Clinical Utility of CellSearch

1. Clinical Utility of CellSearch Richard A. Bender,MD,FACP Medical Director-Hematology/Oncology Quest Diagnostics Nichols Institute

2. What is CellSearch™ • A new technology developed by the Veridex Division of Johnson & Johnson and licensed by the FDA for predicting progression-free and overall survival and response to chemotherapy in patients with metastatic breast cancer • A method to quantitate the number of circulating tumor cells (CTC) contained in 7.5 ml of peripheral blood • Specimens are stable at room temperature for 72 hrs. post collection in special tubes and are be processed at Nichols Institute in Chantilly, Va. or San Juan Capistrano, Ca.

3. Epithelial Cell Kit MagNestTM Molecular Analysis CellSearch™ System

4. Positive for CK-PE (Cytokeratin) • Morphology: Round to oval; sometimes elongated • At least 4 m in size (should fit in the white square inside the measuring box) • In-tact (the cell & all edges are in-tact) • Positive for DAPI (Nucleus): • Must have a nucleus • Should fit in the cytoplasm • Should be at least 50% inside the cytoplasm • Negative for Blank Channel: • Good, Bright cell in Ctrl column = Control Positive • < 3 spots = Control Negative • NOTE: All patient sample tumor cells should be negative in Control Column • Negative for CD45/APC (Leukocyte cytoplasm): • The tumor cells should be negative for CD45/APC • Composite: • TUMOR CELL CK-PE CK-PEDAPI COMP or CD45-APC COMP CD45-APC CK-PEDAPI Defining a Tumor Cell

5. CellSpotter Images For Review COMP CD45-APC CK-PEDAPI

7. CTC’s in Various Carcinomas

8. Recovery of CTC’s Processed By CellSearch Instrumentation

9. Overall Survival of Pts. (n=177) With <5 or > 5 CTC Quantitated by CellSearch-TM

10. In addition, … • The # CTC’s can be used to monitor a patient’s response to chemotherapy as early as 3-4 weeks following the initial dose to determine if Rx affected the #CTC’s as a surrogate to its antitumor affect. • This assay “… is intended for use in adjunctively monitoring and predicting cancer disease progression and response to therapy” for patients with advanced breast cancer according to the FDA.

11. Does a Reduction in CTC’s at First Follow-Up Predict For Improved PFS?

13. References • Allard WJ, Matera J, Miller, MC et al. (2004). Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects of patients with nonmalignant diseases. Clinical Cancer Research, 10: 6897-6904. • Cristofanilli M, Budd GT, Ellis M et al. (2004). Circulating tumor cells, disease progression , and survival in metastatic breast cancer. The New England Journal of Medicine, 351: 781-791. • Cristofanilli M, Hayes DF, Budd T et. al. (2005) Circulating Tumor cells: A novel prognostic factor for newly diagnosed metastatic breast cancer. Journal of Clinical Oncology 23: 1420-1430.

14. CellSearch Information • Name: • CellSearch™ Circulating Tumor Cells, Breast Cancer • Test Code: • 16011X • CPT codes: • 88346 (X2); 88361 • Specimen requirements: • 10mL room temperature whole blood collected in CellSave Preservative Tube™ • Specimen must be received at Nichols Institute within 72 hours from draw.

15. Additional Information If you would like to receive additional information on this assay please contact: • Richard Bender, MD, FACP • Phone: 949-728-4086 • Richard.A.Bender@QuestDiagnostics.com • Jamie Arking, Ph.D., MBA • Phone: 703-802-7168 • Edward.J.Arking@QuestDiagnostics.com

16. Thank You and Questions ?