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.

Molecular Analysis MagNest TM Epithelial Cell Kit CellSearch™ System

Defining a Tumor Cell 1.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) 2.Positive for DAPI (Nucleus): Must have a nucleus Should fit in the cytoplasm Should be at least 50% inside the cytoplasm 3.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 4.Negative for CD45/APC (Leukocyte cytoplasm): The tumor cells should be negative for CD45/APC 5.Composite: TUMOR CELL COMP CD45-APC CK-PE DAPI CK-PE DAPI COMP CD45-APC CK-PE or

5 COMP CD45-APC CK-PE DAPI CellSpotter Images For Review

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7 CTC’s in Various Carcinomas

8 Recovery of CTC’s Processed By CellSearch Instrumentation

9 Overall Survival of Pts. (n=177) With 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: 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: 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:

14 CellSearch Information Name: –CellSearch™ Circulating Tumor Cells, Breast Cancer Test Code: –16011X CPT codes: –88346 (X2); 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: Jamie Arking, Ph.D., MBA –Phone:

16 Thank You and Questions ?