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

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

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)

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,