Molecular diagnostics is a field of medicine that applies molecular biology techniques to the diagnosis of disease. Molecular diagnostics includes traditional methods such as cytogenetics, as well as encompassing the emerging fields of genomics and proteomics in personalized medicine. This chapter reviews the pertinent molecular methods that are commonly used in molecular diagnostics, and their most common applications in the field of gastrointestinal pathology.
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Polymerase chain reaction
The polymerase chain reaction (PCR) is a method that takes one small region among a 3-billion base-pair genome and produces a billion copies of it. Two major advances that permitted the widespread application of PCR were the use of thermostable DNA polymerases such as Taq polymerase and the development of an instrument to cycle between temperatures, a so-called thermocycler. PCR is a fundamental method used in a variety of molecular diagnostic assays and is briefly reviewed here.
The requirements for a PCR reaction include genomic DNA (the template), two primers (short sequences of DNA approximately 18 to 20 nucleotides long) that are complementary to the genomic DNA and that flank the genetic region of interest, a DNA polymerase, and free nucleotides. A thermocycler carries out the three requisite reaction steps: denaturation, annealing, and extension ( Fig. 14-1 A). During the denaturation phase, the sample is elevated to a high temperature to denature the double-stranded genomic DNA. The thermocycler then lowers the temperature to allow primers to anneal to these single-stranded complementary DNA molecules. The temperature is then raised to a middle temperature for the polymerase to extend the primers by incorporating nucleotides as it replicates the template strands. These steps are sequentially repeated 25 to 40 times where each cycle produces two copies for every input copy, thereby resulting in exponential amplification of the desired region (see Fig. 14-1 B). PCR is a required step to identify disease-causing mutations in patients with hereditary gastrointestinal cancer syndromes, to identify carcinomas with microsatellite instability, and to identify gene mutations that influence response to therapies, among others.
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KRAS and BRAF genotyping
KRAS and BRAF are signaling molecules that play critical downstream roles in the epidermal growth factor receptor (EGFR) signaling pathway by regulating cellular proliferation, motility, and survival. In normal cells, activation of the EGFR receptor leads to phosphorylation of tyrosine residues within its intracytoplasmic domain, and via intermediary proteins such as Grb and Sos leads to activation of KRAS and other downstream signaling components with resultant changes in cellular proliferation and migration. KRAS activation is then rapidly deactivated to its inactive form, thus terminating the signals initiated by ligand binding to the EGFR receptor. By contrast, mutations in KRAS or BRAF in cancer cells lead to the constitutive and uncontrolled activation of this signaling pathway independent of binding by growth factor ligands to EGFR, resulting in uncontrolled proliferation and growth ( Fig. 14-2 ). Point mutations at codons 12, 13, and 61 in KRAS or at codon 600 in BRAF mimic the activated state of these signaling pathway intermediates, and for this reason these mutations are associated with resistance to and lack of clinical benefit from anti-EGFR immunotherapy. Thus determinations of the KRAS and BRAF mutation status in a colorectal carcinoma has become common practice to guide therapeutic options regarding anti-EGFR immunotherapy in patients with metastatic colon cancer.
Commonly used laboratory methods to detect these and other disease-associated mutations include conventional Sanger sequencing, pyrosequencing, and real-time PCR with melting curve analysis ( Fig. 14-3 ). Sanger sequencing, named for its developer Frederick Sanger, is considered to be the gold standard because it produces short sequences of genes to highlight the precise mutations present and has a limit of detection of mutated DNA to normal DNA of between 10% and 20%. Pyrosequencing is a relatively new technique that also generates precise sequence data, but has an improved limit of detection compared with Sanger sequencing. Real-time PCR with melting curve analysis is another emerging approach that has the advantage of speed and reduced cross contamination. However, rather than identifying the precise mutation, it delineates, based on quantitative data, whether there is a mutation present and has a limit of detection that is slightly improved over that of Sanger sequencing. At this time, specific mutation subtyping is not known to impact therapeutic choices, and the clinical significance of mutant subtypes on treatment outcomes and prognosis is unknown.
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Molecular evaluations of hereditary gastrointestinal cancer syndromes
Several genetic syndromes predispose to gastrointestinal cancer ( Table 14-1 ). Most of these syndromes are caused by classic tumor suppressor gene mutations wherein one mutant copy of the gene is inherited at birth (a “germline” mutation) and loss or mutation of the second copy of the gene is acquired in the tumor. Although most gastrointestinal cancer syndromes are inherited in an autosomal dominant manner (i.e., hereditary diffuse gastric cancer or familial adenomatous polyposis), some syndromes are associated with recessive inheritance of a mutant gene from both parents (e.g., MutY polyposis). The histopathologic and clinical features of neoplasms that arise in association with gastrointestinal cancer syndromes are discussed in their respective chapters of the stomach (see Chapter 4 ), pancreas (see Chapter 17 ), and colorectum (see Chapter 13 ).
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