Genetic and Epigenetic Alterations in Cancer

Recurrent Mutational Targets in Cancer

Oncogenic variant alleles that are present in cancer are generated from the normal counterpart proto-oncogenes by various mutational mechanisms, including point or localized mutations, gross chromosomal rearrangements, or gene amplification. Some representative oncogene mutations in human cancer are summarized in Table 14.1 . From a brief review of the data in Table 14.1 , some generalizations can be offered. First, the mutations affect proteins functioning in various compartments of the cell, including growth factor receptors, cytoplasmic signal transducers, and nuclear proteins, such as transcription factors. Second, although some oncogene mutations may be unique to cancers of a particular type, such as the specific chromosomal translocations and resultant fusion proteins that are seen in cancers of hematopoietic origin (e.g., the BCR-ABL translocation that is seen in chronic myelogenous leukemia and a subset of acute lymphoid leukemias and the PML-RARα translocation that is seen in acute promyelocytic leukemia), other mutations, such as those affecting the KRAS, β-catenin, and c-MYC genes, are found in a broad spectrum of different cancer types. Third, oncogene mutations in cancer are nearly always somatic, because only a very limited number of germline mutations in proto-oncogenes have been linked to cancer predisposition thus far. Fourth, some proto-oncogenes, such as KRAS or BCL2, are somatically altered in cancer by a single mutational mechanism, namely, point mutations in the KRAS gene and chromosomal translocations affecting the BCL2 gene. In contrast, other proto-oncogenes, such as c-MYC, may be activated by more than one mechanism in cancer, including chromosomal translocation, gene amplification, and, more recently discovered, association with superenhancer. These mutational mechanisms lead to increased levels of c-MYC transcripts and protein. In fact, specific missense mutations at threonine 58 of the c-MYC gene in some lymphomas may further enhance c-MYC protein levels by abrogating a phosphorylation-ubiquitination mechanism targeting c-MYC for proteosomal degradation. However, enhanced c-MYC protein levels can also result from alterations in c-MYC–specific microRNAs, methylation, and other noncoding regulatory elements functioning to regulate c-MYC transcription and translation.

In contrast to the activating mutations that generate oncogenic alleles from proto-oncogenes, inactivation of the normal function of tumor suppressor genes is critical in tumorigenesis. Akin to the proto-oncogenes, the functions of tumor suppressor genes are diverse, and proteins that are encoded by these genes reside in practically all subcellular compartments ( Table 14.2 ). Many tumor suppressor genes were identified by virtue of the fact that they are mutated in the germline of persons who are affected by a known mendelian cancer syndrome or who at the very least display a markedly elevated risk of cancer. The link between a germline-inactivating mutation in a purported tumor suppressor gene and increased cancer predisposition provides very persuasive evidence of the functional significance of the gene in the cancer process. Nevertheless, for the vast majority of tumor suppressor genes, in terms of their magnitude, somatic inactivating mutations play a far more significant role in cancer development than do germline mutations.

Another important point to consider is that much of the attention for tumor suppressor genes has been focused on demonstrating that cancer cells carry biallelic inactivating mutations. Clearly, a diverse array of mechanisms can inactivate gene function, including nonsense, frameshift, and nonconservative missense mutations, as well as splicing mutations and gross deletions of the gene or even the chromosome region that contains the gene. In a number of cases, studies of the chromosomal mechanisms associated with tumor suppressor gene inactivation in cancer tissues, such as loss of the parental heterozygosity (i.e., loss of heterozygosity) that is present in normal tissues, have even been used to infer the existence of tumor suppressor genes, in particular, chromosomal regions, before the actual identification of the tumor suppressor gene of interest. The emphasis on defining biallelic inactivating mutations in tumor suppressor genes has been stimulated in large part by the Knudson hypothesis, which predicted that recessive genetic determinants played a critical role in retinoblastoma and many other cancers and that inactivation of both alleles of a tumor suppressor gene was needed to abrogate tumor suppressor gene activity. Nevertheless, as will be discussed in a bit more detail in the following sections, a variety of observations indicate that epigenetic (nonmutational) mechanisms might play a prominent role in inactivating tumor suppressor gene function in sporadic tumors. Furthermore, for certain tumor suppressor genes, inactivation of only one of the two alleles of a tumor suppressor gene might significantly impair cell growth regulation or programmed cell death. For example, a number of mutant p53 proteins that carry missense mutations likely potently interfere via dominant negative mechanisms with the wild-type p53 protein in the cell because p53 functions as a homotetrameric protein and all subunits must be wild-type for fully intact p53 function in transcriptional regulation. For other tumor suppressor proteins, such as the cyclin-dependent kinase inhibitory protein p27, reduction of protein levels to 50% of the levels present in normal cells might result in significant detrimental effects on the ability of the cell to appropriately regulate growth.

Cancers Arise From the Accumulation of Multiple Genetic and Epigenetic Defects

On the basis of a simple consideration that a cancer involves a large number of genetic and epigenetic alterations that arise in normal cells during the many years of life, it would seem quite unlikely that any adult cancer may arise as the result of single gene defect. Even in persons who are strongly predisposed to cancer as a result of a germline mutation in a specific oncogene or tumor suppressor gene, the vast majority of cells in a person never develop into cancer or even display definitive morphologic changes akin to those that are seen in benign tumors. Therefore, any model for cancer must incorporate these data, which suggest that cancers likely arise as the result of the accumulation of multiple genetic and epigenetic defects in an affected cell. Another issue to consider is that clinical and histopathologic data indicate that the development of nearly all cancers, regardless of the organ site, is often, if not invariably, preceded by precancerous phases or stages in which the neoplastic cells manifest increasingly disordered patterns of differentiation and morphology.

Given this background, it would appear that cancers arise from accumulated defects affecting multiple genes and pathways, and precancerous (benign) precursor lesions harbor fewer of the key gene defects than established cancers. One question, therefore, is how many rate-limiting defects or “hits” are required for cancer development. Although a definitive answer to this question cannot be given at this point, some estimates can be offered. Most common cancers show dramatically increased incidence with increasing age. On the basis of analysis of the age-specific incidence of a number of common cancers and some straightforward assumptions about the rate of mutations and the size of the target cell population, it was argued as early as the mid-1950s that most common epithelial cancers arise as the result of four to seven rate-limiting events. It was inferred that these rate-limiting events might represent mutational events. Moreover, benign lesions harboring fewer gene defects are often shown to have an age-incidence distribution that was shifted roughly one to two decades earlier in life than cancers arising in the corresponding organ or tissue site. Nevertheless, confounding the use of age-incidence data to model the number of rate-limiting mutations were questions about the weight of importance of certain key biologic pathways underlying the multiple hits models. On the other hand, studies of the genetic alterations of pediatric cancers that occur much earlier and are perhaps less complex in terms of their genetic signature may offer insights into key cancer target genes (see Chapter 92 on pediatric solid tumors).

Clonal Selection and Evolution in Cancer

As previously noted, most if not all cancers arise from preexisting precancerous populations of cells, and multiple genetic and epigenetic defects are likely needed for conversion of a normal cell to a cancerous cell. Molecular studies of various cancer types and their corresponding associated precancerous lesions have yielded some fundamental insights into the processes likely to be critical in the emergence of the cancer. First, although normal tissues and tissues from noncancerous disease states display polyclonal (balanced) cell populations, the neoplastic component that is present in benign lesions and cancers displays a clonally related cell population, consistent with the notion that neoplastic transformation of one or a number of different cells within a tissue give rise to all daughter cells or subclones that are present in the tumor. This is further established by NGS deep sequencing in studying intratumor heterogeneity. Second, in tumors in which it has been possible to analyze both cancer cells and associated precancerous cell populations, a subset of the somatic gene defects present in the cancer are clonally represented in the precancerous cell population. Other somatic gene defects appear to be acquired during progression from the precancer subclones to the dominant subclones in the cancer.

These molecular findings in benign and malignant tumors are essentially consistent with a model initially proposed by Foulds and subsequently advanced by Nowell ( Fig. 14.1 ). In brief, the clonal evolution model predicts that cancers arise as the result of successive expansions of clonally related cell populations. The successive expansions are driven by the gradual or punctuated acquisition of mutations and gene expression changes that endow a particular cell and its progeny with a selective advantage over cells that do not harbor the gene defects. In essence, clonal selection is an evolutionary process that allows the outgrowth of precancerous and cancerous cells that carry mutations and gene expression changes that confer the most potent proliferative and survival properties on the cancer cells. However, it is important to note that the specific constellation of genetic and epigenetic changes that are present in precancerous and cancerous cells is context-dependent and certainly varies considerably from one cancer type to another and even most certainly varies to a significant degree among patients whose cancers display quite similar clinical and histopathologic features. The clonal evolution model has biologic and clinical ramifications, just a few of which will be mentioned here. First, the clonal gene defects that are present in a cancer can be traced in precancerous lesions from the same organ site with a goal of attempting to clarify the preferred order in which gene defects arise in the natural history of a particular cancer type. The particular order in which defects accumulate during the initiation and subsequent progression of one cancer type often differs from that in another cancer type. As a result, a genetic or epigenetic change that is critical in tumor initiation in one cancer type might contribute to tumor progression in another tumor type and vice versa. Second, defects that arise at “early” stages of tumorigenesis might play a vital role not only in tumor initiation but also in the aggressive behavior of advanced stage cancers. Third, the model predicts that the acquisition of further genetic heterogeneity will be a common and important factor in primary cancer lesions and metastases. It is becoming more evident that genetic heterogeneity plays a significant role in resistance to chemotherapy and targeted therapy and the emergence of aggressive cell populations in patients with advanced cancer.

Recent comprehensive sequence-based analyses of cancer cell populations from individual patients in which the tumor cells under study were distinct from one another in location and/or time has begun to reveal that quite dramatic intratumoral genetic heterogeneity may be a “rule” in cancer, rather than an exception. Certain, perhaps key, initiating genetic lesions (e.g., truncal mutations) might be shared among all neoplastic clones, but geographically distinct regions of large primary tumors may have very distinct mutation profiles (e.g., branched mutations) from those in other portions of the primary tumor, and the metastatic cell populations may have considerable mutational divergence from the nonmetastatic cells. Accordingly, it seems that quite extensively branched evolutionary growth may be an important feature in both primary tumors and metastatic lesions, with multiple competing clonal populations evolving through divergent and convergent mutational mechanisms. This more recent view of the potentially quite extensive intratumoral genetic heterogeneity in any given cancer and the contributions of intratumoral heterogeneity to tumor progression contrasts with some earlier views. Before the more recent studies, it was suspected that the cell populations in many primary cancers might be more homogeneous, wherein somatic mutations were accumulated in a more stepwise fashion as a result of multiple sequential clonal sweeps of each variant cell population in the primary cancer, with metastases perhaps most often arising from the clonally dominant cell population in the primary tumor.

It is important to note that clonal somatic mutations are often presumed to have a causal role in promoting further tumor outgrowth or progression because somatic mutations can become clonal (i.e., present in all neoplastic cells) by only a limited number of mechanisms. For instance, the genetic alteration itself could have been selected for because it provided the neoplastic cell with a growth advantage, allowing it to become the predominant cell type in the tumor (clonal expansion). Genes with critical roles in promoting clonal outgrowth in a given cancer have been termed drivers. Genes that are mutant in a significant fraction of cancers and for which other lines of evidence link them to the cancer process can more readily be classified as drivers . Alternatively, a somatic mutation, when detected, might have arisen essentially coincident with another, perhaps undetected, alteration that was the crucial change underlying clonal outgrowth. Somatic mutations of this latter type have been termed passengers. However, studies have shown that some of these passengers, which serve as metabolic and housekeeping genes, can be targeted therapeutically, leading to tumor suppression. Therefore, on the basis of extensive data from large-scale sequencing analyses that reveal large numbers of distinct genes that are each mutated in only a minority of cancers of a given type, sorting out drivers and passengers might not be entirely straightforward based solely on sequencing data and will likely require a significant body of functional studies and data. Indeed, increasingly even noncoding alterations have been shown to play important roles in causing and maintaining the cancer phenotypes by introducing new cancer-causing promoters and enhancers that lead to altered gene expression (see the later discussion of noncoding mutations). Given this discussion about the potential uncertainties associated with linking specific somatic mutations to cancer development, it is even more challenging to assign causal significance to any given gene expression change in precancerous and cancerous cells. In particular, the ambiguities arise when considering whether a gene expression or epigenetic change merely reflects the relative difference between cancer and its matched normal tissue or is truly causally involved in the cancer process. Nonetheless, if a specific gene or epigenetic alteration can be shown to promote tumorigenesis or neoplastic transformation in a variety of in vitro and in vivo tumor models or if the same gene or chromosomal region is recurrently altered by particular epigenetic changes in tumors, then it might be reasonable to infer that the particular defect might indeed have a causal (driver) role in tumorigenesis.