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.
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.
The mutational topography of colorectal cancer. The two-dimensional landscape represents the positions of the individual genes along the chromosomes. The height of each peak represents the mutation frequency at that locus. The top map is a representation of many sequenced colorectal cancers. The taller peaks represent the genes that are commonly mutated in colon cancer, while the smaller hills indicate the genes that are mutated at lower frequency. On the lower map, the mutations of two individual tumors are indicated. Note that there is little overlap between the mutated genes of the two colorectal tumors shown. These differences represent Type I heterogeneity, as noted in the test, which is the foundation for personalized medicine in cancer. (From LD Wood et al: Science 318:1108, 2007, with permission.)
TABLE 67-4Signaling Pathways Altered in Cancer ||Download (.pdf) TABLE 67-4 Signaling Pathways Altered in Cancer
|Process ||Pathway ||Representative Driver Genes |
|Cell survival ||Cell cycle regulation/apoptosis ||RB1, BCL2 |
|RAS ||KRAS, BRAF |
|PIK3CA ||PTEN, PIK3CA |
|JAK/STAT ||JAK2, FLT3 |
|MAPK ||MAP3K. ERK |
|TGF-β ||BMPR1A, SMAD4 |
|Cell fate ||Notch ||NOTCH1, FBWX7 |
|Hedgehog ||PTCH1, SMO |
|WNT/APC ||APC, CTNNB1 |
|Chromatin modification ||DNMT1, IDH1 |
|Transcriptional regulation ||AR, KLF4 |
|Genome maintenance ||DNA damage signaling and repair ||ATM BRCA1 |
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.
The four types of tumor heterogeneity. Tumor heterogeneity is the inevitable result of cell proliferation, as new mutations are introduced during clonal expansion. This concept is illustrated by a primary tumor in the pancreas and two metastatic tumors in the liver. The tumors of the founding populations are shown in the middle of each circle, while the distinct subclones are shown around the periphery. Type I: the heterogeneity of tumors that occur among different patients. Type II: the heterogeneity among the cells of a primary tumor, also known as intratumoral heterogeneity. Type III: the heterogeneity among the founding cells of distinct metastatic lesions (marked as 1 and 2) that arise in the same patient, also known as intermetastatic heterogeneity. Type IV: heterogeneity among the cells of each metastasis that develops as each tumor grows, also known as intrametastatic heterogeneity.