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