and characteristics in metastatic cancer � ARv7 positive CTCs predict progression in mCPCR � Comparisons of ctDNA detection methods (dPCR & targetted NGS) � Standard operation procedures/sample collection � Examples of NGS in mBC and CRC � ESR1 detection in CTCs and ctDNA � Conclusions and outlook Outline of the presentation � State-of-the-art of genomics research (focus BC) � Introduction about liquid biopsies (CTCs, ctDNA, EVs) � Application of CTC count and characteristics in metastatic cancer � ARv7 positive CTCs predict progression in mCPCR � Comparisons of ctDNA detection methods (dPCR & targetted NGS) � Standard operation procedures/sample collection � Examples of NGS in mBC and CRC � ESR1 detection in CTCs and ctDNA � Conclusions and outlook 2
Personalized cancer treatment � Giving: � The right drug, to the right patient, at the right dose, from the right moment onwards, till the right moment � Personalized cancer treatment will yield: � Better outcome and less toxicity � Less patients receive treatments, more will benefit � More favorable cost-effectiveness of cancer treatments � To get there: early markers for response, prognostic and in particular predictive factors Lessons learned from NGS of BC genomes 1. Mutations are recurrent in a few common and many rare driver genes (>93 breast cancer genes) Breast tumors Mutated genes ER-positive ER-negative Stephan PJ et al Nature. 2009 Dec 24;462(7276):1005-1010 Signatures in 560 breast cancer genomes (Nik-Zainal, nature 2016 3
Lessons learned from NGS of BC genomes 2. Cancer evolves over time (cancer evolution) Metastases can derive from subclones (un)detectable in the primary tumor Yates LR et al Nat Medicine 2015 Introduction � Breast tumors evolve over time during “natural” disease progression and in response to treatment � Example(s) include: � the appearance of activation ESR1 mutations during endocrine treatment � Discrepancy between ER and/or Her-2 expression and PIK3CA mutation status between primary tumor and metastasis � Evidence of clonal hierarchical relations within heterogenous tumors � Drivers of diversity (APOBEC; BRCAness) are operational during tumor progression � Repeated sampling during tumor progression is likely benefitial for optimal patient care 4
Outline of the presentation � State-of-the-art of genomics research (focus BC) � Introduction about liquid biopsies (CTCs, ctDNA, EVs) � Application of CTC count and characteristics in metastatic cancer � ARv7 positive CTCs predict progression in mCPCR � Comparisons of ctDNA detection methods (dPCR & targetted NGS) � Standard operation procedures/sample collection � Examples of NGS in mBC and CRC � ESR1 detection in CTCs and ctDNA � Conclusions and outlook Liquid biopsies (CTCs/ctDNA) • In the circulation of patients: - tumor cells are present (circulating tumor cells (CTCs)) - tumor DNA is detectable (circulating tumor DNA (ctDNA) ER, and HER2 status can differ between the primary tumor and metastases (or CTCs) (Tewes et al. BCRT 2009; 115: 581-590) Specific mutations are acquired in metastatic disease (EGFR; KRAS; ESR1) CTCs Q: Can these be “surrogate biopsies” of metastatic tissue? 5
CTCs vs cell-free DNA (cf-DNA) CTCs cf-DNA Intact, living tumor cells cell-free DNA, RNA and protein DNA only (histones?) Present in 65% of MBC Present in 82% of MBC More complex processing Relatively easy processing (EpCAM-based enrichment) (plasma isolation) Both: Present in extreme low frequency Calls fo sensitive and specific assays Circulation tumor cells (CTCs) 6
CTC detection � Circulating tumor cells (CTCs) � From peripheral blood � CellSearch System � Median 1 to 9 cells / 7,5 mL � In 30 - 80% � 1 CTC CTC isolation and detection: CellSearch™ 7
CTC isolation and detection: CellSearch™ CTC definition -EpCam positive -CD45 negative -CK 8/18/19 positive -DAPI positive CD45 - CK + DAPI + CTC count and prognosis � Prognostic factor for OS: De Bono, J.S., et al., Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res, 2008. 14 (19): p. 6302-9. 8
CTC count and treatment response � Early response marker: � Already 2 – 5 weeks after start treatment De Bono, J.S., et al ., Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res, 2008. 14 (19): p. 6302-9. CTC characterization � CTC characterization � Use as liquid biopsy? Adapted from Beije N., et al. Circulating tumor cell enumeration: the clinician's guide to breast cancer treatment? Cancer Treat Rev , 2014. 9
Characterization of CTCs Analyse multiple transcripts in limited material B. Nucleic acid ampliciation required One human cell contains 5 5- -10 pg 10 pg total RNA. 5 5 - - 10 pg 10 pg Needed for traditional qRT-PCR: 5 5- 5 5 - - -20 ng 20 ng 20 ng 20 ng per assay. MAGEA3 35 TERT With pre-amplification With pre-amplification KRT20 ESR2 CGB TFF1 30 TACSTD2 (EpCAM check) BST1, blood_check TWIST Ct-values after pre-amplification GALGT 25 ALDH1A1 ESR1 MET MDM2 20 SERPINB5 SCGB2A2 HMBS EGFR CXCL1 15 HPRT PTPRC (CD45),blood_che GUSB TACSTD1 (EpCAM check) 10 TFF3 SPDEF RPL13A MUC1 input RNA equivalent to 5 ERBB2 approximately one cell KRT19 ACTIN TMSB10 0 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 Sieuwerts et al, Breast Cancer Res Treat 2009 Characterization of CTCs Generating profiles Final panel: Input for screening: • 3 reference genes � Reference genes � Epithelial specific control genes (HMBS, HPRT1, GUSB) • 2 leukocyte markers � Leukocyte specific control genes � Subtypes specific genes (PTPRC, BST1) • 55 CTC-specific mRNAs � Prognosis/predictive markers • 10 CTC-specific miRNAs � Targets for therapy � � � � 250 candidates mRNAs -can reliably be measured in 7.5 ml � � � � & 436 miRNAs for qRT-PCR with 5 CTCs vs. 0 CTCs or HBD - CTC fractions of 50 metastatic breast cancer patients (collected before starting first line systemic therapy), - primary tumors of 8 of the patients Sieuwerts, submitted, 2011 10
Characterization of CTCs G enerating profiles CTCs versus the primary tumor: ER and Her-2 mRNA discrepancy ER Her-2 CTCs CTCs primary tumor primary tumor . Sieuwerts, submitted, 2011 Epithelial CTC-specific gene expression patterns in CTC-enriched samples from metastatic BC patients unsupervised hierarchical cluster GFR enriched . ER-related Epithelial Proliferation Sieuwerts, submitted, 2011 11
CTC characterization � Molecular profiling of multiple genes is feasible if: � linear pre-amplification is applied � genes transcripts at least 10/100-fold higher expressed in CTCs compared to leukocytes can be considered � Difference in Her-2/ER between primary tumor and CTC � CTCs not always cluster along with their primary tumor � Profiling CTC identifies three CTC positive subclusters � driven by growth factor receptor, ER & proliferation Outline of the presentation � State-of-the-art of genomics research (focus BC) � Introduction about liquid biopsies (CTCs, ctDNA, EVs) � Application of CTC count and characteritics in metastatic cancer � ARv7 positive CTCs predict progression in mCPCR � Comparisons of ctDNA detection methods (dPCR & targetted NGS) � Standard operation procedures/sample collection � Examples of NGS in mBC and CRC � ESR1 detection in CTCs and ctDNA � Conclusions and outlook 12
CRPC treatment dilemma � Which agent to which patient at which moment? Docetaxel Cabazitaxel � Cross-resistance! - Prostatectomy - Androgen deprivation Local therapy (ADT) disease - ADT - Docetaxel Castration- - Abiratone? sensitive Enzalutamide? mPC Sipuleucel-T? - Docetaxel - Cabazitaxel Castration- - Abiratone resistant - Enzalutamide mPC - ... AR-V7 in CTCs � Predictive value of AR-V7 in CTCs � 62 mCRPC patients: � Abiraterone: N = 31 � Enzalutamide: N = 31 � AR-V7 in CTCs before treatment � AdnaTest � Enza group: 39% positive (65% docetaxel/abi) � Abi group: 19% positive (16% docetaxel/enza) 13
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