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MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls
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MCO 2011 - Slide 35 - F. Blackhall - Spotlight session - Circulating tumour cells: Challenges and pitfalls

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  • Circulating tumour cells are attracting increasing attention as less invasive, virtual biopsies that have potential to be examined serially before, after and during treatment. I imagine that many of you here want to know the answers to these questions posed. Using our experience in Manchester of analysis of CTCs I will explore these questions and illustrate where progress has been made and where future research needs to focus Their promise is of course to provide a surrogate for tissue before, during and after treatment providing real-time information There are new and evolving technologies to facilitate CTC enumeration, and to a relevant standard for incoporation into clinical trials A number of studies have been reported for various types of cancer that have demonstrated CTC number to have utility as a prognostic, predictive and pharmacodynamic biomarker Emerging potential for molecular characterisation of CTCs and for drug-target evaluation in trials Improved understanding of CTC biology may provide new insights into biology of metastasis and novel targets for drug discovery Particular relevance where access to ….
  • Allard’s paper in CCR described enumeration of CTCs in patients with lung cancer but it was not clear whether these patients included those with small cell histological subtype
  • Mention HR is univaraite cox regression analysis
  • Further exploration of the 2 nd time point. Not only using a cut-off of 5 CTCs but looked at any changes in CTC number from 1 st to second sample. Exciting findings. As early as 3 weeks can have indication of how a patient is doing. Accept these are small numbers of patients and needs to be validated in a larger cohort. ? How does it compare to the CTC scan or CXR at cycl
  • Are there technologies to increase yield ?
  • Novel technique – only 2 publications in literature, Opportunity for us to assess new CTC technology essentially from scratch. Not restricted to EpCAM positivity. So CellSearch and ISET for CTC detection and range of tools available to us for CTC characterisation that will concentrate on thie technology as potentially easier to obtain the cells and potentially less white cell contamination with ISET. Rnage of standard tools will be used for molecular characterisation Initial focus has been on exploring whether these methods of characterisation are possible using protocols that have been provided by Metagenex – company that market this product. Rnage of standard tools will be used for molecular characterisation
  • Striking that the number of cells isolated by ISET were dramatically higher then the number of cells isolated by CellSearch and begs the question as to whether cells isolated by filtration truly were malignant cells. Spent much of the rest of the PhD proving that they were....so let me show you the evidence
  • So we believe these are a very real phenomenon.
  • Do CTM provide a survival advantage ? Can CTCs and CTMs tell us anything about anoikis ?
  • Circulating tumour cells are attracting increasing attention as less invasive, virtual biopsies that have potential to be examined serially before, after and during treatment. I imagine that many of you here want to know the answers to these questions posed. Using our experience in Manchester of analysis of CTCs I will explore these questions and illustrate where progress has been made and where future research needs to focus Their promise is of course to provide a surrogate for tissue before, during and after treatment providing real-time information There are new and evolving technologies to facilitate CTC enumeration, and to a relevant standard for incoporation into clinical trials A number of studies have been reported for various types of cancer that have demonstrated CTC number to have utility as a prognostic, predictive and pharmacodynamic biomarker Emerging potential for molecular characterisation of CTCs and for drug-target evaluation in trials Improved understanding of CTC biology may provide new insights into biology of metastasis and novel targets for drug discovery Particular relevance where access to ….
  • Transcript

    • 1. © Paterson Institute for Cancer Research Fiona Blackhall, PhD FRCP Medical Oncologist The Christie NHS Foundation Trust Circulating Tumour Cells : challenges & pitfalls 10 th ESO-ESMO Masterclass 2011
    • 2. <ul><li>PCR Based Methods – 1990’s </li></ul><ul><li>Numerous publications 1,2 </li></ul><ul><li>Sensitive but target transcripts not specific </li></ul><ul><li>Difficult to reproduce across laboratories – many different variables </li></ul><ul><li>Unable to enumerate cells, assess morphology or further characterise </li></ul>Thomas Ashworth, May 1869, Australian Medical Journal Concept of CTCs is not new 1 Paterlini-Brechot .P et al, Cancer Lett, 2007; 253:180-204 2 Pantel. K et al, Nat Rev Cancer, 2008; 8(5):329-40 Tumour Blood sample CellSearch® 2004 3 CellSearch® - Prognostic Studies 3 Allard et al, Clin Cancer Res 10 (20), 6897-904, 2004 Cristofanilli et al, NEJM 351 (8), 781-94, 2004 Budd et al, Clin Cancer Res 12 (21), 6403-09, 2006 Hayes et al, Clin Cancer Res 12 (14), 4218-24, 2006 Cohen et al , JCO 26 (19), 3213-21, 2008 Cohen et al, Ann Oncol 20 (7), 1223-39, 2009 de Bono et al, Clin Cancer Res 14 (19), 6302-09, 2008 Scher et al, Lancet Oncoolgy, 10 (3), 233-39, 2009 Hou et al, Am J Path 175 (2), 808-16, 2009
    • 3. CIRCULATING TUMOUR CELLS (CTCs) IMPORTANT QUESTIONS © Paterson Institute for Cancer Research Are CTCs a surrogate for tissue ‘virtual biopsies’ ? What are the practical applications & current limitations of existing technologies ? What can CTCs tell us about patient survival, chance of response to therapy, target inhibition – Prognostic ? Predictive? Pharmacodynamic? Can CTCs be used for Drug-target evaluation in clinical trials ? At what time points should they be evaluated ? Can CTCs enlighten us on biology of metastases and provide novel targets for drug discovery ? OTHER QUESTIONS ?
    • 4. THE VERIDEX CELLSEARCH SYSTEM © Paterson Institute for Cancer Research FDA Approval for prognosis in metastatic Breast, Prostate and Colorectal Cancer
    • 5. Methods for CTC Detection and Enumeration CellSearch – automated cell isolation system CTCs are immunomagnetically enriched : EpCam labelled Ferrofluid Fluorescently labelled: DAPI (nucleus) Cytokeratin (epithelial) CD45 (white cell) 4 th channel – Ab of choice -> Enumeration -> Morphology Limited to EpCAM + / Cytokeratin + cells FDA Approved DAPI CK-PE Spare CD45-APC Comp
    • 6. © Paterson Institute for Cancer Research Jian Mei Hou et al Am J Pathology 2009 EpCAM, CK and CD 56 positive cells FIRST CLINICAL STUDY SCLC CTCs USING CELLSEARCH Microembolus *
    • 7. © Paterson Institute for Cancer Research Large range and high CTC# (compared to Breast, Prostate and CRC cut off of 3-5 CTCs used for FDA approved prognosis) ES- mean  sd, 1795  4620 LS - mean ± sd, 255 ± 737 CTC #, DISEASE PROGRESSION AND PROGNOSIS 13/76 patients had no CTCs. Baseline CTC# 50 CTC cut off derived Hou et al, JCO submitted
    • 8. © Paterson Institute for Cancer Research CTC# fell after cycle 1 platinum/etoposide therapy, mirroring disease response ~ 50% of patients become CTC negative by day 22 For PD biomarker need to examine CTCs early – potentially in first week of treatment A PHARMACODYNAMIC (PD) BIOMARKER Hou et al Am J Path 2009
    • 9. Phospho H2AX is a marker of DNA dsb damage & a pharmacodynamic biomarker of response to topoisomerase I inhibitors eg topotecan
    • 10. Phase I application of CTC expression of  H2AX as a serial PD biomarker
    • 11. CHARACTERISATION OF SCLC CTCS FOR DEVELOPMENT of PRO-APOPTOTIC BH3 MIMETICS © Paterson Institute for Cancer Research Predictive Biomarkers for ABT 263 Utility for patient stratification? ABT 263 - Navitoclax BH-3 mimetic targeting to anti-apoptotic Bcl-2 family proteins in Phase II trials for SCLC Jian Mei Hou with E McKeegan at Abbott, Gandhi et al JCO 2011 amplification triploid translocation x100 BCL2 IGH translocation amplification amplification triploid x100 BCL2 IGH normal x100 BCL2 IGH translocation FISH analysis of BCL2 in SCLC CTCs Sensitivity? 18q21-23 Bcl-2 Mcl-1 Resistance?
    • 12. CTCs DETECTED USING CELLSEARCH IN NSCLC Positive associations (p<0.01) with Stage IV disease , presence of liver &/or bone metastases 32% of stage IV patients positive for CTCs at baseline CTCs may be useful in management of stage IV patients Krebs et al. JCO 2011 32% ADC, 31 % SQ, 5% undifferentiated, 32% NOS
    • 13. PROGNOSTIC SIGNIFICANCE OF BASELINE CTCs IN NSCLC PATIENTS Krebs et al JCO 2011 Independent prognostic factor in MV analysis
    • 14. <ul><li>70 patients had second time point (TP) </li></ul><ul><li>blood sample available for CTC analysis </li></ul><ul><li>18/70 patients had  2 CTCs at </li></ul><ul><li>baseline. </li></ul>Of the 52 patients who had no CTCs at baseline, only 2 (4%) became positive at the second time point, both with 2 CTCs/7.5ml blood CTC# as a PHARMACODYNAMIC BIOMARKER * ** ** * progressed within 1 month ** died before cycle 2 Can change in CTCs predict for outcome ? Change Median OS 72% (13/18) Decrease 8.3 months 17% (3/18) Increase 3.3 months 11% (2/18) No change 3.9 months NSCLC Patients with CTC ≥2 before treatment Pre-Treatment Post Cycle 1 * * ** ** * progressed within 1 month ** died before cycle 2 (n=18)
    • 15. Molecular characterisation of CTCs <ul><li>Microfluidic protein chip </li></ul><ul><li>EpCAM dependent </li></ul><ul><li> CTC isolation compared to CellSearch </li></ul><ul><li>Fall in CTC# post EGFR-TKI </li></ul><ul><li>Rise in CTC# on progression </li></ul><ul><li>1 st generation chip difficult to scale up </li></ul><ul><li>2 nd generation ‘herringbone’ chip </li></ul>
    • 16. TMPRSS2-ERG fusion transcript detected using RT-PCR from RNA extracted from prostate cancer CTCs in chip
    • 17. © Paterson Institute for Cancer Research CTCS and EPITHELIAL TO MESENCHYMAL TRANSITION (EMT)? “ For most carcinomas, progression toward malignancy is accompanied by a loss of epithelial differentiation & shift towards mesenchymal phenotype ( EMT ) exacerbates motility and invasiveness” “ a pre-requisite for tumour infiltration & metastasis?” “ Numerous examples of advanced carcinomas with mesenchymal features that retained characteristics of well differentiated epithelial cells” “… incomplete EMT , collective migration ” Christiansen & Rajasekaran, Cancer Res, 66:8319, 2006 Do tumour cells in the circulation of lung cancer patients fit this description? Vimentin? (mesenchymal) Cytokeratin? (epithelial) E-Cadherin? (epithelial)
    • 18. ISOLATION BY SIZE OF EPITHELIAL TUMOUR CELLS (ISET) <ul><li>Enrichment based on cell size </li></ul><ul><li>Polycarbonate membrane </li></ul><ul><li>with 8µm pores </li></ul><ul><li>Flexibility for CTC characterisation </li></ul>© Paterson Institute for Cancer Research EpCam negative CTCs? Matt Krebs, Jian Mei Hou RareCells Inc. Paris. CD45 negatives = CTCs Criteria CTC selection Cell size ≥12um CD45 -ve High nuclear-cytoplasmic ratio Irregular nuclear shape Basophilic cytoplasm Hyperchromatic nucleus Clusters of cells
    • 19. 40 patients with NSCLC had paired blood samples available for analysis by CellSearch and ISET CELLSEARCH VS ISET CTC ENUMERATION There was no association between the number of CTCs detected by the two systems (Bland-Altman Plots) More CTCs detected by ISET than CellSearch Are all ISET CTCs, CTCs? CTC Number per 7.5ml Blood CellSearch Mean±SE Median Range Stage IIIA 0.4±0.2 0 0-1 Stage IIIB 0.4±0.3 0 0-3 Stage IV 6±3.6 1 0-78 CTC Number per 7.5ml Blood ISET Mean±SE Median Range Stage IIIA 15±13.1 4 0-68 Stage IIIB 41±16.3 8 0-188 Stage IV 98±44.9 38 0-1045
    • 20. All images 40x magnification PATIENT 122 PATIENT 122 PATIENT 121 HEALTHY DONOR CD45 CD45 CD144 Controls- CD144 Huvecs– Positive Control H1299 – Negative Control PBMCs– Negative Control Skin Cells EXCLUSION OF CIRCULATING ENDOTHELIAL CELLS AND SKIN CELLS 10µm ENDOTHELIAL CELLS
    • 21. © Paterson Institute for Cancer Research PATIENT 101 NSCLC – CIRCULATING TUMOUR MICROEMBOLI Parallel analysis – CellSearch and ISET No CTCs in CellSearch in 7.5ml . In 1ml blood by ISET, 20 single CTCs and 15 CTMs (so an extrapolated total of 885 individual CTCs in 7.5ml ) Patient died within 3 days of blood sample, No evidence of visceral metastases, no clinical features to suggest such a poor prognosis CTCs CIRCULATING TUMOUR MICROEMBOLI Wbc sitting over a pore pore CTM were observed in 15 (38%) of the 40 patients assessed by ISET
    • 22. COMPARISON OF CTM WITH PRIMARY TUMOUR BIOPSIES PATIENT 116 PATIENT 109 PATIENT 101 CD45 Stained CTM Tumour Biopsy – H&E CD45 Stained CTM Tumour Biopsy – H&E CD45 Stained CTM Tumour Cytology– PAP Stain 10µm All images 40x magnification
    • 23. Exploring Epithelial to Mesenchymal Transition Hypothesis Vimentin EpCam E-Cadherin N-Cadherin Christiansen & Rajasekaran, Cancer Res, 66:8319, 2006
    • 24. © Paterson Institute for Cancer Research CTC/CTM heterogeneity? (4 colour assay in development) EMT and/or E and M – collective cell migration ? M only s.c. no mets i.v. no mets E only s.c. no mets, i.v. mets M and E s.c mets, i.v. mets
    • 25. © Paterson Institute for Cancer Research LUNG CANCER CTCs/CTM and ANOIKIS? Anoikis (Frisch & Francis, JCB 1994) &quot;...the state of being without a home” apoptosis in absence of cell-matrix, cell-cell interactions. DO CELLS IN groups / microclusters / circulating tumour microemboli SURVIVE BETTER THAN SOLITARY CTCS? In preclinical studies tumour fragments form more metastases than the same number of single tumour cells injected iv.
    • 26. © Paterson Institute for Cancer Research SCLC CTCs, CTM & Apoptosis BUT So far, no apoptotic nuclei seen within CTM in SCLC or NSCLC patient blood using either CellSearch or ISET Correlation between apoptotic CTC# & circulating levels of apoptotic biomarker M30 (cleaved CK18) in 13 evaluable patients (p<0.05) Apoptotic nuclei frequently seen in CTCs Sheets, strands, clusters, rings reminiscent of collective migration ‘ predominant mode of invasion in a wide range of tumours ’ Friedl & Wolf Nature Reviews Cancer 2003
    • 27. SCLC CTC/CTM PROLIFERATION? Pilot Data – analysis on going in SCLC and NSCLC Ki67 positives more prevalent in CTC than CTM Hypothesis – CTM cells are out of cycle and protected from apoptosis
    • 28. © Paterson Institute for Cancer Research LUNG CANCER CTC SURVIVAL IN A HARSH CLIMATE? New technologies are paving the way to increased molecular knowledge of CTCs CTCs/CTMs have utility as biomarkers in lung cancer & other malignancies CTCs not yet ready for routine genetic testing but promising Proliferation Apoptosis? No proliferation Survival?
    • 29. CIRCULATING TUMOUR CELLS (CTCs) IMPORTANT QUESTIONS ANSWERED ? © Paterson Institute for Cancer Research Are CTCs a surrogate for tissue ‘virtual biopsies’ ? Yes…….. What are the practical applications & current limitations of existing technologies ? Complementary techniques are evolving What can CTCs tell us about patient survival, chance of response to therapy, target inhibition – Prognostic  Predictive ? Pharmacodynamic  Advanced disease setting vs early stage disease ? Can CTCs be used for Drug-target evaluation in clinical trials ? Yes At what time points should they be evaluated ? Early (depends on question) Can CTCs enlighten us on biology of metastases and provide novel targets for drug discovery ? Yes but technically challenging Studies of CTCs obtained serially through treatment and on relapse are now needed
    • 30. The Clinical and Experimental Pharmacology Group Manchester Cancer Research Centre Funding from Cancer Research UK, AstraZeneca The CTC Team: Matt Krebs, Jian Mei Hou, Tim Ward, Lynsey Priest , Rob Sloane, Karen Morris, Lee Lancashire,

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