Clonal Expansion of Malignant Tumors

A fundamental concept in cancer is the clonal expansion of tumors, with each step in tumor formation based on adding another mutation to the tumor genome. Figure 102-1A illustrates this concept. The process starts with a single mutation in a single cell. As mutations in critical genes accumulate in a cell, a cancer develops in stepwise fashion. In brain cancers, it is not clear how long it takes for mutations to accumulate and in what order all the mutations might occur because the early stages of the tumors are not easily observed during tumorigenesis. It appears, however, that many mutations and changes in copy number, in any one of many different combinations, accumulate to form a GBM.⁷ Because this clonal expansion presumably arises from a single cell, all the cells in the tumor should have the same mutation. This is normally true, except in a few situations, such as the presence of unstable amplifications that give rise to heterogeneity for this change in the tumor or the acquisition of a late-occurring mutation in the latter stages of tumor cell clonal expansion. For the vast majority of mutations, however, the same mutations will appear in all parts of the tumor.

FIGURE 102-1 Basic concepts in cancer genetics. A, Clonal expansion of tumors. Cancers are thought to start with a single mutation in a single cell and then accumulate the necessary mutations in critical genes to grow in an uncontrolled manner. Each of the cells in the tumor will therefore have the same complement of mutations and will be “clones” with respect to mutation sequences. In this diagram, progressive mutations are shown by darker shades of pink. Tumor growth may not occur appreciably in many tumors until there are numerous mutations. In this diagram only three steps are shown, although for the highest grade malignancy many more mutations per cell are observed. B, Common mechanisms of gene mutations and alteration. Oncogenes are activated with a gain of function, whereas tumor suppressors are inactivated. Point mutations and other small intragenic sequence changes can activate or inactivate a gene. Entire genes can be lost or gained with homozygous deletion and genomic amplification. Parts of two genes can be fused during chromosomal amplification to form a new oncogene.

Inherited Mutations and Familial Syndromes

Cancer genetics started with identification of the inherited gene mutations responsible for familial clustering of cancer through linkage or association studies. Linkage and association studies can locate the approximate region of the chromosome responsible for a disease phenotype based on finding a polymorphic marker that cosegregates in families with individuals affected by the disease. These studies of inheritance are laborious because they involve identifying large families with the disease, collecting blood from family members, and studying markers throughout the genome to find one that is on the same chromosome, close enough to the gene of interest so that the marker and mutant gene are not separated by DNA crossover during meiosis.

The prototype cancer gene syndrome involves the inheritance of a tumor suppressor mutation in which there is one mutated allele in the germline and a second spontaneous mutation inactivates the second allele. Other spontaneous mutations occur subsequently in the cell as part of the cell’s transformation to a malignant state. This first hit dramatically increases the chance of tumor formation in someone with the germline mutation, but by itself it is not sufficient.

Because the first hit is inherited as a germline mutation, it can be transmitted to offspring. Although most malignant tumors observed in the clinic do not have an evident hereditary basis, it is important to recognize the possibility of familial clustering of brain tumors and consider genetic counseling and further evaluation if evident. Table 102-1 lists some of the more common cancer-associated mendelian disorders that have brain tumors as part of the phenotype. Mendelian disorders are genetic diseases that have a clear pattern of inheritance within families, such as dominant or recessive inheritance. Most of the syndromes that involve brain tumors have an autosomal dominant mendelian inheritance pattern. A complete catalogue and literature review of all the mendelian disorders identified is readily accessible at the Online Mendelian Inheritance in Man (OMIM) website.⁸

In the laboratory, the key way to distinguish a somatic mutation from a hereditary mutation is to sequence the suspected gene mutation in both the tumor and normal tissue. In most cases, lymphocyte DNA from a blood sample is used as the normal control. If the mutation appears only in the tumor and not in normal tissue, it is evidence for a somatic mutation acquired during tumor development.

The classic scenario for a brain tumor phenotype with mendelian inheritance is the dominant inheritance of a mutation in a tumor suppressor gene, with an average of 50% of offspring inheriting one copy of the mutated gene from the affected parent. In the case of tumor suppressor genes, both copies need to be inactivated to initiate the process of tumor formation. When one mutation is inherited as the first “hit,” there is a high probability of the second being inactivated. Inheritance of this first hit greatly increases the risk for cancer.

To find evidence that a DNA sequence is an inherited mutation, DNA samples from affected families are studied via linkage or association studies. If the DNA mutation associates with affected members in the pedigree significantly more frequently than dictated by chance, the DNA sequence studied is probably at or near the chromosomal locus responsible for the phenotype.

Although most mendelian disorders that include in the phenotype an increased risk for brain tumors are relatively rare in the general population, there are some exceptions. Neurofibromatosis is the most common of the syndromes with brain tumors as a phenotypic feature. Germline mutations in the neurofibromatosis type 1 (NF1) gene result in a syndrome characterized by café au lait spots on the skin, fibromatous tumors, and gliomas and meningiomas. Approximately 1 person in 4000 is affected with NF1 mutations. About half the cases are spontaneous and the other half are hereditary. In approximately 10% or less of these patients, a malignancy will develop from one of their multiple benign tumors.

The mismatch repair (MMR) cancer syndrome is an important syndrome that involves several different types of cancers, including some GBMs, meningiomas, and medulloblastomas.^8,11,12 It has an autosomal dominant pattern of inheritance, as do all the syndromes listed in Table 102-1. There are several DNA repair enzymes that are necessary for correct DNA MMR in a normal cell. In MMR cancer syndrome, an inactivating mutation or deletion of the MLH1, MSH2, MSH6, or PMS genes reduces the ability of the cell to identify and correctly repair DNA mismatches that occur during DNA replication. Therefore, a mutation in any of these genes can lead to an increased risk for brain and other cancers. The mutations in these “mutator genes” increase the cell’s mutation rate and accelerates the acquisition of mutations elsewhere in the genome, thus leading to a significantly increased risk for the development of cancer at an early age.

Adenomatous polyposis coli (APC) syndrome increases the risk for colon cancer and brain tumors, similar to MMR syndrome. However, APC syndrome, or familial adenomatous polyposis syndrome, is molecularly and phenotypically distinct from MMR cancer syndrome. This disorder increases the risk for medulloblastomas rather than gliomas and is due to a mutation in the APC gene. Turcot’s syndrome was originally applied to any syndrome with colon cancer and brain tumor but, when used now, applies only to MMR cancer syndrome.

Genetic inheritance of cancer risk is of more than scientific interest. Inherited patterns help researchers locate the genes that can initiate brain tumors, and identifying a familial risk for cancer is also good clinical practice. A new patient with familial clustering of brain tumors or other types of tumors will often want to know whether there is a gene responsible and what the risk is for their family members. Identifying the gene responsible gives the patient options for genetic counseling and family planning, as well as provides the opportunity for early diagnosis and treatment of other family members carrying the disease-causing gene allele. In some cases, identifying a cancer syndrome in a family can be lifesaving for a family member.

Tumor Suppressors, Oncogenes, and Mutator Genes

Cancer-causing gene mutations can be classified by how the mutation contributes to tumor formation. Gain-of-function mutations are changes that either enhance the normal gene function or add a new function. Gain-of-function mutations are activating and change a proto-oncogene to an oncogene. For oncogenes, only one copy of the gene needs to be mutated for activation. Activation of an oncogene then contributes functionally to tumor progression, in concert with other gene mutations. Oncogenes are typically activated either by genomic amplification of the region containing the gene or by small mutations that alter the protein sequence. Occasionally, splicing-related mutations that add or delete gene exons can activate an oncogene.

Loss-of-function mutations inactivate tumor suppressors. A tumor suppressor is a gene that normally keeps malignant progression in check. Mutations that produce a new stop codon (truncating mutation), delete all or part of the gene, disrupt the gene promoter, alter splicing, or change an amino acid or acids rendering the protein nonfunctional are all common ways to inactivate a tumor suppressor.

An important third class of cancer-causing genes is the mutator gene. This is a gene that when mutated, increases the mutation rate in the cell’s DNA. This elevated mutation rate then leads to critical mutations in oncogenes and tumor suppressors, although many new mutations will occur throughout the entire genome. Most of the mutator genes identified to date are DNA repair enzymes; when these genes are lost because of inactivating mutations, the cell subsequently loses its ability to edit and repair errors that occur during DNA replication. The resulting accelerated mutation rate then leaves the cell primed to activate oncogenes and inactivate tumor suppressors at an accelerated rate. This class of cancer gene includes the genes responsible for MMR syndrome, mentioned earlier. In GBMs, about 5% of tumors will have MMR instability, thus indicating a mutation in one of the DNA MMR enzymes.

Different Types of DNA Mutations and Alterations

DNA damage is the basis of cancer and can occur in several different ways. Mutations are changes in the DNA sequence that are acquired during the life of the cell, although in common usage they are sometimes thought of as inherited disease-causing sequences. However, mutations can occur in somatic cells and be inherited in germline cells. Mutations are more accurately and broadly defined as any DNA change that increases risk for a disease or directly promotes disease formation.

DNA alterations can range from single base pair changes all the way to entire chromosome gains or losses, as well as any size change in between. There does not seem to be any restriction to the DNA sequence changes that can occur during the development of cancer. In addition to DNA sequence changes, it also appears that epigenetic changes can contribute to tumor progression by altering gene expression without altering the nucleotide sequence.¹⁷

Point mutations are single base pair changes. In the coding regions of genes, a point mutation that alters the three-letter genetic code in such a way that the amino acid is changed is referred to as a nonsynonymous change. A synonymous (silent) mutation is one that does not alter the amino acid at that position. Normally, these silent changes are thought to be nonfunctional, but there may be hidden regulatory sequences within the coding region that can cause a functional change. Point mutations may also change an amino acid to a stop codon and, along with other mutations that induce early termination of protein translation, are referred to as truncating mutations. Point mutations and other changes can also alter gene regulatory regions in the gene or at regulatory regions distant from the gene. Other common small mutations can alter gene splicing, alter transcript levels, or form new proteins.

Insertion and deletion of one or more bases can have the same effect as point mutations. New amino acids can be added or deleted to a protein and thereby either activate a new function or delete the normal function.

If both copies of a gene are deleted, it is known as a homozygous deletion. Homozygous deletions are sometimes observed in a cancer genome and are frequently a signal to the researcher that a tumor suppressor gene was located in the lost region of the genome. More frequently, only one copy of a gene is deleted in its entirety, and this occurs in regions of loss of heterozygosity (LOH). LOH is often the first hit in inactivating a tumor suppressor in sporadic cancers, with the second inactivating smaller mutation occurring in a tumor suppressor gene within the region of LOH.

When the number of alleles is increased substantially beyond the normal two copies, it is known as genomic or gene amplification. The presence of increased copy numbers of a gene as a result of genomic amplification is a reliable indication that an oncogene is located in the amplified region. Genes such as Mouse Double Minute 2 homolog (MDM2) and epidermal growth factor receptor (EGFR), for example, are frequently amplified in GBMs. Either the normal gene can be found to be amplified (and simply increases its normal function to pathologic levels), or a mutated oncogene can be found in the amplified region. If a normal gene sequence is genomically amplified, it is still regarded as an oncogene if it results in increased expression of the gene’s protein and the increased levels promote tumor growth. Mutated genes can also arise in genomically amplified regions, and the mutation might occur either before or after the amplification.