AFTER THE SURGERY:
Rogue Tumor Cells - Are They Out There?

Developed by: B. Ancell
© American Association of Immunologists 1998

Abstract
Despite having their tumors discovered and removed in the early stages many people will still die within a few years of their cancer surgery. One reason may be the presence of undetected cancer cells circulating in their blood stream. These rogue tumor cells may go on to establish malignancies elsewhere in the body. The certainty of the existence of such cells would be invaluable knowledge for physicians trying to determine the extent of post-operative cancer treatment.

Because the numbers of these rogue cells are quite small an extremely sensitive test is needed to detect their presence. Such a test is the Polymerase Chain Reaction (PCR). In this test double-stranded DNA from a tumor cell is ‘unzipped and each resulting single-stranded template is copied repeatedly until there is enough to detect.

Because the DNA of a tumor cell could be different for every individual it is difficult to find a common standard for comparison. Many cancer cells, however, send out messages that healthy cells would never be expected to send. One of these messages that is common to many types of cancer cells is for the production of Carcinoembryonic Antigen (CEA). The messenger RNA (mRNA) sequence for CEA is known and can be used as a standard for comparison.

The polymerases used for PCR cannot amplify mRNA so it must first be converted to cDNA. This is done with the help of an enzyme called Reverse Transcriptase. Since this extra step is required the reaction is known as the Reverse Transcriptase Polymerase Chain Reaction, or RT-PCR for short.

The presence of this copied DNA is visualized by gel electrophoresis; a process whereby DNA migrates through a gel matrix under the influence of an electric current. Larger pieces of DNA move slower than smaller pieces resulting in a banding pattern that can then be compared with a known standard.

Students already familiar with the structure of DNA, the function of RNA, and the processes of transcription and translation learned about PCR and DNA electrophoresis technology. Students performed a PCR to amplify DNA samples from three cancer patients and then visualized the DNA on an agarose gel. The three DNA samples were compared with the known banding pattern for CEA. Students determined if any of the three cancer patients had CEA in their blood stream based on an analyses of the PCR product. One patient was found to have CEA present.

Objectives
At the end of this lesson students should:

  1. Be familiar with the concept and process of PCR.
  2. Be able to explain the difference between PCR and RT-PCR.
  3. List and explain the three basic steps of PCR.
  4. Explain the function and Taq Polymerase in PCR.
  5. Explain the role of primers in PCR.
  6. Explain what a dNTP is, list the four dNTPs, and explain their function in PCR.
  7. Explain what a Thermal Cycler is and its role in PCR.
  8. Explain the process by which DNA is copied in PCR.
  9. Explain what the letters CEA stand for any where CEA comes from.
  10. Explain why RT-PCR is used to detect the presence of CEA rather than just PCR
  11. Define the terms metastasis, malignancies, denatured, amplified, oligonucleotides, template, annealing.
  12. Name and give the function of the parts of an electrophoresis box.
  13. Explain the process by which banding patterns appear on a gel.
  14. Explain the use of a standard when examining banding patterns.
  15. Define the terms agarose, casting tray, combs, wells, migrate, matrix, base pairs.
  16. Perform a simple PCR analysis when given a DNA sample.
  17. Load PCR product onto an agarose gel and run an electrophoresis.
  18. Read, interpret, and draw conclusions from the banding pattern on an agarose gel.

To receive a copy of this curriculum, please contact: The American Association of Immunologists, telephone: (301) 634-7178, fax: (301) 634-7887, email infoaai@aai.org.