Chapter 67: Cancer Genetics

Cancer arises through a series of somatic alterations in DNA that result in unrestrained cellular proliferation. Most of these alterations involve subtle sequence changes in DNA (i.e., mutations). The somatic mutations may originate as a consequence of random replication errors or exposure to carcinogens (e.g., radiation) and can be exacerbated by faulty DNA repair processes. While most cancers arise sporadically, clustering of cancers occurs in families that carry a germline mutation in a cancer gene.

The idea that cancer progression is driven by sequential somatic mutations in specific genes has only gained general acceptance in the past 30 years. Before the advent of the microscope, cancer was believed to be composed of aggregates of mucus or other noncellular matter. By the middle of the nineteenth century, it became clear that tumors were masses of cells and that these cells arose from the normal cells of the tissue from which the cancer originated. The molecular basis for the uncontrolled proliferation of cancer cells was to remain a mystery for another century. During that time, a number of theories for the origin of cancer were postulated. The great biochemist Otto Warburg proposed the combustion theory of cancer, which stipulated that cancer was due to abnormal oxygen metabolism. Others believed that all cancers were caused by viruses, and that cancer was in fact a contagious disease.

In the end, observations of cancer occurring in chimney sweeps, studies of x-rays, and the overwhelming data demonstrating cigarette smoke as a causative agent in lung cancer, together with Ames’s work on chemical mutagenesis, were consistent with the idea that cancer originated through changes in DNA. However, it was not until the somatic mutations responsible for cancer were identified at the molecular level that the genetic basis of cancer was definitively established. Although the viral theory of cancer did not prove to be generally accurate (with the exception of human papillomaviruses, which can cause cervical and other cancers), the study of retroviruses led to the discovery of the first human oncogenes in the late 1970s. Oncogenes are one of the two major classes of cancer driver genes. The study of families with genetic predisposition to cancer was instrumental to the discovery of the other major class of cancer driver genes, called tumor-suppressor genes. Current technologies permit the sequence analysis of entire cancer genomes, and provide a comprehensive view of the genetic changes that cause tumors to arise and become malignant. The field that studies the various types of mutations, as well as the consequences of these mutations in tumor cells, is now known as cancer genetics.

Nearly all cancers originate from a single cell; this clonal origin is a critical discriminating feature between neoplasia and hyperplasia. Multiple cumulative mutational events are invariably required for the progression of a tumor from normal to fully malignant phenotype. The process can be seen as Darwinian microevolution in which, at each successive step, the mutated cells gain a growth advantage resulting in the expansion of a neoplastic clone (Fig. 67-1). Based on observations of cancer frequency increases during aging, the epidemiologists Armitage and Doll and Nordling independently proposed that cancer is a result of three discrete cellular changes. Remarkably, this early model has been validated by extensive sequencing of cancer genomes. These studies revealed that just three causal mutations are required for the development of several of the most common cancers. Overall, it is currently believed that most common solid tumors require a minimum of three mutated cancer driver genes (either oncogenes or tumor suppressor genes) for their development. One or two mutations is sufficient for benign tumorigenesis, but not for the invasive capacity that distinguishes cancers from benign tumors. Less common tumors, such as liquid tumors (leukemias or lymphomas), sarcomas, and childhood tumors, require two driver gene alterations for malignancy. Note that a cancer driver gene is best defined as one containing a mutation that increases the selective growth advantage of the cell containing it. Normally, cell birth and cell death are in perfect equilibrium; every time a cell is born, another in the same lineage dies. Cancer driver gene mutations alter this equilibrium, so that more cells are born than die. The imbalance is often slight, so that the difference between cell birth and cell death is <1%. This explains why tumorigenesis—the journey from a normal cell to a typical malignant, solid tumor—often takes decades.

We now know the precise nature of the genetic alterations responsible for nearly all malignancies and are beginning to understand how these alterations promote the distinct stages of tumor growth. The prototypical example is colon cancer, in which analyses of genomes from the entire spectrum of neoplastic growths—from normal colon epithelium through adenoma to carcinoma—have identified mutations that are highly characteristic of each type of lesion (Fig. 67-2).

As briefly mentioned above, there are two major types of cancer genes. The first type comprises genes that positively influence growth and are known as tumor-suppressor genes. Both oncogenes and tumor-suppressor genes exert their effects on tumor growth through their ability to determine cell fates, influence cell survival and contribute to genome maintenance. The underlying molecular mechanisms can be extremely complex. While tightly regulated in normal cells, oncogenes acquire mutations that typically relieve this control and lead to increased activity of the gene products. This activating mutational event occurs in a single allele and acts in a dominant fashion. In contrast, the normal function of tumor-suppressor genes is usually to restrain cell growth, and this function is lost in cancer. Because of the diploid nature of mammalian cells, both alleles must be inactivated for a cell to completely lose the function of a tumor-suppressor gene. Thus, it requires two genetic events to inactivate a tumor-suppressor gene mutation, while only one genetic event is required to activate an oncogene.

A subset of tumor-suppressor genes controls the ability of the cell to maintain the integrity of its genome. Cells with a deficiency in these genes acquire an increased number of mutations throughout their genomes, including those in oncogenes and tumor-suppressor genes. This “mutator” phenotype was first hypothesized by Loeb to explain how the multiple rare mutational events required for tumorigenesis can occur in the lifetime of an individual. A mutator phenotype underlies several forms of cancer, such as those associated with deficiencies in DNA mismatch repair. The great majority of cancers do not harbor repair deficiencies, and their rate of mutation is similar to that observed in normal cells. Many of these cancers, however, appear to harbor a different kind of genetic instability, affecting the loss or gains of whole chromosomes or large parts thereof (as explained in more detail below).

Work by Peyton Rous in the early 1900s revealed that a chicken sarcoma could be transmitted from animal to animal in cell-free extracts, suggesting that cancer could be induced by an agent acting positively to promote tumor formation. The agent responsible for the transmission of the cancer was a retrovirus (Rous sarcoma virus, RSV) and the oncogene responsible was identified 75 years later as V-SRC. Other oncogenes were also discovered through their presence in the genomes of retroviruses that are capable of causing cancers in chickens, mice, and rats. The non-mutated cellular homologues of these viral genes are called proto-oncogenes and are often targets of mutation or aberrant regulation in human cancer. Whereas many oncogenes were discovered on the basis of their presence in retroviruses, other oncogenes, particularly those involved in translocations characteristic of particular leukemias and lymphomas, were identified through genomic approaches. Investigators cloned the sequences surrounding the chromosomal translocations observed cytogenetically and identified the genes activated at the breakpoints (see below). Some of these were oncogenes previously found in retroviruses (like ABL, involved in chronic myeloid leukemia [CML]), whereas others were new (like BCL2, involved in B-cell lymphoma). In the normal cellular environment, proto-oncogenes have crucial roles in cell proliferation and differentiation. Table 67-1 is a partial list of oncogenes known to be involved in human cancer.

The normal growth and differentiation of cells is controlled by growth factors that bind to receptors on the surface of the cell. The signals generated by the membrane receptors are transmitted inside the cells through signaling cascades involving kinases, G proteins, and other regulatory proteins. Ultimately, these signals affect the activity of transcription factors in the nucleus, which regulate the expression of genes crucial in cell proliferation, cell differentiation, and cell death. Oncogene products have been found to function at critical steps in these pathways (Chap. 68). Inappropriate activation of these pathways can lead to tumorigenesis.

POINT MUTATION

Point mutation (alternatively known as single nucleotide substitution) is a common mechanism of oncogene activation. For example, mutations in KRAS are present in > 95% of pancreatic cancers and 40% of colon cancers but are less common in other cancer types, although they can occur at significant frequencies in leukemia, lung, and thyroid cancers. Remarkably—and in contrast to the diversity of mutations found in tumor-suppressor genes—most of the activated KRAS alleles contain point mutations in codons 12, 13, or 61. These mutations reduce RAS GTPase activity, leading to constitutive activation of the mutant RAS protein. The restricted pattern of mutations observed in oncogenes compared to that of tumor-suppressor genes reflects the fact that gain-of-function mutations must occur at specific sites, while a broad variety of mutations can lead to loss of activity. Indeed, inactivation of a gene can in theory be accomplished through the introduction of a stop codon anywhere in the coding sequence, whereas activations require precise substitutions at residues that can somehow lead to an increase in the activity of the encoded protein under particular circumstances within the cell.

DNA AMPLIFICATION

The second mechanism for activation of oncogenes is DNA sequence amplification, leading to overexpression of the gene product. This increase in DNA copy number may cause cytologically recognizable chromosome alterations referred to as homogeneous staining regions (HSRs) if integrated within chromosomes, or double minutes (dmins) if extrachromosomal. The recognition of DNA amplification is accomplished through various DNA sequence-based methods for copy number analysis. With both microarray and sequencing technologies, the entire genome can be surveyed for gains and losses of DNA sequences, thus pinpointing chromosomal regions likely to contain genes important in the development or progression of cancer.

Numerous genes have been reported to be amplified in cancer. Several of these genes, including NMYC and LMYC, were identified through their presence within the amplified DNA sequences of a tumor and had homology to known oncogenes. Because the region amplified often includes hundreds of thousands of base pairs, multiple oncogenes may be amplified in a single amplicon in some cancers (particularly in sarcomas). Indeed, MDM2, GLI, CDK4, and TPSPAN31 at chromosomal location 12q13-15 have been shown to be co-amplified in several types of sarcomas and other tumors. Amplification of a cellular gene is often a predictor of poor prognosis; for example, ERBB2/HER2 and NMYC are often amplified in aggressive breast cancers and neuroblastoma, respectively.

CHROMOSOMAL REARRANGEMENT

Chromosomal alterations provide important clues to the genetic changes in cancer. The chromosomal alterations in human solid tumors such as carcinomas are heterogeneous and complex and occur as a result of the frequent chromosomal instability observed in these tumors (see below). In contrast, the chromosome alterations in myeloid and lymphoid tumors are often simple translocations, that is, reciprocal transfers of chromosome arms from one chromosome to another. The breakpoints of recurring chromosome abnormalities usually occur at the site of cellular oncogenes. Table 67-2 lists representative examples of recurring chromosome alterations in malignancy and the associated gene(s) rearranged or deregulated by the chromosomal rearrangement. Translocations are often observed in liquid tumors in general and are particularly common in lymphoid tumors, probably because these cell types have the capability to rearrange their DNA to generate antigen receptors. Indeed, antigen receptor genes are commonly involved in the translocations, implying that an imperfect regulation of receptor gene rearrangement may be involved in their pathogenesis. In addition to transcription factors and signal transduction molecules, translocation may result in the overexpression of cell cycle regulatory proteins or proteins such as cyclins and of proteins that regulate cell death. Recurrent translocations have more recently been identified in solid tumors such as prostate cancers. Fusions between TMPRSS2 and ERG, which are normally located in tandem on chromosome 21, contribute to about one-third of all prostate cancers and correlate with more aggressive disease.

The first reproducible chromosome abnormality detected in human malignancy was the Philadelphia chromosome detected in CML. This cytogenetic abnormality is generated by reciprocal translocation involving the ABL oncogene on chromosome 9, encoding a tyrosine kinase, being placed in proximity to the breakpoint cluster region (BCR) gene on chromosome 22. Figure 67-3 illustrates the generation of the translocation and its protein product. The consequence of expression of the BCR-ABL gene product is the activation of signal transduction pathways leading to cell growth independent of normal external signals. Imatinib (marketed as Gleevec), a drug that specifically blocks the activity of Abl tyrosine kinase, has shown remarkable efficacy with little toxicity in patients with CML. The successful targeting of BCR-ABL by imatinib is the paradigm for molecularly targeted anti-cancer therapies.

Solid tumors generally contain an abnormal number of chromosomes, a state known as aneuploidy; chromosomes from aneuploid tumors exhibit structural alterations such as translocations, deletions, and amplifications. These abnormalities reflect an underlying defect in cancer cells known as chromosomal instability. While aneuploidy is a striking cellular phenotype, chromosomal instability is manifest as only a small increase in the tendency of cells to gain, lose, or rearrange chromosomes during any given cell cycle. This intrinsically low rate of chromosome aberration implies that cancer cells become aneuploid only after many generations of clonal expansion. The molecular basis of aneuploidy remains incompletely understood. It is widely believed that defects in checkpoints, the quality-control mechanisms that halt the cell cycle if chromosomes are damaged or misaligned, contribute to chromosomal instability. This hypothesis emerged from experimental observations that the tumor suppressor p53 controls checkpoints that regulate the initiation of DNA replication and the onset of mitosis. These processes are therefore defective in many cancer cells. The mitotic spindle checkpoint, which ensures proper chromosome attachment to the mitotic spindle before allowing the sister chromatids to separate, is also altered in some cancers, irrespective of p53 status. The precise relationship between checkpoint deficiency and chromosomal instability remains unclear, but it is believed that even a subtle perturbation of the highly orchestrated process of cell division can impact the ability of a cell to faithfully replicate and segregate its complement of chromosomes. From a therapeutic standpoint, the checkpoint defects that are prevalent in cancers have been proposed as vulnerabilities that may be exploited by novel agents and combinatorial strategies.

In contrast to the genome-wide cytogenetic changes that are typical indications of an underlying chromosomal instability, more focal patterns of chromosomal rearrangement have been recurrently detected in several cancer types. A curious phenomenon known as chromothripsis causes dozens of distinct breakpoints that are localized on one or several chromosomes. These striking structural alterations are thought to reflect a single event in which a chromosome is fragmented and then imprecisely reassembled. While the exact process that underlies chromothripsis remains obscure, and its effects on driver genes is not yet clear, a transient period of extreme instability stands in contrast to the gradual loss, gain and rearrangement of chromosomes that is typically observed in serially cultured cancer cells.

The first indication of the functional existence of tumor-suppressor genes came from experiments showing that fusion of mouse cancer cells with normal mouse fibroblasts led to a nonmalignant phenotype in the fused cells. The normal role of tumor-suppressor genes is to restrain cell growth, and the function of these genes is inactivated in cancer. The three major types of somatic lesions observed in tumor-suppressor genes during tumor development are point mutations, small insertions and/or deletions known as indels, and large deletions. Point mutations or indels in the coding region of tumor-suppressor genes will frequently lead to truncated protein products or allele-specific loss of RNA expression by the process of nonsense-mediated decay. Unlike the highly recurrent point mutations that are found in critical positions of activated oncogenes, known as mutational hotspots, the point mutations that cause tumor-suppressor gene inactivation tend to be distributed throughout the open reading frame. Large deletions lead to the loss of a functional product and sometimes encompass the entire gene or even the entire chromosome arm, leading to loss of heterozygosity (LOH) in the tumor DNA compared to the corresponding normal tissue DNA (Fig. 67-4). LOH in tumor DNA often indicates the presence of a tumor-suppressor gene at a particular chromosomal location, and LOH studies have been useful in the positional cloning of many tumor-suppressor genes. The rate of LOH is increased in the presence of chromosomal instability, a relationship that would account for the high prevalence of aneuploidy in late-stage cancers.

Gene silencing, an epigenetic change that leads to the loss of gene expression, occurs in conjunction with hypermethylation of the promoter and histone deacetylation, and is another mechanism of tumor-suppressor gene inactivation. An epigenetic modification refers to a covalent modification of chromatin, heritable by cell progeny that may involve DNA but does not involve a change in the DNA sequence. The inactivation of the second X chromosome in female cells is an example of an epigenetic silencing that prevents gene expression from the inactivated chromosome. Genomic regions of hypermethylated and hypomethylated DNA can be detected by specialized techniques, and a subset of these regional modifications has consequences on the cell’s behavior.

A small fraction of cancers occurs in patients with a genetic predisposition. Based on studies of inherited and sporadic forms of retinoblastoma, Knudson and others formulated a hypothesis that explains the differences between sporadic and inherited forms of the same tumor type. In inherited forms of cancer, called cancer predisposition syndromes, one allele of a particular tumor suppressor gene is inherited in mutant form. This germline mutation is not sufficient to initiate a tumor, however; the other allele, inherited from the unaffected parent, must become somatically mutated in a normal stem cell for tumorigenesis to be initiated. In sporadic (non-inherited) forms of the same disease, all cells in the body start out with two normal copies of the tumor suppressor gene. A single cell must then sequentially acquire mutations in both alleles of the tumor suppressor gene to initiate a tumor. Thus bi-allelic mutations of the same tumor suppressor gene are required for both inherited and non-inherited forms of the disease; the only difference is that individuals with the inherited form have a “head-start”: they already have one allele mutated, from conception, and only need one additional mutation to initiate the process (Fig. 67-4). This distinction explains why those with inherited forms of the disease develop more cancers, at an earlier age, than the general population. It also explains why, even though every cell in an individual with a cancer predisposition syndrome has a mutant gene, only a relatively small number of tumors arise during his/her lifetime. The reason is that the vast majority of cells within such individuals are functionally normal because one of the two alleles of the tumor suppressor gene is normal. Mutations are uncommon events, and only the rare cells that develop a mutation in the remaining normal allele will exhibit uncontrolled proliferation. The same principle applies to virtually all types of cancer predisposition syndromes, though the particular genes differ. For example, inherited mutations in RB1, WT1, VHL, APC, and BRCA1 lead to predispositions to retinoblastomas, Wilms’ tumors, renal cell carcinomas, colorectal carcinomas, and breast carcinomas, respectively (Table 67-3). Also note that the biallelic inactivation of any of these genes is not sufficient to develop cancer; it requires other, additional somatic mutations for the initiating cells to evolve to malignancy, as noted above.

Roughly 100 familial cancer syndromes have been reported; the great majority are very rare. Most of these syndromes exhibit an autosomal dominant pattern of inheritance, although some of those associated with DNA repair abnormalities (xeroderma pigmentosum, Fanconi’s anemia, ataxia telangiectasia) are inherited in an autosomal recessive fashion. Table 67-3 shows a number of cancer predisposition syndromes and the responsible genes.

The next section examines inherited colon cancer predispositions in detail because several lessons of general importance have been derived from the study of these syndromes.

Familial adenomatous polyposis (FAP) is a dominantly inherited colon cancer syndrome caused by germline mutations in the adenomatous polyposis coli (APC) tumor-suppressor gene on chromosome 5. Affected individuals develop hundreds to thousands of adenomas in the colon. In each of these adenomas, the APC allele inherited from the affected parent has been inactivated by virtue of a somatic mutation (Fig. 67-2). This inactivation usually occurs through a gross chromosomal event resulting in loss of all or a large part of the long arm of chromosome 5, where APC resides. In other cases, the remaining allele is inactivated by a subtle intragenic mutation of APC, which as a single base substitution resulting in a nonsense codon. Gross chromosomal losses occur more commonly than point mutations in normal cells, explaining why these are the predominant mechanism underlying the inactivation of the normal allele of APC. The same is true for other cancer predisposition syndromes caused by other inherited tumor suppressor gene mutations; gross chromosomal events are generally responsible for inactivation of the tumor suppressor gene allele inherited from the non-affected parent. Several thousand adenomas form in FAP patients, and a small subset of the billions of cells within these adenomas will acquire a second mutation, leading to tumor progression, that is, a larger adenoma. A third mutation in such a larger adenoma may convert it to a carcinoma. If untreated (by colectomy), at least one of the adenomas will progress to cancer by the time patients are in their mid-40s. APC can be considered to be a gatekeeper for colon tumorigenesis in that the absence of mutation of this gatekeeper (or a gene acting within the same pathway), a colorectal tumor simply cannot be initiated. Figure 67-5 shows the germline and somatic mutations found in the APC gene. A negative regulator of a signaling pathway that determines cell fate during development, the APC protein provides differentiation and apoptotic cues to colonic epithelial cells as they migrate up the crypts. Defects in this process can lead to abnormal accumulation of cells that would otherwise differentiate and eventually undergo apoptosis.

In contrast to patients with FAP, patients with hereditary nonpolyposis colon cancer (HNPCC, or Lynch’s syndrome) do not develop polyposis, but instead develop only one or a small number of adenomas that rapidly progress to cancer. HNPCC is due to inherited mutations in one of four DNA mismatch repair genes (Table 67-3) that are components of a repair system responsible for correcting errors in newly replicated DNA. Germline mutations in MSH2 and MLH1 account for more than 90% of HNPCC cases, and mutations in MSH6 and PMS2 account for the remainder. When a somatic mutation inactivates the remaining wild-type allele of a mismatch repair gene, the cell develops a hypermutable phenotype characterized by profound genomic instability that is most readily apparent in short repeated sequences called microsatellites and is sometimes called microsatellite instability (MSI). The high rate of mutation in such cells impacts all genes, including oncogenes and tumor suppressor genes, and thereby accelerates the activation of the former and the inactivation of the latter (Fig. 67-2). HNPCC can be considered a disease of tumor progression; once tumors are initiated (by an inactivating mutation of APC or by some other gene in the APC pathway), tumors rapidly progress because of the accelerated mutation rate. Progression from a tiny adenoma to carcinoma takes only a few years in HNPCC patients instead of the two or three decades this progression takes in patients with FAP (or in patients with sporadic colorectal tumors). Approximately half of HNPCC patients develop colorectal cancers by the time they are in their mid-40s—similar to that of FAP patients. This coincidence in age of onset emphasizes that both tumor initiation (abnormal in FAP patients) and tumor progression (abnormal in HNPCC patients) are the two pillars of cancer development and are equally important for cancer development.

Another general principle is apparent from the comparison between FAP and HNPCC patients. The tumors in FAP patients, like those in patients without hereditary predisposition to cancers, are chromosomal instability. MSI and chromosomal instability appear to be mutually exclusive in colon cancers, suggesting that they represent alternative mechanisms for the generation of genomic instability (Fig. 67-2). Other cancer types rarely exhibit MSI. Chromosomal instability is far more prevalent than MSI among all cancer types, perhaps explaining why nearly all cancers are aneuploid.

Although most autosomal dominant inherited cancer syndromes are due to mutations in tumor-suppressor genes (Table 67-3), there are a few interesting exceptions. Multiple endocrine neoplasia type 2, a dominant disorder characterized by pituitary adenomas, medullary carcinoma of the thyroid, and (in some pedigrees) pheochromocytoma, is due to gain-of-function mutations in the proto-oncogene RET on chromosome 10. Similarly, gain-of-function mutations in the tyrosine kinase domain of the MET oncogene lead to hereditary papillary renal carcinoma. Interestingly, loss-of-function mutations in the RET gene cause a completely different disease, Hirschsprung’s disease (aganglionic megacolon [Chaps. 321 and 381]).

Although the heritable forms of cancer have taught us much about the mechanisms of growth control, most forms of cancer do not follow simple Mendelian patterns of inheritance. The majority of human cancers arise in a sporadic fashion, solely as a result of somatic mutation, and in the absence of any mutations in cancer-predisposing genes in their germlines.

The discovery of cancer susceptibility genes raises the possibility of DNA testing to predict the risk of cancer in individuals of affected families. An algorithm for cancer risk assessment and decision making in high-risk families using genetic testing is shown in Fig. 67-6. Once a mutation is discovered in a family, subsequent testing of asymptomatic family members can be crucial in patient management. A negative gene test in these individuals can prevent years of anxiety in the knowledge that their cancer risk is no higher than that of the general population. On the other hand, a positive test may lead to alteration of clinical management, such as increased frequency of cancer screening and, when feasible and appropriate, prophylactic surgery. Potential negative consequences of a positive test result include psychological distress (anxiety, depression) and discrimination, although the Genetic Information Nondiscrimination Act (GINA) makes it illegal for predictive genetic information to be used to discriminate in health insurance or employment. Testing should therefore not be conducted without counseling before and after disclosure of the test result.

Recent technological developments have made it feasible to obtain high-quality sequence of all of the protein-coding DNA sequences, and even of the entire genome, in any given individual. The redundant nature of modern DNA sequencing provides an extremely high level of sensitivity, such that mutations and polymorphisms will inevitably be identified in every subject. In patients lacking a clear family history, the significance of these DNA sequence findings will not be apparent. Even mutations in tumor suppressor genes are difficult to interpret unless there is an obvious functional implication, such as the truncation of the open reading frame, or that particular mutation has previously been associated with cancer. Such germline mutations are very rare in the general population. Vastly more common are variants of unknown significance (VUS). VUS that are found during genetic testing cannot be used to evaluate the relative risk of cancer, but may nonetheless cause anxiety because they represent a deviation from the reference allele that is established as “normal.” Because of the low yield of informative mutations that modify cancer risk and the frequent identification of VUS, it is generally not appropriate to use DNA sequencing to assess cancer risk in individuals unless the family history is suggestive of a germline mutation. Conversely, testing may be appropriate in some subpopulations with a known increased risk, even without a defined family history. For example, two mutations in the breast cancer susceptibility gene BRCA1, 185delAG and 5382insC, exhibit a sufficiently high frequency in the Ashkenazi Jewish population that genetic testing based on ethnicity alone may be warranted.

It is important that genetic test results be communicated to families by trained genetic counselors, especially for high-risk high-penetrance conditions such as the hereditary breast and ovarian cancer syndrome (BRCA1/BRCA2). To ensure that the families clearly understand its advantages and disadvantages and the impact it may have on disease management and psyche, genetic testing should never be done before counseling. Significant expertise is needed to communicate the results of genetic testing to families.

Several human malignancies are associated with viruses. Examples include Burkitt’s lymphoma (Epstein-Barr virus; Chap. 189), hepatocellular carcinoma (hepatitis viruses), cervical cancer (human papillomavirus [HPV]; Chap. 193), and T cell leukemia (retroviruses; Chap. 196). There are several types of HPV, including the high-risk types 16 and 18 that are strongly associated with the development of cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancer. The mechanisms of action of all these viruses involve inactivation of tumor suppressor genes. For example, HPV proteins E6 and E7 bind to and inactivate cellular tumor suppressors p53 and pRB, respectively. This is the reason that HPV is such a potent initiator of cancer: infection with a virus is tantamount to having two of the three mutant driver genes required for cancer, that is, one viral oncogene inactivates p53 and the other inactivates Rb. Though these two inactivated gene products are not sufficient for tumorigenesis, only one additional mutant gene is required to develop a malignancy.

CANCER GENOMES

The advent of relatively inexpensive technologies for rapid and high-throughput DNA sequencing has facilitated the comprehensive analysis of numerous genomes from many types of tumors. This unprecedented view into the genetic nature of cancer has provided remarkable insights. Most cancers do not arise in the context of a mutator phenotype, and accordingly the number of mutations in even the most advanced cancers is relatively modest. Common solid tumors harbor 30–70 subtle mutations that are non-synonymous (i.e., result in an amino acid change in the encoded protein). Liquid tumors such as lymphomas and leukemias, as well as pediatric tumors, typically have fewer than 20 mutations. The vast majority of the mutations detected in tumors are not functionally significant, they simply arose by chance in a single cell that gave rise to an expanding clone. Such mutations, which provide no selective advantage to the cell in which they occur, are known as passenger mutations. As noted above, only a small number of the mutations confer a selective growth advantage and thereby promote tumorigenesis. These functional mutations are known as driver mutations, and the genes in which they occur are called driver genes.

The frequency and distribution of driver mutations within a single tumor type can be represented as a topographical landscape (Fig. 67-7). The picture that emerges from these studies reveals that most genes that are mutated in tumors are actually mutated at relatively low frequencies, as would be expected of passenger genes, whereas a small number of genes (the driver genes) are mutated in a large proportion of tumors. There are a total of ~200 driver genes that are responsible for the development of all solid tumors, representing only ~1% of the total number of human protein-encoding genes. The majority of the mutations in these driver genes provide a direct selective growth advantage by altering the signaling pathways that mediate cell survival or the determination of cell fate. The remaining driver gene mutations indirectly provide a selective growth advantage by accelerating the mutation rate of proto-oncogenes and tumor suppressor genes. The functions of all these driver genes can be organized into a dozen signaling pathways, as shown in Table 67-4.

TUMOR HETEROGENEITY

The mutant cells that compose a single tumor are not genetically identical. Rather, cells obtained from different sites on a tumor will harbor common mutations as well as mutations that are unique to each sample. Genetic heterogeneity results from the ongoing acquisition of mutations during tumor growth. Each time a genome is replicated, there is a small but quantifiable probability that a mutation will spontaneously arise as a result of a replication error and be passed on to the cellular progeny. This is true in normal cells or in tumor cells. Any randomly chosen cell from the skin of one individual will harbor hundreds of genetic alterations that distinguish it from a different randomly chosen skin cell, and the same is true for all organs of self-renewing tissues. Tumors are actually less genetically heterogeneous than normal cells; any two randomly chosen cells from a tumor of an individual will have fewer differences than any two randomly chosen cells from that individual’s normal tissues. The reason for this decrease in heterogeneity is clonal expansion, the fundamental feature of tumorigenesis. Every time a clonal expansion occurs, a genetic bottleneck wipes out heterogeneity among the cells that didn’t expand; these unexpanded cells either die or form only a minute proportion of the total cells in the expanding tumor.

The mutations that vary between cells of a given tumor are invariably passenger mutations that arose since the last evolutionary bottleneck, that is, those mutations that arose during the expansion of the founder cell that gave rise to the final clonal expansion. In contrast, the passenger mutations that were present in the founder cell will be uniformly present in every cell in the tumor. In that respect, these passenger mutations that are not heterogeneously distributed, that is., those that are present in every cancer cell, are like the driver gene mutations, which are also present in virtually all cancer cells. The total number of mutations and their distribution within tumor cells therefore represents a complex interplay between the age of the patient (the older the patient, the more passenger mutations will have accumulated in the founding cell of the first clonal expansion) and the evolutionary history of the cancer (its age and number of clonal expansions it experienced).

Tumor heterogeneity has been recognized for decades at the cytogenetic, biochemical, and histopathologic levels. However, it is only recently, with the advent of a deep understanding of cancer genetics that genetic heterogeneity can be interpreted in a medically relevant fashion. The first important point to recognize about tumor heterogeneity is that it is only the variation in driver gene alterations that is important; the cellular distribution of passenger gene mutations is completely irrelevant. In this discussion of heterogeneity, we can expand the definition of “driver genes” to include those that provide a selective growth advantage in the face of therapy in addition to those that provide a selective growth advantage during tumor evolution, prior to treatment.

Type I heterogeneity refers to that among tumors of the same type from different patients (Fig. 67-8). Though adenocarcinomas of the lung generally harbor mutations in three or more driver genes, the genes differ among the patients and the precise mutations within the same gene can vary considerably. Type I heterogeneity is the basis for precision medicine, where the goal is to treat patients with drugs that target the proteins encoded by genetic alterations within their specific tumors. Type 2 heterogeneity refers to the genetic heterogeneity among different cells from the same primary tumor. Tumors continue to evolve as they grow, and different cells of the same cancer, in its original site (e.g., the colon), may acquire another driver gene mutations that are not shared among the other cells of the tumor. Such a mutation can result in a small clonal expansion that may or may not be important biologically. In cases in which the primary tumor can be surgically excised, such mutations are unimportant unless they give rise to Type III heterogeneity (described below). The reason they are important is because all primary tumor cells, whether homogeneous or not, are removed by the surgical procedure. In primary tumors that cannot be completely excised (such as most advanced brain tumors and many pancreatic ductal adenocarcinomas), heterogeneity is biomedically important because it can give rise to drug resistance, analogously to that described for Type IV heterogeneity (see below). Type III heterogeneity refers to the genetic differences among the founder cells of the metastatic lesions from the same patient. For example, a patient with melanoma may have 100 different metastases distributed throughout various organs. Only if a mutant BRAF is present in every founder cell of every metastasis, then the patient has a chance at a complete response to a BRAF inhibitor. There have been several recent detailed studies of the metastases from various tumor types. Fortunately, these studies suggest there is very little, if any, Type III heterogeneity among driver genes, a necessary prerequisite for the successful implementation of future targeted therapies. Finally, Type IV heterogeneity refers to that among cells of individual metastatic lesions. As the founder cell of each metastasis expands to become detectable, it acquires mutations, a small number of which can act as “drivers” if the patient is exposed to therapeutics. This type of heterogeneity is of major clinical importance, as it has been shown to be responsible for the development of resistance in virtually all targeted therapies. The development of such resistance is a fait accompli based simply on known mutation rates and known genetic resistance mechanisms. The only way to circumvent acquired resistance is to treat metastatic tumors earlier (i.e., in adjuvant setting, before much tumor expansion has occurred) or to treat with combinations of drugs for which cross-resistance is genetically impossible.

High-throughput DNA sequencing has led to an unprecedented understanding of cancer at the molecular level. A comprehensive mutation profile provides a molecular history of a given tumor and insights into how it arose. Because tumor cells and tumor DNA are shed into the blood and other bodily fluids, common driver mutations can be used as highly specific biomarkers for early detection. For diagnosed tumors, tumor-specific mutations can be used to estimate tumor burden, to assess treatment responses and to detect recurrence.

In some cases, information regarding specific genes and pathways that are altered provides patients and physicians with options for personalized therapy. This general approach is sometimes referred to as precision medicine. Because tumor behavior is highly variable, even within a tumor type, personalized information-based medicine can supplement and perhaps eventually supplant histology-based tumor assessment, especially in the case of tumors that are resistant to conventional therapeutic approaches. Conversely, molecular nosology has revealed similarities in tumors of diverse histotype. The success of the precision medicine approach in any given patient depends on the presence of tumor-associated genetic alterations that are actionable (i.e., can be targeted with a specific drug). Examples of currently actionable changes include mutations in BRAF (targeted by the drug vemurafenib) and RET (targeted by sunitinib and sorafenib), and ALK rearrangements (targeted by crizotinib). At present, the proportion of tumors that can be treated with such precision medicine approaches is small, but future therapeutic development will hopefully change this situation. The development of new targeted agents is at present hindered by the fact that such agents can only target activated oncogenes, while the great majority of genetic alterations in common solid tumors are those that inactivate tumor suppressor genes. Because all drugs, whether for use in oncology or any other purpose, can only inhibit protein actions, drugs cannot be used to directly target the proteins encoded by inactivated tumor suppressor genes; these proteins are already inactive. More information about the pathways through which tumor suppressor genes act may provide a way around this obstacle. For example, when a tumor suppressor gene is inactivated, some downstream component of the pathway is likely to be activated, thereby presenting a realistic target. An example of this is provided by PARP-1 inhibitors, which have been successfully used to treat patients whose tumors have inactivating mutations of genes involved in DNA repair processes, such as BRCA1. Patterns of global gene expression can be used to help unravel such pathways and are already being used to predict drug sensitivities and provide prognostic information in addition to that provided by DNA sequence analysis. Evaluation of proteomic and metabolomics patterns may also prove useful.

THE FUTURE

A revolution in cancer genetics has occurred in the past 30 years. Most types of cancer are now understood at the DNA sequence level and this accomplishment has led us to an increasingly refined understanding of tumorigenesis. Cancer gene mutations have proven to be reliable biomarkers for cancer detection and monitoring as well as for informing therapeutics through precision medicine approaches. Gene-based tests are already standard of care for certain tumor types, such as melanoma, colorectal and pancreatic cancers, and the utility of these tests will undoubtedly be expanding greatly in the coming years as new therapies and ways of predicting responses to therapies are developed. While effective treatment of advanced cancers remains difficult, it is expected that breakthroughs in these areas will continue to emerge and be applicable to an ever-increasing number of cancers. Moreover, with the hoped-for advances in diagnostics, particularly in the earlier detection of cancers, the new and old therapies for cancer can be expected to have a much greater impact on reducing cancer deaths.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the past contributions of Pat J. Morin, Jeff Trent, and Francis Collins to earlier versions of this chapter.