1. 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

2. Background There are approximately 1,000,000 new cases of colorectal cancer and 500,000 deaths worldwide each year Multiple therapeutic options render the management of patients with metastatic disease complicated and nuanced A noninvasive prognostic and predictive marker could guide therapeutic decisions Circulating tumor cells can be isolated from the blood of patients with metastatic colorectal cancer (SJ Cohen et al, Clin Colorectal Cancer 2006)

3. Hypothesis: Circulating tumor cells can be used to guide therapy Baseline –prognostic During treatment –early indication of treatment effect –complementary to radiographic imaging –prognostic –Non-invasive source of tumor for in vivo pharmacodynamic monitoring

4. Objectives Primary Objective To measure agreement between circulating tumor cell number and objective antitumor response defined by imaging Secondary Objective To determine the association between circulating tumor cell number and progression-free and overall survival

5. Eligibility Adults with measurable metastatic colorectal cancer Initiating first-, second-, or third-line therapy (if EGFR inhibitor) ECOG performance status 0-2 Hemoglobin >8 g/dL Written informed consent

6. 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

7. CTC Sample Preparation (1) 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. CTC Sample Preparation (2) 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. Training and Validation Sets Preplanned analysis after 100 patients to select cutoff for “favorable” vs. “unfavorable” CTC at 3-5 weeks Criteria for selection of cutoff: –CTC threshold must be above normal background –Percent accuracy for agreement of CTC with imaging and median PFS and OS must reach a plateau –Frequency of CTC unfavorable patients must be at least 10% Based upon these criteria, > 3 CTC per 7.5 ml of blood was defined as “unfavorable”

10. Statistical Considerations for Primary Endpoint >99% power one-sided type I error (  ) = 0.025 Ho of <60% overall agreement between CTC (“favorable” vs. “unfavorable”) and radiographic response Ha of >75% agreement N = 400

11. Patient Characteristics Training Set (N=109) Validation Set (N=321) pAll Patients (N=430) Median Age (Range)65 (25-86)63 (22-92)0.2964 (22-92) Female42%45%0.5845% Performance Status 0 1 2 Unknown 46% 39% 9% 6% 45% 7% 3% 0.4946% 43% 7% 4% First-line Second-line Third-line 55% 35% 10% 77% 18% 5% <0.000172% 22% 6% Liver Metastasis71%74%0.6273% Median Followup, months (mean+/- SD) 14.5 (14.9 + 8.6) 9.7 (9.9 + 5.2) 0.00110.2 (11.2 + 6.6)

12. 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)

13. CTCs at 3-5 Weeks and Response Radiographic Response CTC at 3-5 Weeks N (%) Total < 3 cells> 3 cells CR/PR/SD228 (93%)18 (7%)246 (77%) PD or death54 (73%)20 (27%)74 (23%) Total282 (88%)38 (12%)320 (100%) Sensitivity = 27% Specificity = 93% Overall Agreement = 78%

14. 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

15. 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

16. 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)

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

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

24. Change in CTC at 3-5 Weeks is Predictive of Overall Survival %Probability of 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 CTC22717.7 (14.7 - 19.9) ≥3 CTC<3 CTC5311.0 (8.7 - 18.1) <3 CTC≥3 CTC910.9 ( 0.6 - ----) ≥3 CTC≥3 CTC303.7 (2.4 – 8.4) vs. P = 0.002 vs. P = 0.0002

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 Elevated CTC number at 3-5 weeks has high specificity but low sensitivity for early disease progression 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. Participating Clinical Investigators N. Iannotti, Hematology Oncology Associates of the Treasure Coast, Port Saint Lucie, FL; B.H. Saidman, Medical Oncology Associates, Kingston, PA; T.M. Fynan, Medical Oncology and Hematology, P.C., New Haven, CT; J. Picus, Washington University School of Medicine, St. Louis, MO; N. Gabrail, Canton, OH; M. Morse, Duke University Medical Center, Durham, NC; S.J. Cohen and N.J. Meropol, Fox Chase Cancer Center, Philadelphia, PA; M. Schwartz, Richmond VA; E. Mitchell, Thomas Jefferson University, Philadelphia, PA; F. Swan, Cancer Outreach Associates, Abingdon, VA; H.S. Jhangiani, Compassionate Cancer Care Medical Group, Inc, Corona, CA; K. Patel, Little Rock Hematology Oncology Associates, Little Rock, AR; R. Pelley, Cleveland Clinic Foundation, Cleveland, OH; C. Farber, Hematology-Oncology Associates of Northern New Jersey, Morristown, NJ; R.L. Robles, Bay Area Cancer Research Group, Concord, CA; V. Jones, Yakima Valley Memorial Hospital/North Star Lodge Cancer Center, Yakima, WA; H. Ghazal, Kentucky Cancer Center, Hazard, KY; P. Ross, St. Thomas Hospital, London, UK; G.A. Fisher, Stanford University Medical Center, Stanford, CA; R. Rangineni, St. Joseph Oncology, Inc, St. Joseph, MO; L. Bertoli, Clinical Research Consultations; Birmingham, AL; R.O. Giudice, New Mexico Oncology Hematology Consultants, Ltd., Alruquerque, NM; A. Khojasteh, Columbia Comprehensive Cancer Care Clinic, Jefferson City, MO; M.V. Pillai, Virginia Oncology Care, P.C., Richlands, VA; A. Pippas, John B. Amos Cancer Center, Columbus, GA; S. Berk, Hematology Oncology Associates of South Jersey, P.A., Mount Holly, NJ; A. Rakkar, Palo Verde Hematology Oncology, Inc, Glendale, AZ; M.B. Wax, Summit Medical Group/Overlook Oncology Center, Summit, NJ; K. Nahum, Oncology Physicians Network, Howell, NJ; A. Gonzalez, Cancer Specialists of South Texas, P.A., Corpus Christi, TX; R.K. Gamble, Cancer Care Specialists, Houma, LA; J.W. Jakub, Lakeland Regional Cancer Center, Lakeland, FL; R.J. Jacobson, Palm Beach Cancer Institute, Palm Beach, FL; J. Fleagle, Rocky Mountain Cancer Center – Thornton, Thornton, CO; G. E. Newman, Knoxville, TN; M. Woodson, Hematology Oncology Consultants, Inc., St. Louis, MO; P. Cobb, Hematology/Oncology Centers of the Northern Rockies, Billings, MT; D. Mukhopadhyay and R. Prabhu, Redrock Medical Group, Las Vegas, NV; A.M. Wierman, Nevada Cancer Center, Las Vegas, NV; W. Ho, St. Joseph Hospital, Orange, CA; A. Cohn, Rocky Mountain Cancer Center – Lone Tree, Lone Tree, CO. Dutch Colorectal Cancer Group (DCCG): C.J.A. Punt, Radboud University Medical Center, Nijmegen; G.J. Creemers, Catharina Ziekenhuis, Eindhoven; P. Depres t, F.A. Valster and T.J. Roding, Ziekenhuis de Lievensberg, Bergen op Zoom; M.B. Polee, Medisch Centrum Leeuwarden, Leeuwarden; A.J. ten Tije, Ziekenhuis Gooi-Noord, Blaricum; D.J. Richel, University of Amsterdam, Amsterdam; M. Legdeur, Medisch Spectrum Twente, Enschede, J.J.M. van her Hoeven, Amstelveen, A. Honkoop, Isala Klinieken, Zwolle; Z. Erjavec, Delfzicht Ziekenhuis, Delfzijl; R.S. de Jong, Martini Ziekenhuis, Groningen, P. Slee, St. Antonius Ziekenhuis, Nieuwegein; J. Douma, Ziekenhuis Rijnstate, Arnhem; J. Sleeboom, Ziekenhuis Leyenburg, Den Haag.