QUICK CELL CAPTURE AND CHARACTERIZATION GUIDE FOR CELLSEARCH CUSTOMERS

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1 QUICK CELL CAPTURE AND CHARACTERIZATION GUIDE FOR CELLSEARCH CUSTOMERS CellSave EDTA Blood sample Rare cell capture Enumeration Single protein marker Cell capture for molecular characterization CELLSEARCH Profile Kit (RUO) CELLTRACKS Circulating Endothelial Cell Profile Kit (RUO) Further characterization of CTCs through CRS Services FISH Multiple protein markers (Cheshire) Extraction and characterization of DNA, RNA, and proteins Gene Expression Mutation Detection Sequencing

2 Cellular Kits for Rare Cell Capture and Staining Circulating Tumor Cells CELLSEARCH Circulating Tumor Cell Kit (IVD) product code: CTC-identification marker: Cytokeratin (PE) Free channel: FITC Please refer to the Instructions for Use at CELLSEARCH CXC Kit (RUO) product code: CTC-identification marker: Cytokeratin (FITC) Additional moderately expressed marker: PE CELLSEARCH Epithelial Cell Kit (RUO) product code: CTC-identification marker: Cytokeratin (PE) Circulating Endothelial Cells CELLSEARCH Circulating Endothelial Cell Kit (RUO) product code: Capture antigen: CD146 CEC-identification marker: CD105 (PE) Circulating Melanoma Cells CELLTRACKS Circulating Melanoma Cell Kit (RUO) Capture antigen: CD146 product code: 9594V CMC-identification marker: HMW-MAA (PE) White blood cell and endothelial cell markers: CD45 (APC), CD34 (APC) Please note that all of the above kits are commercially available and can be run on any installed CELLSEARCH instrument.

3 Cellular Kits for Rare Cell Capture and Staining Control Cell Kits CELLSEARCH CTC Control Kit (IVD) product code: Please refer to the Instructions for Use at Each bottle contains both high & low control CELLSEARCH Circulating Epithelial Cell Control Kit (RUO) product code: Each bottle contains both high & low control CELLSEARCH CXC Control Kit (RUO) product code: Each bottle contains a high control CELLSEARCH CEC/CMC Control Kit (RUO) Each kit contains 24 single use bottles of fixed cells from SK-Mel28 product code: 9572V Each bottle contains a high & low control Tumor Phenotyping Reagents (TPRs) CELLSEARCH Tumor Phenotyping Reagent HER-2/neu (RUO) product code: Fluorescein-conjugated monoclonal antibody that is to be used in conjunction with the CELLSEARCH Epithelial Cell Kit to phenotype circulating epithelial cells for the presence of HER-2/neu CELLSEARCH Tumor Phenotyping Reagent EGFr (RUO) product code: Fluorescein-conjugated monoclonal antibody that is to be used in conjunction with the CELLSEARCH Epithelial Cell Kit to phenotype circulating epithelial cells for the presence of EGFr CELLSEARCH Tumor Phenotyping Reagent IGF-1R (RUO) product code: 9597 Allows for the qualitative determination of the presence of the IGF-1R antigen on circulating epithelial cells in the blood, and is to be used in conjunction with the CELLSEARCH CXC Kit

4 Molecular Characterization of Rare Cells Overview Using the profile kits, CTCs and CECs are captured and presented in a volume of 900 microliters. No fixation, staining or permeabilization is performed during capture. Cells are captured alive and can be maintained and potentially propagated under appropriate tissue culture conditions. The enriched sample also contains ferrofluid and a few thousands leukocytes which were non-specifically retained during the enrichment procedure. Samples should be processed as soon as possible to maximize nucleic acid integrity and recovery. Blood samples may be stored or transported in EDTA tubes at room temperature for up to 36 hours prior to processing. Note however that immediate processing is preferred since a loss of mrna from epithelial cells may result if processing is delayed. Captured cells can be lysed and stored in Trizol or RLT buffers for future extraction of total RNA (including mirna), DNA and proteins. Simultaneous extraction of RNA, DNA and proteins can be performed from each sample using: Trizol protocol from Invitrogen ( AllPrep kits from Qiagen ( All-in-One Purification kit from Norgen ( Numerous commercial kits can be used for separate isolates of RNA, mirna, DNA and proteins. We recommend utilizing kits that are designed for small samples since CTC-enriched samples rarely contain more than 10,000 cells. Molecular Kits for Rare Cell Capture CELLSEARCH Profile Kit (RUO) product code: CTCs are captured using EPCAM CELLSEARCH CEC Profile Kit (RUO) CECs are captured using CD146 product code: 9564V Molecular Characterization Methods Analyte RNA DNA Proteins Methods RT-PCR Microarray RNA Seq Next Gen Sequencing Mutation Detection Proteomics Platforms Taqman (ABI) BioMark (Fluidigm) HiSeq/Miseq (Illumina) Cast PCR (ABI)

5 CLINICAL RESEARCH SOLUTIONS (CRS) SERVICES Rare Cell Enumeration and Single Marker Protein Expression Using any of our commercially available kits Other rare cell testing available (e.g. CMMC) A variety of Protein Markers (e.g. AR, ER, Ki-67 and more) Custom Assay Development Ferrofluid and Antibody Development For the development of other Rare Cell Capture and Protein Expression assays not currently commercially available Assay verification/pilot studies Fluorescent in situ hybridization (FISH) Custom FISH assay development Unique probes prepared for each study Utilizes special instrumentation for rare cell fixation in CELLSEARCH cartridges If desired, FISH can be performed following enumeration kits that were described above. Rare cell FISH allows interrogation of CTCs at the genetic level. Janssen Diagnostics was the first company to create REPEAT-FREE probes, which provide a clearer signal and are easier to score. By removing the repeat sequences, Janssen Diagnostics made it possible to deliver a level of performance key to understanding complex diseases. Numerous FISH assays have been developed for CTCs. Examples include Her-2/SE17, cmet/se7, EGFR/SE7, AR/SEX, ERG break, AURK-A/Chr20, AURK-B/SE17, P53/SE17, IGF1-R/Chr15, cmyc/se8 and PTEN/SE10. Additional assays can be developed upon request. Please note that subsequent FISH analysis requires special equipment and fixation of captured stained cells in cartridges. Contact CRS for additional information: Multiple Protein Markers (Cheshire) Fluorescent signals from an initial set of markers can be erased using a proprietary Cheshire procedure and re-stained with a second set of biomarkers. For details about this protocol please contact CRS: Molecular Characterization of Rare Cells Profile Kit Enrichment followed by lysis and storage of samples DNA & RNA Extraction Method development to enable rare cell characterization via downstream molecular platforms Assay Development Process Feasibility 1. Potential antibody selection 2. Antibody evaluation by specific performance criteria 3. Final antibody selection 4. Antibody conjugation Assay Verification 1. Create prototype assay 2. Test prototype assay to specific agreed-upon criteria 3. Identify assay controls Assay Transfer 1. Development report 2. Train clinical lab scientists 3. Protocols Assay Validation 1. Test with clinical samples 2. Final report CELLSEARCH and related products are covered by one or more patents as described on CELLSEARCH, CELLTRACKS, and REPEAT-FREE are registered trademarks of Janssen Diagnostics, LLC Janssen Diagnostics, LLC VX121019rev1

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