1. AGA/ASCO/ASTRO/SSO Gastrointestinal Cancers Symposium Orlando, FL January 26, 2008 Circulating tumor cells: are they predictive markers? Neal J. Meropol, M.D. Fox Chase Cancer Center

2. “Predictive” vs. “Prognostic” Predictive: explains variability in response to treatment –Traditional application before treatment Prognostic: explains variability irrespective of treatment

3. Natural History of Circulating Tumor Cells Paterlini-Brechot and Benali, Cancer Letters, 2007

4. Potential advantages of circulating tumor cells (CTCs) compared to other blood markers Representative of tumor access to circulation Permits multiple simultaneous analyses –Enumerate, phenotype, gene expression Cytopathology in vivo pharmacodynamic assessment, gene expression profiling Sensitivity/Specificity

5. Why Study CTCs? Prognosis Monitor disease course –Minimal residual disease –Early relapse –Response to therapy Treatment selection Drug development (pharmacodynamics) –Target acquisition –Down stream effects

6. Methods for CTC Detection Density gradient Immunomagnetic separation (beads, ferrofluid) Size-based filtration Count Cytopathology RT-PCR Genotyping 20%-70% of patients with colorectal cancer have detectable CTCs

7. Immunomagnetic Separation Magnetic Incubations - 7.5 ml Blood + 6.5mL Buffer Plasma Aspiration & Addition of EPCAM Ferrofluid Station 1Stations 2 & 3 Primary Magnetic Separation & Resuspension Station 4 - Addition of Cytokeratin-PE CD45-APC, & DAPI Station 5 Centrifuge Place on Instrument Described in: WJ Allard et al, Clin Cancer Res 10: 6897-6904, 2004

8. Immunomagnetic Separation Stations 6,7, & 8Station 9b Staining Incubation, Magnetic Wash & Free Particle Removal Final Resuspension Station 9a Image Gallery Automatic Transfer of Sample for Fluorescent Image Analysis

9. Characterization of CTCs by Flow Cytometry SJ Cohen et al. Clin Colorectal Cancer, 2006 CTC, green and red EGFR+, red WBC, blue Beads, yellow

10. N. J. Meropol, S. J. Cohen, N. Iannotti, B. H. Saidman, K. D. Sabbath, M. C. Miller, G. V. Doyle, H. Tissing, L. W.M.M. Terstappen, C.J.A. Punt Fox Chase Cancer Center, Philadelphia, PA; Hematology Oncology Associates, Port St. Lucie, FL; Medical Oncology Associates, Kingston, PA; Medical Oncology and Hematology, PC, Waterbury, CT; Immunicon Corporation, Huntingdon Valley, PA; Radboud University Medical Center, Nijmegen, The Netherlands Circulating tumor cells (CTC) predict progression free (PFS) and overall survival (OS) in patients with metastatic colorectal cancer ASCO 2007

11. Objectives and Eligibility Overall Objective To determine the association between circulating tumor cell number and clinical outcomes in patients with metastatic colorectal cancer Eligibility Adults with measurable metastatic colorectal cancer, initiating first-, second-, or third-line therapy

12. Methods Multicenter international study Radiographic tumor measurement at baseline and every 6-12 weeks after treatment initiation (RECIST criteria) Peripheral blood was collected for CTC enumeration at baseline and subsequently at 1-2, 3-5, 6-12, and 13-20 weeks after treatment initiation Blood mailed overnight at room temperature, and processed at 1 of 4 laboratories within 96 hours

13. 11%12%13%14%16%18%21%26%33%47%53% 0 2 4 6 8 10 12 14 16 18 20 22 24 26 0>= 1>= 2>= 3>= 4>= 5>= 6>= 7>= 8>= 9>= 10 CTC / 7.5mL of Blood (Baseline Draw) Median OS from Baseline (Months) Median OS for Patients with Metastatic Colorectal Cancer Based Upon number of CTC Prior to the Initiation of Therapy (N=413) ASCO 2007

14. Baseline CTC: Progression Free Survival % Progression Free Time from Baseline Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 P = 0.0002 CTC/7.5 mLMedian in Months (95% CI) <3 CTCs7.9 (7.0 - 8.6) ≥3 CTCs4.5 (3.7 - 6.3) <3 CTCs ≥3 CTCs 305 108 269 84 229 60 187 42 138 28 88 16 44 8 32 3 20 2 15 2 8 1 6 0 3 0 0 0 0 0 0 0 ASCO 2007

15. Baseline CTC: Overall Survival % Survival 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Time from Baseline Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 20 <3 CTC ≥3 CTC 305 108 289 102 276 86 252 66 227 49 180 36 134 24 107 13 78 12 60 11 43 7 32 4 22 2 11 1 4 1 2 0 CTC/7.5 mLMedian in Months (95% CI) <3 CTC18.5 (15.5 - 21.2) ≥3 CTC9.4 (7.5 - 11.6) P < 0.0001 ASCO 2007

16. CTC During Treatment: PFS %Probability of Progression Free Survival Time from Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 NMedian PFS in Months (95% CI) <3 CTC ≥3 CTC 1-2 wks3157.3 (6.5 - 8.1)3.8 (1.9 - 5.1) 3-5 wks3296.8 (6.1 - 7.6)1.9 (1.2 - 4.4) 6-12 wks2846.5 (5.8 - 7.7)2.0 (0.5 - 2.5) P < 0.0001 at each timepoint ASCO 2007

17. CTC During Treatment: Overall Survival %Probability of Survival Time from Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Median OS in Months (95% CI) N<3 CTC ≥3 CTC 1-2 wks35715.7 (14.3 - 18.4)6.1 (4.9 - 8.9) 3-5 wks33316.4 (14.1 - 18.3) 4.4 (2.6 - 8.7) 6-12 wks31015.8 (13.8 - 19.2)3.3 (1.8 - 5.6) P < 0.0001 at each timepoint ASCO 2007

18. Predictors of PFS and OS: Multivariable Model – Baseline (N=373) VariablePFSOS HRP P CTC number 31.80.0002.40.000 Line of therapy 1 st vs. 2 nd vs. 3 rd 1.60.0001.40.007 Age 65 1.50.0011.90.000 PS0 vs. 1 vs. 2 1.20.0841.50.001 ASCO 2007

19. Predictors of PFS and OS: Multivariable Model – 3-5 Weeks (N=302) VariablePFSOS HRP P CTC number 32.20.0004.50.000 Line of therapy 1 st vs. 2 nd vs. 3 rd 1.70.0001.70.001 Age 65 1.60.0002.10.000 PS0 vs. 1 vs. 2 1.20.1091.30.032 ASCO 2007

20. Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR 27118.8 (17.0 - 25.1) <3 CTCPD/Death648.3 (5.8 - 11.2) >3 CTCS/PR/CR137.1 (5.4 - 10.8) >3 CTCPD/Death163.1 (2.0 - 4.4) vs. P < 0.0001 ASCO 2007

21. Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR 27118.8 (17.0 - 25.1) <3 CTCPD/Death648.3 (5.8 - 11.2) >3 CTCS/PR/CR137.1 (5.4 - 10.8) >3 CTCPD/Death163.1 (2.0 - 4.4) vs. P = 0.0001 ASCO 2007

22. Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR 27118.8 (17.0 - 25.1) <3 CTCPD/Death648.3 (5.8 - 11.2) >3 CTCS/PR/CR137.1 (5.4 - 10.8) >3 CTCPD/Death163.1 (2.0 - 4.4) ASCO 2007

23. Decrease in CTC at 3-5 Weeks is Associated with Improved PFS in Patients with > 3 CTC at Baseline % Probability of Progression Free Survival Time from 3-5 Week Blood Draw (Months) 0 246 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Baseline3-5 wksNMed PFS (95% CI) ≥3 CTC<3 CTC526.2 (4.6 - 7.0) ≥3 CTC≥3 CTC281.6 (1.2 - 2.7) P = 0.02 ASCO 2007

24. CTC Association with PFS and OS by Lines of Therapy Cohen et al. AGA/ASCO/ASTRO/SSO GI Cancers Symposium, 2008 1 st line 2nd line PFS OS HR=1.44 (1.04-1.98) HR=2.22 (1.49-3.30) HR=1.90 (1.26-2.85) HR=2.98 (1.85-4.77)

25. Conclusions In patients with metastatic colorectal cancer, CTC number before and after initiation of treatment is a significant independent predictor of progression free survival and overall survival CTC enumeration is complementary to imaging, and may provide early evidence of treatment success or failure Further research is required to determine whether change in therapy based upon elevated CTC number at early followup will improve patient outcomes

26. Are CTCs Predictive Markers? Maybe Traditional application before treatment –no data yet regarding role of phenotyping/genotyping CTCs and response to therapy Alternative application early after treatment initiation –suggestive evidence that CTCs indicate resistance to treatment

27. Clinical validation study design requirements Standardized assay platform for clinical decision making Prospective randomized trial –archival well-annotated clinical specimens do not exist Randomized population of adequate size to answer clinical question –Requires previous validation with assay platform

28. Metastatic disease study design: early change in therapy Begin Treatment A Assess at 1-3 weeks Favorable CTCs? Continue Treatment A Yes* Continue Treatment A Change to Treatment B R No *assumes that continuation of Treatment A is best in Favorable CTC group

29. Metastatic disease study design: selection of initial therapy Favorable CTCs? Standard initial therapy Yes* Standard initial therapy More aggressive initial therapy R No *assumes that standard therapy is “best” in Favorable CTC group

30. Surveillance study design 1: randomize early Resected patients at risk for recurrence Routine surveillance Routine surveillance + CTC evaluation, with aggressive intervention if CTC “recurrence” R

31. Surveillance study design 2: randomize later Resected patients at risk for recurrence Routine surveillance plus CTC evaluation CTC “Recurrence” Continue routine surveillance Aggressive intervention R

32. Many Questions Remain What are “circulating tumor cells”? Are CTCs the same as in situ cancer? How does cell separation process affect gene expression? How can CTCs be used in the drug development process? How can CTCs be integrated into routine patient care?

33. The End