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Breast and Thyroid Cancer Detection Using Immunomagnetic Beads

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

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Breast and Thyroid Cancer Detection Using Immunomagnetic Beads

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  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 Storage Chemicals Centrifuge Workstation

  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

  12. Polymerase Chain Reaction (PCR)

  13. Lab Materials Incubator PCR Machines PCR Workstation Cold Room

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

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

  16. RT-PCR Results in the Detection of Breast CTC Positive Result: + Negative Result: -

  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/) • 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.: 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/) • 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)

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

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