All methods for detecting mutations rely on the manipulation of DNA, the basic building block of heredity in the cell. DNA consists of two long strands of polynucleotides that twist around each other clockwise in a double helix (Fig. 1-1). Nucleic acid bases attached to the sugar groups of each strand face each other within the helix, perpendicular to its axis. Only four bases exist: the purines adenine and guanine (A and G) and the pyrimidines cytosine and thymine (C and T). During assembly of the double helix, stable pairings of nucleotides from either strand are made between A and T or between G and C. Each base pair (bp) forms one of the billions of rungs in the long, unbroken ladder of DNA that forms a chromosome.

Figure 1-1 **DNA structure.** DNA, the cell’s genetic material, is contained in single compacted strands comprising chromosomes within the cell nucleus. In the DNA double helix, the two intertwined components of its backbone are composed of sugar (deoxyribose) and phosphate molecules, which are connected by pairs of molecules called bases. The sequence of four bases (guanine, adenine, thymine, and cytosine) in the DNA helix determines the specificity of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs with complementary bases on the opposing strand for specific recognition. The arrangement of chemical groups is unique for each base pair, allowing base pairs to be specifically targeted by transcription factors, polymerases, restriction enzymes, and other DNA-binding proteins. (From Chalquist C. http://www.terrapsych.com.)

The functional unit of inherited information in DNA, the gene, usually is represented by a discrete section of sequence necessary to encode a particular protein structure. Gene expression is initiated by forming a copy of the gene with use of messenger RNA (mRNA); the gene is constructed base by base from the DNA template by a polymerase enzyme. Once transcribed, an mRNA transcript is modified and the processed product is transported out of the nucleus. In the cytoplasm, proteins are then synthesized, or translated, in macromolecular complexes called ribosomes that read the mRNA sequence and convert the nucleic acid code, based on three-base segments or codons, into a 20 amino acid code to form the corresponding protein.

Generating Diversity with Alternate Splicing

In higher organisms, most protein-coding gene sequences are interrupted by stretches of noncoding DNA sequences, called introns. In the nucleus, these introns are removed after mRNA transcription to produce a continuous chain of coding sequences, or exons, which subsequently undergo translation into protein. The splicing process requires absolute precision, because the deletion or addition of a single nucleotide at the splice junction would throw the three-base coding sequence out of frame or lead to exon skipping or addition, thus creating abnormal proteins.

The dramatic increase in genetic complexity conferred by alternate RNA splicing is underscored by the multiple splice patterns of many medically relevant genes, in which different combinations of exons are chosen for the final mRNA transcript, such that one gene can encode many different proteins (Fig. 1-2). The choice of protein isoform to be expressed from a gene with multiple splicing possibilities is a decision that can be perturbed in disease. To date, errors in splicing mechanisms have been associated with a large group of cancers. These errors include mutations in several transcription factors, cell signaling, and membrane proteins. These include the oncogene p53 in more than 12 different types of cancer and mutL homolog 1 protein mutation in hereditary nonpolyposis colorectal cancer. When mutations in the splicing site lead to insertion of novel sequences in the mRNA, the encoded protein can be used as a potential clinical marker, as seen for the transcription factor NSFR in persons with small cell lung cancer. Because of their unique expression in cancer cells, these markers can be further explored as new cancer-specific therapeutic targets.

Figure 1-2 **RNA splicing.** Alternate splicing produces multiple related proteins, or isoforms, from a single gene. (From Guttmacher AE, Collins F. Genomic medicine—a primer. N Engl J Med 2002;347:1512–20.)

The Genomics of Cancer

The complete set of DNA sequences carried on all the chromosomes is known as the genome. Although the general map of the genome is shared by all members of a species, the recent sequencing of thousands of individual human genomes has given rise to the new field of genomics, providing us with new tools to reveal the more subtle variations that arise between individuals. These variations are critical, both as a natural engine driving heterogeneity within a species and as a source of predisposition to cancer types. The most common forms of human genetic variations, or alleles, arise as single-nucleotide polymorphisms, or SNPs. Because these allelic dissimilarities are abundant, inherited, and dispersed throughout the genome, SNPs can be used to track racial diversity, personal traits, and susceptibility to common forms of cancer (Fig. 1-3).

Figure 1-3 **Determining cancer susceptibility with single-nucleotide polymorphisms** (**SNPs).** Millions of SNPs exist between individuals, as depicted by the red arrows and the SNP density map of human chromosome 11 (*right*). By contrast, point mutations, deletions, insertions, and rearrangements between normal tissues and tumors or between primary and secondary tumors probably number in the tens to hundreds (or potentially thousands), as depicted by the spectral karyotype image at the bottom of the figure. Because the constitutional genetic polymorphisms are present in all of the tissues of the body, it might be possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissue before those metastatic cells develop into a solid tumor. (From Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer 2006;6:141–6.)

How do SNPs arise between individuals? One source of variation in DNA sequence derives from deviations in the strict base-pairing rule underlying the structure, storage, retrieval, and transfer of genetic information. The duplicated genetic information in the two strands of DNA not only permits the repair of a damaged coding sequence but also forms the basis for the replication of DNA. During cell division, polymerase enzymes unwind the DNA strands and copy them, using the base sequences as a template for constructing a new helix so that the dividing cell passes its entire genetic content on to its progeny. Errors in this process are rare, and person-to-person differences constitute only about 0.1% of the human genome. SNPs are inherited if they occur in the germline. Many genetically inherited variations occur in regions that do not encode protein or alter the regulation of nearby genes. Given the disruptive effects that even subtle genetic changes may have on cell function, it is important to distinguish SNPs that represent true mutations from benign polymorphisms.

Our ability to monitor hundreds of thousands of SNPs simultaneously is one of the most important advances in modern medical genetics. Relatively simple genotyping technologies for SNP detection rely largely on the polymerase chain reaction (PCR). In this procedure, two chemically synthesized single-stranded DNA fragments, or primers, are designed to match chromosomal DNA sequences flanking the segment in which an SNP is positioned. With the addition of nucleotide building blocks and a heat-stable DNA polymerase, the primer pairs, or amplicons, initiate synthesis of new DNA strands using the chromosomal material as a template. Each successive copying cycle, initiated by “melting” the resulting double-stranded products with heat, doubles the number of DNA segments in the reaction (Fig. 1-4). The technique is exceptionally sensitive; millions of identical DNA copies can be generated in a matter of hours with PCR using a single DNA molecule as the starting material.

Figure 1-4 **Amplification of DNA by polymerase chain reaction.** The DNA sequence to be amplified is selected by primers, which are short, synthetic oligonucleotides that correspond to sequences flanking the DNA to be amplified. After an excess of primers is added to the DNA, together with a heat-stable DNA polymerase, the strands of both the genomic DNA and the primers are separated by heating and allowed to cool. A heat-stable polymerase elongates the primers on either strand, thus generating two new, identical, double-stranded DNA molecules and doubling the number of DNA fragments. Each cycle takes just a few minutes and doubles the number of copies of the original DNA fragment.

Other novel methods for large-scale SNP detection include single nucleotide primer extension, allele-specific hybridization, oligonucleotide ligation assay, and invasive signal amplification, which detect polymorphisms directly from genomic DNA without the requirement of PCR amplification. The International HapMap project has been established with the objective of identifying those variations (commonly thought to be in the order of 10 million in our genome) in the human population. This project is already in its third phase (HapMap3) and now includes both SNPs and copy number variations observed in 1184 samples from 11 different human populations. Regardless of the method used to characterize them, the collective SNPs in a selected genomic region characterize a haplotype, or a specific combination of alleles at multiple linked genetic loci along a chromosome that are inherited together.

Even when the SNPs within a given haplotype are not directly involved in a disease, they provide markers for clonality and for the loss or rearrangement of specific chromosomal segments in growing tumors. In the human nucleus, each of the 23 tightly compacted chromosomes has a characteristic size and structure and a distinctive base sequence that carries unique protein coding information. Other noncoding DNA sequences are used for directing the transcription of neighboring genes through complex regulatory circuits involving protein binding and modification of the DNA itself, or shifting of its chromosomal packaging. Although genomic instability generally is considered a consequence of tumor formation rather than the initial trigger of cancer, the loss, gain, or rearrangement of chromosomal segments through deletion or translocation is a common form of neoplastic mutation, as protein-coding segments from different genes are combined or regulatory sequences are brought into new proximity to genes they do not normally control, as is seen in persons with chronic myeloid leukemia. In persons with chronic myeloid leukemia, recombination events lead to the fusion of BCR and ABL genes (Philadelphia chromosome). This process results in constitutive activation of the fused gene, leading to loss of proliferative control in myeloid cells and, consequently, cancer. Gross changes in DNA arrangement can be detected by cytogenetic analysis of chromosomal features on metaphase spreads. Fluorescent in situ hybridization provides greater resolution by localizing specific chromosomal DNA sequences corresponding to fluorescently labeled probes (Fig. 1-5) and can be used to track specific alterations in chromosomal structure where known genes are involved.

Figure 1-5 **Detection of chromosomal translocations.** Fluorescent in situ hybridization technology uses a labeled DNA segment as a probe to search homologous sequences in interphase chromosomes for the t(9;22)(q34;q11) translocation, which is associated with chronic myeloid leukemia. On the left, patient nuclei were hybridized with probes for chromosome 9 (labeled with SpectrumRed fluorophore) and chromosome 22 (labeled with SpectrumGreen). (Modified from Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. Oncologist 2003;8:45–58.)

The plethora of data arising from genome-wide association studies using currently available techniques poses particular challenges to cancer researchers. Discerning the causal genetic variants among genotype-phenotype associations requires extensive replication, control for underlying genetic differences in population cohorts, and consistent classification of clinical outcomes. New technologies must be met with equivalently sophisticated and rigorous analytical methodologies for the true genetic cause of cancer to be teased out from our variable and often unstable heredity.

Building Gene Libraries

The engineering of genes by recombinant DNA technology evolved from methods initially devised to provide sequences in amounts sufficient for biochemical analysis. The original protocol involves clipping the desired segment from the surrounding DNA and inserting it into a bacterial or viral vector, which is then amplified millions of times in a host bacterium. With use of recombinant DNA technology, genetic engineering routinely can produce industrial quantities of pure, clinically useful products in a cost-effective manner. For diagnostic purposes, it is easier and faster to amplify a known genomic DNA sequence directly from a patient sample with PCR, but the classic approach is still applied to the construction of recombinant DNA libraries.

To be useful, a DNA library must be as complete as possible, with recombinant members, or clones, sufficiently numerous to include all the sequences in an individual genome. For certain kinds of gene-linkage analysis that require long, uninterrupted stretches of DNA, special vectors, such as bacterial or yeast artificial chromosomes, can carry foreign DNA fragments of enormous lengths. Chromosomal segments represented in genomic DNA libraries can contain the structure of an entire gene, including the information that regulates its expression, and formed the starting material for sequencing the human genome.

Many genes associated with cancer originally were identified using partial DNA libraries, which contain only the DNA sequences transcribed by a particular tissue or type of cell. The starting material in this case is mRNA. For cloning purposes, the enzyme reverse transcriptase can convert mRNA into complementary DNA (cDNA). The number of clones in a cDNA library is much smaller than in a genomic library, because a cDNA library represents only the genes expressed by the tissue of interest and contains exclusively the coding portion of genes. For this particular reason, this technique has become obsolete for organisms whose genome has now been fully sequenced. New advances in PCR chemistry allow for the direct cloning of increasingly larger cDNA fragments with high specificity and low error rates. Highly accurate PCR technology, coupled with the constant evolving generation of genomic sequence maps in humans and models organisms, has expanded exponentially the availability of candidate genes to be tested in cancer biology.

Losing Control of the Genome

Mutations that lead to oncogenic transformation of a cell invariably affect the expression of the cell’s genetic information that specifies functional products—either RNA molecules or proteins used for various cellular functions. The primary level of gene control is the transcription of DNA into RNA. Gene regulation, or the control of RNA synthesis, represents a complex process that itself is a frequent target of neoplastic mutation.

DNA regulatory sequences do not encode a product, and yet without them, a cell could not coordinate the expression of the hundreds of thousands of genes in its nucleus, select only certain genes for expression, and activate or repress them in response to precise internal or external signals. These control centers of the genome contain binding sites for multiple proteins, called transcription factors, which interact to form regulatory networks that control gene transcription. Their function can be altered by signals that induce modifications such as phosphorylation or by interactions with other regulators such as steroid hormones. Many of the cell’s responses to a wide variety of external stimuli, such as neurotransmitters, antigens, cytokines, and growth factors, are mediated through transcription factors binding to DNA regulatory sequences.

Certain regulatory DNA sequences common to many genes are positioned upstream of the transcription start site (Fig. 1-6). Collectively called the “promoter” of a gene, these proximal sequences constitute binding sites for the RNA polymerase and its numerous cofactors. Whereas the position of the promoter with regard to the transcription start site is relatively inflexible, other DNA regulatory elements, known as enhancers, occur in unpredictable locations, often at a considerable distance from the genes they control. Some transcription factors bind to particular regions of enhancers and drive their associated genes in many types of cells, whereas others, which are active in only a limited variety of cells, maintain a tissue-specific pattern of gene expression. Enhancers often are responsible for the aberrant expression of genes induced by chromosomal translocation-associated specific forms of cancer; for example, a normally quiescent gene promoting cell growth that is dislocated to a position near a strong enhancer may be activated inappropriately, resulting in loss of control of growth.