Molecular pathology and biotechnology in clinical diagnosis

An anatomic pathology laboratory uses formalin-preserved, paraffin-embedded tissue as the main source of material used to study and diagnose diseases. Clinical pathology, on the other hand, is involved in measuring serum enzyme levels (AST and ALT), blood constituents (hemoglobin and hematocrit) and other serum metabolic byproducts (BUN and Creatinine) in order to monitor organ or tissue damage in disease processes.

DNA and RNA analysis (genomics) and total protein analysis (proteomics) are now at the forefront of molecular pathology as new tools for diagnosis. Forensic pathology uses DNA analysis as part of its armamentarium for investigations. Paraffin-embedded tissues can now be analyzed for specific genes of interest by using probes. At the Institute of Ophthalmology of the UP-PGH, this technique is used to study the genetics of retinoblastoma, a congenital tumor of the eye in children.

Indeed, molecular pathology has come of age. The use of the electron microscope (EM) has revolutionized histology and anatomic pathology because of its ability to see beyond the resolution of the light microscope. But even beyond the resolution of the EM is the molecular level. State-of-the-art technology allows the pathologist a view of the sub-organelle and molecular levels. How is this possible?

Immunopathology is a field that employs immunostaining, a method that arose from immunochemistry and molecular biology research. This technique allows the pathologist a glimpse of disease at the molecular level: antigens are demonstrated in certain tumor cells by means of monoclonal antigen-antibody complexes, generated to bind with specific cell surface, cytoplasmic or nuclear receptors. A positive result (through chromogen staining) indicates binding, which provides a clue to the cells’ origins. An example of this is the HMB45 antibody, which identifies the receptors on the cell membrane of malignant melanoma, a type of skin cancer. This is not necessarily specific for this type of disease, but provides important clues to the cell of origin of the malignant transformed cell.

Microarray analysis is now at the forefront of clinical molecular pathology. Microarray technology uses a specialized “chip,” composed of tiny array probes. Tissue microarray is a high-throughput technique that allows rapid gene expression and copy-number surveys of large numbers of different tissue specimens. In addition, it represents a technical revolution for the effective use of human tissue specimens, which are both difficult and expensive to obtain in sufficient amount and case number.

Tissue microarrays are usually produced by re-locating tissues from conventional histological blocks. Typically, 40 to 1,000 cylindrical formalin-fixed and paraffin-embedded tissue cores can be densely and precisely arrayed into a single paraffin block. From the block, up to 300 serial 4-8 µm thick sections can be produced and placed on individual glass microscopic slides. Tissue microarray enables parallel in situ detection of DNA, RNA or protein targets in each specimen on an array at cellular and tissue levels; at the same time, the large number of available consecutive arrays allows rapid analysis of multiple molecular markers in the same set of specimens. Essentially the same tests performed on conventional histological samples, including H&E staining, immunohistochemistry and in situ hybridization, can be done separately, or in parallel, on tissue microarrays.

The microarray technique is employed to compare normal and cancerous genes, which can be tested on a vast number or “array” of test materials quickly and efficiently. This technology has also been used to increase our understanding of basic mechanisms in cancer, as in the lymphomas, which are classified into numerous subtypes. It provides a more effective way to determine the diagnosis and prognosis in patients, and eventually, it may be able to lead to better treatment and increase the survival rate of patients.

Microarray technology is also being used for translational medicine like screening for newborn genetic diseases and the study of infectious diseases. Applications in diagnostic and forensic pathology are also at present being tested.

FISH, or Fluorescent In Situ Hybridization, is a method used to determine the presence of certain markers in cells. For example, the HER-2/neu protein, amplified in certain breast cancers, may be demonstrated through FISH. Although immunohistochemistry is the most frequently used method to assess over-expression of HER-2/neu, FISH is recognized as the “gold standard” in determining its status.

Experimental pathology has yielded a treasure trove of information. More recently, a group of scientists has re-evaluated p53, a tumor suppressor gene, whose product is absent or inactive in certain types of cancer. The study was done on genetically engineered mice, which developed an aggressive form of liver cancer through the inactivation of p53. When the researchers reactivated p53, they found that the liver tumors completely disappeared. In due time, such studies may lead to translational research and accelerated clinical trials to cure liver cancer, among others.

In situ PCR (Polymerase Chain Reaction) is another technique used in molecular pathology. An example of one of the most difficult issues in viral pathogenesis today is that presented by the lentiviruses, which include HIV, the causative agent of AIDS in humans. Upon infection with these agents, the provirus frequently integrates into the host genome and establishes a persistent infection, whereby the infected cells are in a transcriptionally quiescent state with respect to the viral antigens. This allows the infected cells to escape host immune surveillance. It is this state which is potentially the most lethal, since these cells provide a reservoir for the future release of active virus. It is these latently infected individual cells, then, which need to be discovered.

The techniques of nucleic acid hybridization and polymerase chain reaction have been used extensively to investigate these issues of pathogenesis. In situ hybridization applies the technology of nucleic acid hybridization to the single cell level, and, in combination with cytochemistry and immunocytochemistry, permits the maintenance of morphology. The identification of cellular markers allows the localization of sequences to specific cells within populations, such as tissues and blood samples. However, with in situ, the technology is limited primarily to the detection of non-genomic material (e.g., RNA), reiterated genes or multiple genomes. Since the viral nucleic acid within these infected cells is below the level of routine detection, the application of amplification technologies, i.e. PCR, is essential.

These are just a few of the molecular pathology techniques that are paving the way for new diagnostic, experimental and treatment modalities for diseases. Pathology, which was traditionally the study of dead or devitalized tissue, is now at the vanguard of understanding the molecular basis of diseases as well as monitoring the course of a disease process. By studying the subtle transformation of cells, tissues and organs from health to disease, the pathologists are leading the way to finding new treatment modalities for chronic illnesses, emerging diseases and diseases with unknown etiology or with complex pathogenesis.

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