Introduction To Molecular Pathology

Infectious Disease

Increasing numbers of microorganisms are detected or characterized by molecular methods, including mass spectroscopy, as discussed in Part 7, which in many cases have become the standard of practice, especially for microorganisms that are difficult to culture or when subsequent culture is needed to identify drug resistance ( ). As the volume of testing has grown, a need has arisen for automation of different steps of testing and standardized kits. There has been a surge of different automation platforms. Automated platforms perform nucleic acid extraction from different types of specimens, automated liquid handling systems set up reactions, and real-time technology monitors and quantifies nucleic acids as they are being amplified. In addition, quantitative molecular methods based on polymerase chain reaction (PCR) amplification and automated rapid sequencing techniques that are essential for viral speciation have become the standard of practice in monitoring response to therapy, especially in patients with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections. With these automated methodologies, the need for confidentiality, especially for patients undergoing HIV testing, has prompted specific federal and state regulations to ensure the protection of patients and their families. In addition, each state might have a list of different organisms that, if detected, must be reported to the state health department.

Cancer

Molecular methods are currently used for hematopoietic neoplasms as well as for solid tumors. The nucleic acid changes, either of DNA or ribonucleic acid (RNA), are present only in the affected populations of cells and are not part of the genetic makeup of the individual. Current technologies—such as real-time PCR, next-generation DNA sequencing, and microarray analysis—have provided greater diagnostic sensitivity and specificity for diagnostic testing, prognosis, treatment selection, monitoring response to therapy, and detection of minimal residual disease. In addition, the exquisite sensitivity of these molecular methods has allowed the testing of very small amounts of nucleic acid from a wide range of sample types. Formalin-fixed paraffin-embedded tissue can provide good quality and quantity of DNA and RNA from processed tissues. However, very specific protocols either for DNA or RNA extraction must be followed. In contrast to inherited genetic testing that needs to be done once per lifetime, molecular oncology tests often are performed repeatedly (e.g., for initial diagnosis and during and after treatment for monitoring response to therapy). Molecular methods that identify rearrangements or mutations that cause specific tumor types may also be used to assess minimal residual disease. Molecular test results should be interpreted in the context of other laboratory testing, such as histopathology, flow cytometry, and clinical findings. The detection of a specific mutation in the tumor cells may also be used to determine the appropriate course of therapy. This is the case for metastatic colon cancer. A number of studies have shown that anti–epidermal growth factor receptor (anti-EGFR) monoclonal antibodies are effective treatments for metastatic colorectal cancer, but only in patients with the wild-type oncogene KRAS . The anti-EGFR monoclonal antibodies panitumumab and cetuximab are approved in the United States for treatment of metastatic colorectal cancer refractory to chemotherapy but are not recommended for use in patients with mutations in KRAS codon 12 or 13. Similarly, panitumumab is approved for the treatment of metastatic colorectal cancer only in patients with wild-type KRAS in Europe and Canada. It is clear that KRAS mutational analysis has become an important aspect of disease management in patients with metastatic colorectal cancer ( ; ). Laboratory issues specific for hematopoietic neoplasms and solid tumor testing include the need for fresh or frozen tissue for RNA-based testing, the use of microdissection to reduce nonmalignant cell population in the specimens, and the need to work with small tissue specimens, such as needle biopsies.

Inherited Disorders

All diseases have a genetic contribution, whether it is a specific genetic disease or an increased likelihood for developing a medical condition. Genetic disorders are primarily the result of a germline mutation or a mutation present in every cell of an individual. For this reason, genetic testing has far-reaching implications, because it could affect family members that might have inherited the same mutation. Molecular genetic testing is currently used for diagnosis, carrier status evaluation, and prenatal and presymptomatic DNA testing. Diagnostic testing is usually performed on affected individuals for establishing or confirming a clinical diagnosis.

Carrier testing is performed in an asymptomatic healthy individual to identify whether the individual is a carrier for an autosomal or X-linked recessive condition and whether the individual is at risk for having an affected child. This application can be used for individuals with a family history of a genetic disorder or for population screening, such as in screening for cystic fibrosis (CF). Carrier testing can be done first by testing an affected family member to identify the specific mutation present in the family. Once a specific mutation is identified, family members can be tested for that particular mutation, thus improving the accuracy of the risk assessment for the individuals in that family with a negative test result. In contrast, population screening focuses on the most common or prevalent mutation, most of the time with different rates of detection for different ethnic populations. Prenatal testing is performed to identify a fetus with a genetic disease or condition. Prenatal testing analyzes fetal cells obtained by amniocentesis or by chorionic villus sampling. Genomic technologies such as noninvasive prenatal testing (NIPT) are capable of detecting abnormalities in fetal DNA from maternal blood samples ( ). This type of testing is usually initiated because family history or maternal factors suggest the need for it. In addition, some laboratories offer preimplantation genetic testing in the setting of in vitro fertilization for couples with a family history of a specific genetic disease. Presymptomatic testing is used primarily for the identification of adult onset of a genetic condition that will occur later in life, such as Huntington disease. Presymptomatic testing is the most problematic and challenging type of testing in terms of its psychological effect on the individual. Hence, it requires extensive protocols for pregenetic and postgenetic testing counseling.