ABSTRACT
In recent years, many oncology institutions have implemented the use of molecular approaches to assess and manage cancer patients. One commonly observed type of genetic alteration in cancer is the loss of heterozygosity (LOH). In the clinical setting, this molecular genetic marker is an important tool for disease prognosis, diagnosis and treatment. For example, the loss of 1p/19q is a classical molecular marker for oligodendroglioma assessment. In addition, this marker is associated with a favorable prognosis and chemosensitivity in oligodendroglial tumors. Interpretation of the clinical significance of molecular markers requires that health professionals and biomedical scientists understand the basic theoretical fundamentals of molecular diagnostic techniques. Although there are different methodologies to assess LOH, including high-performance techniques, this review aims to describe the polymerase chain reaction (PCR)-based LOH assays and fluorescence in situ hybridization (FISH), which are the molecular techniques most used for evaluation of 1p/19q status in pathology laboratories.
Key words:
oligodendroglioma; polymerase chain reaction; fluorescence in situ hybridization; loss of heterozygosity
This molecular procedure consists of a series of repeated cycles for
amplification of microsatellite regions of 1p and 19q chromosomes using
microsatellite primers. A) In the illustrated example, microsatellite loci located in
chromosome 1p were used for the loss of heterozygosity (LOH) assay; B) fluorescent
labeled forward and non-fluorescent reverse primers are used in PCR amplification
producing amplicons at each PCR cycle, so that after 20-30 cycles, greater than 1
million-fold amplification is reached. At the end of PCR, the majority of generated
fragments in the loci 1 and 2 are fluorescent.
A) PCR products from normal DNA (1), tumor DNA (2) and molecular weight standard
(3) are applied to polyacrylamide gel; B) after electrophoretic separation, the DNA
bands are visualized by autoradiography or silver staining; C) data analysis of PAGE.
The normal tissue DNA (lane 1) shows heterozygosity for locus 1 and locus 2 in 1p
chromosomal arm, tumor tissue DNA (lane 2) shows loss of heterozygosity for both loci
(arrows). Molecular weight standard is shown in lane 3.
LOH: loss of heterozygosity; DNA: deoxyribonucleic acid; PCR: polymerase chain
reaction; CCD: charge-coupled device.A) After microsatellite amplification, internal-lane size standard (1 and 3),
PCR products from normal DNA (2) and tumor DNA (4) are subjected to capillary
electrophoresis on an automated DNA sequencer, and DNA fragments of PCR-based LOH
assays are separated; B) illustrated scheme of capillary electrophoresis system. This
system is composed by cathode (-) and anode (+) electrodes, a high-voltage power
supply, silica capillary, detection cell, charge-coupled device (CCD) camera and a
computer workstation. When a high voltage is applied, negatively charged DNA
fragments begin to migrate through the capillary. In the detection cell, the DNA
fragments are intercepted by a laser beam, which excites the attached dye labels
making them fluoresce. Following laser excitation, the incident fluorescence is
captured by a CCD camera, which converts fluorescence into electronic information.
This information is transferred to a computer workstation, where the data are
processed and analyzed by specific software; C) data processing and analysis of
capillary electrophoresis. Two representative capillary electropherograms of two
PCR-amplified microsatellites are shown from normal and tumor tissues. The normal
tissue DNA shows heterozygous peaks for locus 1 (red peaks) and locus 2 (green peaks)
in the 1p chromosomal arm, while loss of heterozygosity is noted for both markers
(red and green peaks) in the tumor tissue DNA. Internal-lane size standard is
represented as a black line.
FISH: fluorescence in situ hybridization; LOH: loss of heterozygosity; DNA:
deoxyribonucleic acid.Eight basic steps are included: deparaffinization in xylene, rehydration in
graded alcohols, pre-treatment/target retrieval, denaturation of target DNA and
probe, hybridization, post-hybridization washes, direct or indirect detection and
image analysis in a fluorescence microscope.