1. Breast and Thyroid Cancer Detection Using Immunomagnetic Beads By: Rami Abdelghafar & Pankaj K. Saha Memorial Sloan-Kettering Cancer Center Dr. Sat Bhattacharya June 30, 2005

2. What is Cancer?  “Silent Killer”  Genetic and Environmental factors  Metastasis  Bloodstream and Lymphatic system

3. Experimental Objectives  Isolation of circulating tumor cells (CTC)  Identifying cancer cells  Study the circulating breast and thyroid cancer cells

4. Methodology  Isolating Polymorphonuclear Cells (PMNC’s)  Isolation of RNA using Immunomagnetic Beads  Polymerase Chain Reaction (PCR)

5. Lab Materials Chemicals Storage CentrifugeWorkstation

6. Procedure for Isolating Polymorphonuclear Cells Blood (Cancer patient) “Buffy Coat” Storage

7. Lab Materials

8. Isolation of RNA Using Immunomagnetic Beads

9. Immunomagnetic Isolation of mRNA

10. Images of Immunomagnetic Isolation of CTC Immunomagnetic Beads Target Ber-Ep4 Epithelial Cell Antigen (ECA)

11. Reverse Transcriptase Polymerase Chain Reaction  Animation Animation

12. Polymerase Chain Reaction (PCR)

13. Lab Materials PCR MachinesIncubator PCR WorkstationCold Room

14. Identifying Type of Cancer Cell  Most cells have three types of genes: 1. Housekeeping Genes (Actin) 2. Tissue-specific Genes (Cytokeratin-19) 3. Organ Specific Genes (Mammaglobin) 4. Cancer Specific Genes (BRCA1 & BRCA 2)

15. Gel Electrophoresis Molecular Markers: 1.Actin (~350 bp) 2.Cytokeratin-19 (~300 bp) 3.Mammaglobin (~280 bp)

16. RT-PCR Results in the Detection of Breast CTC Positive Result: + Negative Result: - Sample #ActinCytokeratin 19MammaglobinOverall 1 ++-+ 2 ++++ 3 +--- 4 +--- 5 +--- 6 ++++ 7 ++++ 8 ++-+ 9 ++-+ 10 +--- 11 ++++ 12 +--- 13 ++-+ 14 ++-+ 15 ++-+ Percentage100%67%27%67%

17. Conclusion  Use of multiple markers enhances sensitivity  Cancer cells can be detected at early stages  Less radiation therapy, chemotherapy, and immunotherapy

18. Future Plans  Use additional molecular markers to enhance sensitivity (Telomerase)  Associate risk factors genes (BRCA1 & BRCA2)

19. References  Cotran, Kumar, Collins: The Pathological Basis of Disease 6th Edition (W.B. Saunders Company)  Hospital Practice (http://www.hosppract.com/)http://www.hosppract.com/  Foss AJ, Guille MJ, Occleston NL, Hykin PG, Hungerford JL, and LightmanS: The detection of melanoma cells in peripheral blood by reverse transcriptase polymerase chain reaction. Br J Cancer 72: 155 – 159, 1995  Ghossein RA, Bhattacharya S, Coit DG. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned? Recent Results Cancer Res. 2001;158:63-77. Ghossein RA, Bhattacharya S, Coit DG.  Ghossein RA, Bhattacharya S.: Molecular detection and characterization of circulating tumour cells and micrometastases in solid tumours. Eur J Cancer. 2000 Aug 36 (13 Spec No):1681-94.  NATIONAL BREAST CANCER FOUNDATION (http://www.nationalbreastcancer.org/)http://www.nationalbreastcancer.org/  Pelkey TJ, Frierson HF and Bruns DE: Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 42:1369 – 1381, 1996  Ronald A. Ghossien, Leo Carusone, and Satyajit Bhattacharya: Molecular detection of micrometastases and circulating tumor cells in melanoma prostatic and breast carcinomas. In vivo 14: 237 – 250, 2000

20. Acknowledgements  Dr. Sat Bhattacharya Memorial Sloan- Kettering Cancer Research Center Harlem Children Society Minimal Residual Lab (MRD Lab)

22. Thank You for Your Cooperation! Don’t be afraid to ask questions Rami Pankaj