PCR in molecular pathology

Yohannes Yesus, M.D.

Major improvements in under-standing the genetic alterations responsible for human disease have been made in recent years, resulting in quicker and more accurate diagnoses. One of the most exciting prospects is assessing disease predisposition based on the inheritance of specific genes. Many diseases are amenable to treatment if they can be diagnosed at an early stage or before the manifestation of symptoms. Genetic testing of individuals to evaluate their susceptibility to particular heritable diseases, including many forms of cancer, will greatly reduce the associated morbidity and mortality.

A new molecular tool
Knowledge in the molecular field has expanded with the development of tools that allow rapid and specific analysis of genetic material from blood or tumor tissues. A vast array of molecular biology techniques have been developed and have made the cloning and characterization of genetic material simple and reliable. One of the most useful and versatile techniques is the polymerase chain reaction (PCR).

Developed in 1985, PCR involves the in vitro amplification of virtually any gene sequence. This process results in an exponential increase of a particular sequence, generating millions of exact copies. By producing such a large amount of material, the subsequent analysis can then be made with relative ease and accuracy. PCR quickly has become an invaluable tool in the laboratory and will play an increasing role in disease diagnosis due to its remarkable sensitivity and specificity.

Detecting genetic changes in cancer
A vast amount of information recently has emerged about the genetic alterations associated with cancers of various types. It now appears likely that all human malignancies may be associated with the mutation or absence of specific tumor suppressor genes and/or with the activation of certain cancer-causing oncogenes. When triggered, oncogenes and tumor-supressor genes can alter protein products within the cell, stimulate cell division or inhibit normal cell death, all of which may initiate or continue the conversion of normal cells into cancer cells.

In many cancers, particularly solid tumors, it is likely that a complex series of genetic alterations must exist before malignant transformation occurs. Conversely, in hematologic malignancies, a single genetic alteration may be sufficient for the development or the initiation of that cancer. This is especially true in certain leukemias and lymphomas, where nonrandom chromosomal re-arrangements often develop, resulting in the activation of oncogenes. The association of specific oncogenes with certain hematologic malignancies has allowed for the development of PCR-based assays to detect these genetic alterations.

PCR in monitoring therapy
In addition to its use in the initial diagnosis of cancer, PCR analysis is changing the ways in which these diseases are monitored and treated. PCR is capable of detecting one cancer cell in more than 100,000 normal cells. With such sen-sitivity, PCR is well suited to monitor disease progression or regression during treatment, to detect disseminated disease and to assess the presence of residual disease following high dose chemotherapy or bone marrow transplantation. In autologous bone marrow transplantation, PCR can be used to detect the presence of extant cancer cells prior to reinfusing the marrow into the patient.

One of the most exciting uses of PCR is the significant role it will eventually play in identifying individuals with cancer susceptibility genes. Presently, several of these genes have been isolated, including the retinoblastoma susceptibility gene, the p53 tumor suppressor gene and the APC tumor suppressor gene whose inactivation is responsible for inherited polyposis colon cancer. Additionally, it is likely that the genes responsible for familial breast cancer and other familial colorectal cancer syn-dromes will be isolated in the near future.

Identifying individuals with genetic susceptibility to cancer should greatly reduce the mortality of these diseases, since many cancers are curable if diagnosed early. When this evaluation becomes part of routine clinical practice, PCR will play a central role.

PCR and infectious disease
Many types of viruses and atypical bacteria such as mycobacteria are difficult or slow to culture. In the area of cancer, infections are a significant problem because the patient's immune system is frequently inhibited by their disease or treatment. The ability to rapidly diagnose certain infectious agents could lead to prompt specific treatments. PCR-based diagnosis has been applied to several infectious diseases for which a specific gene sequences have been identified.

Summary
PCR is revolutionizing the way laboratories diagnose, monitor and help determine treatments in cancer patients. This will clearly have a dramatic effect, not only on cancer, but in all areas of medicine for the next decade. While PCR is the current "workhorse" in the mole-cular pathology laboratory, other mole-cular biology methods also are necessary for complete evaluation of cancers.

Patients or specimens may be readily referred for specialized testing. Samples of blood, bone marrow and tissue samples in a tissue culture fluid may be sent by Federal Express or overnight mail. Referred specimens are reported initially be telephone to the referring physician, followed by a written report. For questions or consultation, call Yohannes Yesus, MD, or Charles Caldwell, MD at 1 (800) 595-6559, Ext. 1305 or 1283.