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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. - PowerPoint PPT Presentation
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Breast and Thyroid Cancer Detection Using
Immunomagnetic Beads
By: Rami Abdelghafar & Pankaj K. SahaMemorial Sloan-Kettering Cancer Center
Dr. Sat BhattacharyaJune 30, 2005
What is Cancer?
“Silent Killer”
Genetic and Environmental factors
Metastasis
Bloodstream and Lymphatic system
Experimental Objectives
Isolation of circulating tumor cells (CTC)
Identifying cancer cells
Study the circulating breast and thyroid cancer cells
Methodology
Isolating Polymorphonuclear Cells (PMNC’s)
Isolation of RNA using Immunomagnetic Beads
Polymerase Chain Reaction (PCR)
Lab Materials
Chemicals Storage
Centrifuge Workstation
Procedure for Isolating Polymorphonuclear Cells
• Blood (Cancer patient)
• “Buffy Coat”
• Storage
Lab Materials
Isolation of RNA Using Immunomagnetic Beads
Immunomagnetic Isolation of mRNA
Images of Immunomagnetic Isolation of CTC
Immunomagnetic Beads Target Ber-Ep4 Epithelial Cell Antigen (ECA)
Reverse Transcriptase Polymerase Chain Reaction
Animation
Polymerase Chain Reaction (PCR)
Lab Materials
PCR Machines Incubator
PCR Workstation Cold Room
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)
Gel Electrophoresis
Molecular Markers:
1. Actin (~350 bp)
2. Cytokeratin-19 (~300 bp)
3. Mammaglobin (~280 bp)
RT-PCR Results in the Detection of Breast CTC
Positive Result: +Negative Result: -
Sample # Actin Cytokeratin 19 Mammaglobin Overall
1 + + - +
2 + + + +
3 + - - -
4 + - - -
5 + - - -
6 + + + +
7 + + + +
8 + + - +
9 + + - +
10 + - - -
11 + + + +
12 + - - -
13 + + - +
14 + + - +
15 + + - +
Percentage 100% 67% 27% 67%
Conclusion
Use of multiple markers enhances sensitivity
Cancer cells can be detected at early stages
Less radiation therapy, chemotherapy, and immunotherapy
Future Plans
Use additional molecular markers to enhance sensitivity (Telomerase)
Associate risk factors genes (BRCA1 & BRCA2)
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
Acknowledgements
Dr. Sat Bhattacharya Memorial Sloan-
Kettering Cancer Research Center
Harlem Children Society
Minimal Residual Lab (MRD Lab)
Thank You for Your Cooperation!Don’t be afraid to ask questions
Rami
Pankaj