Molecular pathology. The study of DNA and RNA sequencing, genes, and genetics.

Introduction:

In one sense, molecular testing has been used in surgical pathology for several decades in the context of immunohistochemistry, in which antibodies are used to detect and quantify the expression of specific proteins. However, in common parlance, molecular testing refers to testing based on the analysis of nucleic acids such as DNA and RNA.

Regardless of whether the disease process is benign or malignant, most genetic testing protocols in surgical pathology are designed to detect somatic or acquired DNA mutations limited to specific cells of the disease process. The recent increase in molecular genetic tests reflects their ability to improve patient care by providing new, independent, or refined information with the potential for clinical stratification of disease subtypes, disease predispositions, prognostic categories, or treatment regimens. While this review will concentrate on the most commonly used molecular techniques in surgical pathology. It is worth noting that almost every testing method has additional applications in other clinical laboratory disciplines.

Molecular Genetic Techniques Currently in Common Use:

Classical Cytogenetics:

Classical cytogenetics is useful in surgical pathology because specific chromosomal abnormalities are associated with morphologically and clinically distinct subsets of lymphoma, leukemia, and soft tissue neoplasms. The advantage of traditional cytogenetic analysis is that it can provide simultaneous analysis of the entire genome without knowing which chromosomal regions are involved in the disease process. In most cases, the type and/or location of the chromosomal abnormality can be used to make a diagnosis or guide further testing.

The technique's primary disadvantages are that it can only be performed on tissue specimens containing viable Tumors cells that will proliferate in vitro, and it has a resolution of only about three million base pairs. As a result, in routine clinical practise, classical cytogenetic analysis is only useful for detecting numerical abnormalities and major structural changes. The method lacks the sensitivity to identify the genes involved in the gross structural changes or to detect mutations such as small deletions, small insertions, or single base pair substitutions.

Tissue in Situ Hybridization:

This technique detects target DNA or RNA sequences in histologic sections of fresh or FFPE tissue, allowing direct correlation of the morphology of individual cells with the presence or absence of specific genetic changes. Because the method detects target DNA or mRNA sequences in interphase cells (that is, cells that are not actively undergoing mitosis), it can be used to study a broader range of patient specimens than traditional cytogenetic analysis or metaphase FISH.

The primary limitations of interphase FISH are resolution; technical limitations on the size of the probes used to detect the target DNA or RNA make the approach unsuitable for evaluating certain classes of mutations such as small insertions, small deleottions, and single base pair substitutions. Furthermore, the technique does not scan the genome for mutations, but rather identifies abnormalities at the probe-targeted sites.

Because interphase FISH can be performed on cytology specimens or histologic sections from routinely processed biopsy or excision specimens, it is well suited for routine use in diagnostic surgical pathology.

Polymerase Chain Reaction (PCR):

Polymerase chain reaction (PCR)-based approaches have become the standard for much clinical molecular genetic testing because PCR is fast (it can be performed in a matter of hours), can be applied to nucleic acids from a variety of substrates (including DNA and RNA from cytology slides, as well as fresh, frozen, or fixed tissue), and is very sensitive.

DNA Sequence Analysis:

Currently, almost all direct DNA sequence analysis is done on PCR templates. However, once the normal and mutant alleles of a gene have been identified, indirect sequencing methods are frequently sufficient to provide the clinically required information. Indirect methods distinguish different alleles based on size, electrophoretic mobility patterns in different gel matrices, the presence of sequences that are targets for various sequence-specific endonucleases (known as restriction endonucleases), and so on. Because almost all indirect methods are ultimately based on PCR template analysis, they can be applied to a wide range of clinical specimens.

Technologies in Development:

Currently, the majority of molecular tests performed in surgical pathology involve the analysis of a single gene or genetic locus, and it is true that single genetic markers can provide information that is diagnostic, prognostic, or therapeutic in many clinical settings. However, for a wide range of hematopoietic, lymphoid, and solid tissue tumours, testing focused on a single gene is more often than not a result of a lack of complete understanding of tumour biology rather than an optimised testing paradigm. It is becoming increasingly clear that single genetic events are usually insufficient to account for all of the features of a neoplasm, 7,8, and thus testing methods that can evaluate multiple loci will likely provide more clinically useful information.

MicroRNA:

The discovery that several classes of short double-stranded or single-stranded RNA molecules (collectively known as miRNA) play a significant role in gene expression regulation was quickly followed by evidence that altered patterns of miRNA expression play a role in the pathogenesis of many benign and malignant diseases. 11,12 Because microarray-based analysis of genome-wide patterns of miRNA expression has already proven useful for documenting the role of miRNA in disease pathogenesis, it is likely that GeneChip analysis of miRNA will soon become a component of tissue specimen evaluation.

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