In this chapter, the practical aspects of mutation assessment are reviewed.
Physicians, surgeons, diagnostic pathologists, laboratory technologists, nurses, epidemiologists, and other health workers need to be prepared for research studies and clinical decision making (17,18). Health professionals
working in developing countries also are encouraged to participate in this field (19). Every cancer patient, whether suspected as representing a familial cancer syndrome or not, should be considered unique and their tissues irreplaceable.
Biopsy and other surgical specimens must be properly handled, and different simultaneous pathological processing procedures should be used to allow for different types of assays. More and more, tissues collected in community hos-pitals, otherwise not involved in research, are serving as a valuable resource in large studies. So, staff members must prevent autolysis. It is helpful to use dif-ferent fixations because these provide difdif-ferent DNA yields. As described later, a success rate for DNA recovery from the paraffin blocks depends on appropriate fixation. For example, long immersion of resected tissues in for-malin fixatives damages tissues for further molecular investigations. Frozen tissue is essential for many genetical assays, but is not sufficient for RNA ana-lysis or cell culture without special processing. Quick fixation is required for electron microscopy, but not for DNA extraction.
Good pathological examination using standard techniques is required to ensure success at subsequent analysis. The histological type of cancer should be confirmed when planning to assay DNA, then microdisection is needed so tumor cells are separated from other cells. This is best done from fixed tissues.
Every step in the process of tumor collection, fixation, and storage affects the ability to perform subsequent genetical assays because they can affect DNA quality and quantity. The most commonly used fixative is for-maldehyde (so-called 1=10, which is 3.6%). Many laboratories recommend neutral buffered formalin. Some institutes use AMEX fixation, which has been asserted to be one of the best ways to keep the tissues as a possible good source of DNA, RNA, and protein (20,21). This procedure takes some extra steps in the routine histopathological laboratory and at least one per-son knowledgable in that procedure is needed. The time between resection and fixation should be short and the fixation time should be short. An alter-nate fixative is ethanol, which is better for subsequent DNA studies. And more laboratories are using OCT with rapid freezing with success.
Other variables that affect DNA quality and quantity occur during DNA extraction, such as insufficient time for dewaxing with xylene, deter-gent (SDS or tween-20) or insufficient time for proteinase K digestion.
Greater quantities of the paraffin embedded tissue are not necessarily better for DNA assays, because this allows for greater amounts of inhibitors from the blocks. Several agents are available for extracting DNA from fixed tissues that are thought to reduce these inhibitors, e.g., Chelex-100 (22).
Table 1 lists the biomarker assays used for risk assessment and diag-nosis. Basic and additional protocols are available elsewhere (23–26).
There are many types of genetical assays in use today. Some of these will be described below. The majority of molecular genetical tests used today begin with the polymerase chain reaction (PCR). There are emerging
Table 1 Biomarkers of Cancer Risk and Diagnosis Biomarkers for disease risk
Markers of inherited susceptibility
Genetical variation causing impaired metabolic activation or excretion of toxins
Genetical variation causing defects in the repair of DNA, cell cycle control, or programmed cell death
Markers of acquired susceptibility Formation of DNA adducts Integration of viral DNA Mutations in critical genes Mutations in noncritical genes
Hypermethylation of gene promoter region Altered gene expression
Clastogenic abnormalities Antibodies to DNA adducts
Altered protein or mRNA expression patterns Biomarkers for preclinical disease
Markers of cellular alteration Altered morphology of cells
Altered phenotypic expression of cells Clonal proliferation of cells
Altered gene expression Antibodies to gene products
Altered protein or mRNA expression patterns Biomarkers for clinical disease
Markers of cellular alteration Altered morphology of cells
Altered phenotypic expression of cells Immunohistochemical staining Clonal proliferation of cells Altered gene expression Antibodies to gene products
Altered protein or mRNA expression patterns Markers of prognosis
Pathological diagnosis Immunohistochemical staining Altered gene expression Cytogenetic abnormalities
Altered protein or mRNA expression patterns
technologies that will eliminate this need, but none have been sufficiently validated for use in the clinical setting. Polymerase chain reaction, in fact, is among the most important recent advances in molecular genetics. The reac-tion is the amplificareac-tion of small amounts of DNA to make lots of DNA, which are then available for subsequent analyses. Polymerase chain reaction is facile and inexpensive. It has been used in forensic medicine for DNA fin-gerprinting from a single hair follicle or blood stain (27); mutation detection in single sperm cells to assess teratogenicity rates (28); and amplification of DNA from paraffin embedded tissue blocks (29), serum (30) or ancient DNA (31). It also forms the basis for microarray technology (32,33). Poly-merase chain reaction relies upon a temperature stable enzyme (Taq polymer-ase) that can replicate DNA when using gene- and site-specific primers that begin the reaction. While PCR is generally used for DNA amplification, it also can be used for RNA amplification using a different enzyme (reverse transcriptase) (34). The major limitations of PCR lie in its sensitivity that allows for contamination by unwanted DNA from other sources. It also is cri-tical to choose primers carefully to ensure specificity and prevent amplifying the wrong gene.
There are many applications for PCR. It is being used directly without other techniques for diagnosing viral infections (e.g., HIV in lymphocytes (35), hepatitis B virus in liver and serum (36), and papilloma virus in uterine cervix (37)). It can be used to amplify mutated and structurally altered regions of a given gene (e.g., translocation of chromosomes by determining the break-point cluster region for the bcr-abl oncogene for the diagnosis of chronic myelogenous leukemia (38). Other applications involve the identification of single-base mutations or genetical polymorphisms by designing primers that anneal only if matched to the unique sequence (e.g., oligo-specific PCR for the identification of polymorphisms in the N-acetyl transferase gene predictive of cancer risk in workers exposed to aromatic amines (39). Polymerase chain reaction also is combined with other techniques whereby PCR amplification products can be subjected to restriction enzyme digestion to identify genetical polymorphisms or mutations [e.g., restriction fragment length polymorphism (RFLP) analysis for cytochrome P450 genetical polymorphisms (40)] or used for hybridization with mutation-specific probes (e.g., oligonucleotide hybridi-zation for the detection of Ras mutations (41). Another important application is the use of PCR to amplify sufficient quantities of DNA fragments for nucleo-tide sequencing. This method allows for the determination of specific sequences from unknown genes or for the detection of mutations (42).
3.1. Genotyping
Genotyping to determine genetical variation (e.g., color of hair, metabolic activity, DNA repair) can be done by using different types of detection methods following PCR. This genetical variation can happen via SNPs, or
multiple-base pair insertions or deletions. A common way is to utilize RFLP analysis. Restriction fragment length polymorphism enzymes identify short, specific DNA sequences, cutting the DNA at those sequences into uniquely sized fragments that can be separated electrophoretically. Restriction enzymes recognize palindromic sites where the sequence on each strand is identical with each other (when read in 50 to 30direction). Restriction frag-ment length polymorphism enzymes are only useful when a palindromic site exists. In other cases, the variant may be determined using single-strand con-formational polymorphism (SSCP) analysis. If the variant results in the insertion or deletion of a base or bases, then electrophoretic methods that separate fragments based on size can be used. The fortunate property of DNA, where each strand is complementary and annealed by nucleic acid base-pairing (guanine to cytosine and adenine to thymine), can be taken advantage of to identify specific genetical sequences. Under experimental conditions the two complementary DNA strands can be separated and rean-nealed. Single-stranded probes of short DNA fragments can be used to iden-tify a specific genetical sequence by exposing DNA to the probe. Using oligo-specific hybridization, a radioactive or fluorescently labeled probe marker will bind to the matched DNA. Two probes are used in tandem that are matched to one variant or the other. This unique property allows for Southern blot analysis of DNA (43), which subjects DNA to restriction enzyme digestion, separation of the resulting fragments by electrophoresis, and then probing the fragments for the genetical sequence and measuring the lengths of the fragments. The method also is used for northern blot analysis of messenger RNA (mRNA) (44), which is almost identical with Southern blot analysis except that RNA is used instead of DNA.
Several new methodologies exist for high through-put genotyping.
These include microarrays that can determine 2000 SNPs following 24 dif-ferent PCR assays, real-time PCR that allows for detection of SNPs without gel electrophoresis, matrix-assisted laser desorbtion=ionization time of flight (Maldi-TOF) mass spectroscopy (45), denatured high-performance liquid chromatography (46), capillary gel electrophoresis, and flourescence detec-tion (47) and pyrosequencing (48).
3.2. Sequencing
DNA sequencing can be used to determine the actual genetical code. This may be used for identifying an inherited code (i.e., sequence of entire gene or SNPs) or mutations in tumors. The dideoxy-mediated chain termination method was among the first established and allows for the determination of the nucleic acid sequence of a gene (49). For example, a PCR fragment is amplified and four dideoxy reactions are carried out for each of the four nucleotides. The amplified product, radiolabeled nucleotides, 2,30- dideoxy-nucleotides, and a polymerase are mixed so that the 2,30-dideoxynucleotide
is randomly incorporated into the DNA. Based on the location of the dideoxynucleotide incorporation, the DNA sequence can be determined after electrophoretic separation. More recent high-throughput methods rely on microarray technology following PCR (32) or capillary electrophoresis.
Because sequencing can be labor intensive, some investigators use methods to screen for mutations. The SSCP analysis was originally devel-oped by Orita et al. (50) as such a screening method. Here, DNA is dena-tured into single strands and analyzed by gel electrophoresis. If there are base changes, then the migratory distance on the gel changes. The basis for this technique is still empirical, thus the sensitivity of the detection of mutation depends on which product you like to screen (51). The electro-phoresis conditions including the glycerol content and gel temperature determine the specifity and sensitivity of the procedures, but there is no gen-eral principle about which condition is the best. For some fragments, only 12.5% glycerol can identify the migrationdifferences while at other times electrophoresis at 4C is needed.
3.3. Gene Loss and Loss of Heterozygosity
Assessing for loss at heterozygosity (LOH) is a major way for determining gene deletions. Using PCR and SNP analysis, we examine tumors in people who are heterozygous for the loci (germline polymorphisms where each allele is different) and determine if both or only one allele is present in the tumor. This only works in persons who have inherited different sequences on the allele from each parent, but then in the tumor only one of those alleles is seen. Southern blotting is the classical technique for LOH, named after the inventor. In this procedure, extracted DNA is enzymatically digested with restriction enzymes and the digested products are transferred to a membrane. The membrane with the products is then hybridized to a
‘‘probe.’’ The probe is a labeled marker matching the gene of interest.
The procedure usually takes 3 days or more including electrophoresis, trans-ferring, hybridization, washing, and exposure to the film. The required DNA amounts are greater than in other procedures described in the follow-ing sections. This method requires that the DNA is of good quality. Pre-viously a few years ago, Southern blotting has essentially been replaced by PCR amplification of several genes assessment for loss of heterozygosity, using the SNP analysis. This allows for greater odds for informative cases, especially if the loci is a minisatellite (tandem repeats of 20–30 DNA bases).
Several methods are available to analyze the gross structure of chromo-somes in metaphase and prophase of mitosis. Chromosome aberrations can be observed by identifying each of the 23 chromosomal pairs for completeness and number (52). Common uses of such analyses include the detection of tri-somy 21, which is diagnostic for Down’s syndrome, and the detection of a translocation between chromosomes 9 and 21, which is diagnostic for chronic
myelogenous leukemia and the Philadelphia chromosome. The availability of specific chromosomal markers now makes this method more specific. Another gross chromosomal change detectable in human cells includes the sister chro-matid exchange (53). In this case, sister chrochro-matids of one chromosome are switched, which can be counted using nonspecific markers and correlated with exposures to tobacco and certain chemicals. A method of detecting DNA damage that does not require cell culture and examination of chromosomes during mitosis is the detection of micronuclei (54). Small chromosomal frag-ments are sometimes found to exist outside the nucleus.
3.4. Microsatellite Instability
The assessment of microsatellite instability is a marker for altered DNA repair. Analysis of tumors indicates that there are increased numbers of repeat DNA sequences that are not present in the patient’s nontumor tis-sues. Thus, there were errors during DNA replication. Mono-, di-, tri-, quadra-, and pentanucleotide repeats are ubiquitous in human genomes, probably due to replication errors through evolution, but then in tumors, these loci are possible sites for more slippage during replication. Microsatel-lite instability is one of the common genetical alterations in human tumors where the repeats might be more or less. They are caused by somatic changes, and also occur in people with genetical mismatch repair deficiencies in hereditary nonpolyposis colorectal cancer. The germline mutations of MLH1 and MSH2 (PMS1, PMS2, MSH6) have been most commonly docu-mented in some, not all, families with high rates of colon cancer (55,56) . Target molecules can be surrogate markers for microsatellite instability (replication error type). These include TGF beta II receptor, MSH3, MSH6, IGF II receptor, and Bax gene (57). The alterations in repetitive sequences in the coding exon of these genes can be predictive of progress.
3.5. Immunohistochemistry
Overexpression of genes relating to DNA damage can be detected with immunohistochemistry. Using tumor tissues fixed on slides, antibodies raised against specific proteins can be labeled and used to bind to the protein on the slide. The more the binding, the more the overexpression. This method, though, can have pitfalls like false positives (the antibody is not specific for the protein of interest) and negatives (the antibody is not good enough to stay bound to the protein during binding). In addition to the quality of the antibody, these can occur because of poor slide preparation, denatured antibodies, or high background.
3.6. Carcinogen–DNA Adducts
There are many types of DNA damage that can be detected using molecular genetical methods, such as carcinogen–DNA adduct detection. Chemicals or
their reactive metabolites can bind to DNA, resulting in promutagenic lesions. The combination of the chemical and the nucleotide is an adduct.
The measurement of DNA adducts allows for the distinction between the measurement of chemicals in the environment and exposures inside the body and in target organs, because the former is not always indicative of the lat-ter. DNA adducts reflect the biologically effective dose of an exposure, resulting from the competition of exposure, absorption, activation, detoxifi-cation, and DNA repair. Thus, the measurement of DNA adducts reflects both exposure and inherited susceptibilities. Elevated levels of DNA adducts have been correlated with cigarette use (58), occupational exposures to poly-cyclic aromatic hydrocarbons (59), and air pollution (60).
Several methods are currently available for the measurement of DNA adducts, although all remain research tools. These include the32 P-postlabel-ing assay that uses hydrolytic enzymes to reduce DNA to individual nucleo-tides and then uses another enzyme to radiolabel the nucleonucleo-tides (61). Any adducts that are present are then resolved chromatographically and quanti-tated by measuring the radioactivity incorporated into the nucleotide. This assay can be used as a screening method to detect unknown adducts (61) or can be combined with purification techniques to identify specific compounds such as adducts formed from polycyclic aromatic hydrocarbons (62) and N-nitrosamines (63). Several important immunological methods are available for the detection of DNA adducts. Using procedures such as enzyme-linked immunoadsorbant assays (ELISA) or radioimmunoassays, adducts for poly-cyclic aromatic hydrocarbons can be measured (64–66). More recent methods utilize improved mass spectroscopy methods (67) and flourescence detection.