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C O N T R I B U T O R S

HISTORICAL OVERVIEW

Cancer is every malignant tumor whose cells have the properties of endless replication, loss of contact inhibition, invasiveness, and the ability to metastasize and whose result, generally, if left untreated, is fatal.1 Cancer cells typically display three distinct phenotypes that are not associated with normal cells:

1.Immortalization: an indefinite proliferative life span

2.Transformation: the loss of response to normal regulation of cell growth

3.Metastasis: the ability to leave the tumor and invade other tissues at another location in the body

A close relationship between chromosomal alterations and cancer was postulated by Theodor Boveri, who conducted experiments on double-fertilized sea urchin eggs. Boveri demonstrated the following:

•Individual chromosomes carry different information.2,3

•The unresticted growth of tumor cells resembles the same abnormal growth of double-fertilized sea urchin eggs carrying the wrong chromosomal complement.4

In his last book, on the origin of malignant tumors, Boveri5 concluded that malignancy is the result of inappropriate balance of instructions (genetic information) in the tumor cells.

The extraordinary relevance of the discoveries made by Boveri in the field of cancer can be fully appreciated only in light of the fact that it took almost 50 years after the publication of his monograph to find out that chronic myeloid leukemia (CML) is associated with a characteristic chromosomal aberration (the Philadelphia chromosome),6 another decade to identify the chromosomes involved in the translocation producing this aberrant chromosome,7 a further decade to identify the genes fused and activated as a result of this translocation,8 and almost two more decades to develop a drug against the fusion protein

(BCR-ABL) produced by the gene activated as a result of the chromosomal translocation.9

At the time of Boveri’s investigations, the concept of the gene was not developed yet. Subsequent research on his observations seemed to demonstrate the following:

•Except for the Philadelphia chromosome in CML, “cancer-specific” aneuploidy is very rare.

•Aneuploidy may not necessarily be associated to cancer development (as shown by the Down Syndrome).

•Cancers can be diploid (in the early stages of cancers of viral origin).10

This last observation originated from studies made by Peyton Rous who in 1911 was able to repeatedly induce tumors in a particular breed of chicken by means of tumor-derived cell-free filtrates, probably containing a virus.11 By the early 1970s, virologist Hidesaburo Hanafusa showed that the Rous sarcoma virus (RSV) contains a gene (called src for sarcoma) that produces a protein necessary for cancer development.12 Removing the gene prevented the virus from inducing cancer.13 These discoveries led researchers to entirely abandon Boveri’s idea that aneuploidy was at the basis of the unregulated growth of tumor cells and to shift the focus of cancer research to genes and gene mutations.

At about the same period, Howard Temin,14–16 together with David Baltimore and Renato Dulbecco, observed that certain viruses were able to synthesize DNA from RNA by using an enzyme called reverse transcriptase (RT). The viruses that produce this enzyme were called retroviruses, and the RSV was found to belong to this group.

This discovery not only definitively disproved the accepted dogma that protein synthesis within the cell proceeds as a one-way process, from DNA to RNA and to protein, it also had relevant consequences for further investigations in the field of cancer research. In the late 1970s, Michael Bishop and Harold Varmus identified cellular src (c-src) homologues in organisms

as diverse as fruit flies, chicken, fish, mammals, and humans. These cellular genes (c-onc) are present in normal cells, where they play an essential role in cell growth regulation (proto–oncogenes). When they become overexpressed or mutated (oncogenes), they are able to confer to the cells the traits of rapid, uncontrolled growth that are typical of many tumors.17–19

ONCOGENES

Oncogenes represent the first identified class of activated human genes responsible for tumor development in humans. A short list of these genes is given in Table 1.1. The distinction between the terms “proto-oncogene” and “oncogene” relates to the activity of the protein product of the gene. A protooncogene is a gene whose protein product plays an essential role in cell growth regulation and has the capacity to induce cellular transformation if it sustains a genetic insult. An oncogene is a gene that has sustained genetic damage and therefore produces a protein capable of cellular transformation.

Many proto-oncogenes code for proteins that relay growth stimulating signals from outside the cell to deep within its interior. Cell-to-cell signaling begins when one cell secretes a protein called growth factor. The growth factor moves into the intercellular space and binds to the receptors located on the target cell membrane. After that binding, the receptor conveys a proliferative signal to proteins located in the cytoplasm, in a cascade of protein activation that, in turn, brings the signal to the nucleus, where other proteins, known as transcription factors (TFs), respond by activating a cohort of genes to usher the cell through its growth cycle (Figure 1.1).

Some oncogenes force cells to overproduce growth factors. Sarcomas and gliomas, of particular interest for this book, overproduce platelet-derived growth

factor (PDGF). Other cancers overproduce transforming growth factor (TGF- ). These growth factors can drive proliferation in the same cell producing them.

Oncogenic versions of growth factors’ receptor genes that induce aberrant receptors to release a flood of proliferative signals within the cell, in the absence of growth factors have been also identified. This is the case, for example, of the Erb-b2 oncogenic protein. In other cases, oncogenes perturb the cytoplasmic signal cascade. This is the case with the RAS oncogene, which normally (proto-oncogene) conveys stimulatory signals from growth factor receptors farther down the line, but, when activated, fires continuously even when growth factors are not prompting it.

Still another family of oncogenes, such as those belonging to the Myc family, alter the activity of transcription factors within the nucleus. Cells normally produce Myc transcription factor when the cell surface is stimulated by growth factors. Myc proteins, in turn, activate genes that stimulate cell growth. However, in many types of cancer, such as blood malignancies, Myc levels are kept high even in the absence of growth factors.20 Since the discovery and characterization of the first oncogene, a great number of other oncogenes have been added to the list. For detailed information regarding the types, mechanisms of action, and related tumors and proto-oncogenes see the Park reference21. For the purposes of this chapter, the list in Table 1.1 is an acceptable synthesis.

From the point of view of Mendelian genetics, oncogenes act in a dominant fashion: that is, a single activated copy (oncogene) of the allelic pair is able to induce cell transformation.

TUMOR SUPPRESSOR GENES

In 1971, while researchers were still identifying new members of the family of oncogenes, Alfred G. Knud-

son,22 by studying several cases of retinoblastoma (Rb) demonstrated that this rare eye tumor affecting infants and young children, is likely to depend on two sequential mutations affecting a key gene, still unknown at that time. The hypothesis, formulated by Knudson to explain the different clinical phenotypes of retinoblastoma, was based on the study of the relationships between age at diagnosis, clinical phenotype (unilateral vs bilateral disease), and number of tumor foci per affected eye. When the first mutation is transmitted genetically from one of the parents, all the somatic cells of the individual will carry it. As a consequence, the individual will be likely to develop, at an early age, a tumor affecting both eyes (bilateral Rb), with multiple foci and, given the first mutation in all somatic cells, an increased susceptibility to develop second nonocular tumors. On the other hand, when both the first and second mutations affect the somatic cell (the retinoblast), the individual will develop, later in life, a retinoblastoma affecting, commonly a single eye (unilateral Rb), with a single tumor focus and no susceptibility to second nonocular tumors.

Molecular studies using genetic markers that are heterozygous in the majority of individuals showed that tumor genotypes of affected patients usually differ from the corresponding constitutional genotype (e.g., the genetic makeup of patient’s blood cells). In

its most simplified expression, this was evidenced as difference in the electrophoretic migration pattern of selected DNA markers. When these markers were found to show a typical two-band model indicating heterozygosity in the patient’s constitutional genotype (e.g., nucleated blood cells), it was common to find a single band, indicating homozygosity, when the tumor DNA of the same patient was comparatively analyzed (Figure 1.2). As described by Cavenee et al.,23 this phenomenon, called loss of heterozygosity (LOH), was considered to be specific to tumors involving the loss or inactivation of a new type of cancer gene and, as shown in Figure 1.2, seemed to represent the physical demonstration of the “two-hit” model hypothesized by Knudson in the genesis of retinoblastoma.

With the introduction of the polymerase chain reaction (PCR), which allows the amplification of large amounts of specific DNA fragments, and the concurrent discovery of a number of new genetic markers from within specific genes, molecular analysis of cancer became available on a larger scale and armed clinical oncologists with a powerful new tool for genetic counseling and prenatal/presymptomatic diagnosis of different types of cancer.24 Further investigations of a suspect gene for the development of this tumor led to the identification of the gene Rb1, located in the long arm of chromosome 13 (13q14), and, most important, opened a completely new line of research on cancer genes. When

considered from the point of view of Mendelian genetics, Rb1, as opposed to the known oncogenes, seemed to behave in a “recessive” way. That is, to develop a fully expressed disease, it seemed necessary for both copies of the gene to be lost or inactivated, thus implying that even a single functional gene is normally sufficient to inhibit the proliferative activity of the cell.25 Therefore, it became evident that the cell contains genes of two different types that regulate its proliferative capacity, one with stimulatory (oncogenes) and the other with inhibitory activity. This last category of cancer genes, of which Rb1 represents the prototype,26,27 was called tumor suppressor genes (TSGs).

TSGs normally function to inhibit, or “put the brakes on,” the cycle of cell growth and division; that is, they function to prevent the development of tumors. As for oncogenes, this task is accomplished by a number of heterogeneous proteins in a number of different ways.

ious to TGFby inactivating a gene that encodes a surface receptor for this substance.

•A variety of cancers discard the p15 gene, which normally codes for a protein needed to shut down the machinery guiding the cell through its growth cycle.

•Some TSGs, such as NF1, the gene for neurofibromatosis type 1, block the flow of signals through the growth stimulatory pathway (RAS oncogene).

•Some other genes, currently considered TSGs, are involved in the repair of DNA mismatches; still others are involved in the apoptotic cascade.

More recently, a simplified classification proposed two different functions for these genes, namely that of caretakers, or guardians of the integrity of the genetic material, and that of gatekeepers, or regulators of tumor growth by inhibition of cell growth or by promotion of cell death. According to this classification, both caretakers and gatekeepers share the characteristics of “recessive” genes (i.e., two mutations are

to neoplasia, leading to genetic instability, requires more mutational events than the gatekeeper pathway.28 A short list of TSGs is given in Table 1.2.

The discovery of TSGs seemed to complete the picture of how the cell can be transformed by endogenous influences of opposite sign on its proliferative activity, that is, the activation of the stimulatory pathway (oncogenes) or the disruption of the inhibitory pathway (TSGs). In both cases, the deregulation implies the loss of the normal control these genes exert on the entrance into the cell cycle or the permanence of the cell in a quiescent state (G0) (Figure 1.1).

DNA MUTATIONS AND CANCER

The discovery and characterization of both oncogenes and TSGs represented outstanding achievements in the understanding of the molecular pathogenesis of cancer. An important step in this process has been the elucidation of the mechanisms by which proto-oncogenes are activated to oncogenes and TSGs are inactivated, resulting in the uncontrolled growth that characterizes cancer cells. Further investigations on oncogenes made it clear that their activation in cancer cells can be ascribed to several different mechanisms, as follows:

1.Structural alterations of the genes:

•Point mutations

•Chromosomal translocations, such as the t(9;22) in chronic myeloid leukemia

2.Gene amplification (e.g., NMYC amplification in neuroblastoma) evidenced as either

•Small separate chromosomes (double minutes) or

•Insertions within normal chromosomes (homogeneously staining regions, or HSR)

3.Loss of appropriate control mechanisms, by either

•Chromosome translocation (translocation of the MYC gene on chromosome 8 to one of the immunoglobulin loci on chromosome 14 in Burkitt’s lymphoma) or

•Insertional mutagenesis (insertion of a DNA copy of a retrovirus into the cellular genome close to a proto-oncogene)29

Despite the reported variety of the mechanisms involved in the activation of proto-oncogenes, the proposed mechanisms of inactivation of TSGs has remained limited to the structural alteration evolving from a single base (point mutation) to wider portions of the genome. With the increasing interest in TSGs and the potential application of genetic testing to the early diagnosis or identification of predisposition to cancer, the role of mutations in cancer development has become increasingly relevant. Indeed, most researchers worldwide acknowledge no “cause” of cancer other than mutation.

In one of the following paragraphs on gene methylation, we will see that the close relationship between mutations and cancer should be viewed with a more relativistic eye, since gene expression may be altered even in the absence of structural alterations of genes. Moreover, considering the complexity of cell structure and function, and the number of different environmental influences a cell undergoes during its vital cycle, limiting the possible molecular pathogenesis of cancer to one or two mutations within a single gene, as in the case of retinoblastoma, appears to be a very restricted view of the problem.

NEW PERSPECTIVES

ON DNA MUTATIONS

Studies on hereditary nonpolyposis colorectal cancer (HNPCC) have shown that affected individuals may inherit an inactive copy of one of the DNA mismatch repair genes. The main function of these genes is to produce proteins whose primary function is to iden-

tify and correct DNA replication errors. Mismatch repair prevents spontaneous mutation; therefore, cells defective in the DNA mismatch repair enzymes system may accumulate mutations at rates several hundredfold higher than normal.

Randomly dispersed throughout the human genome are tens of thousands of microsatellites, long stretches of reiterated monoor dinucleotides: for example An (A adenine; n repeated n times) or (C-A)n (C-A cytosine–adenine dinucleotide; e.g., CACACACAn]. The accurate replication of such repetitive DNA is usually compromised by the tendency of template and daughter DNA strands to misalign during DNA synthesis. In cells with defective DNA mismatch repair enzyme system, the precise control of microsatellite length is lost, and therefore, the cells contain many thousands of altered microsatellites. This accumulation of mutated repeats is commonly known as microsatellite instability (MSI), which is the defining characteristic of mismatch- repair-deficient tumors.

MSI is a continuing process and is a direct consequence of failure to rectify replication errors. In practice, MSI is defined by differences in the lengths of several microsatellites between DNA from tumor and normal tissue of the same individual.30 In principle, the two alleles of a given chromosome of an individual contains microsatellite DNA made of repeats (e.g., CA repeats) of unequal length. In “genetic” terms this means that individuals are most frequently heterozygous (they have two different copies of the same DNA portion in the two homologue copies of the same chromosome) for most of the known microsatellite sequences within their genome. This implies that microsatellites are genetic markers of great value in detecting the LOH process, which characterizes cancers that are due to the loss or inactivation of TSGs. In routine laboratory activity, microsatellite DNA can be easily amplified by using the PCR. The amplified DNA is analyzed by resolving it through a polyacrylamide gel, where it can be evidenced as a band pattern that is characteristic of any single microsatellite in each individual.

While microsatellites have long been considered to be genetic markers useful in detecting LOH in the tumor genotype, as opposed to the somatic genotype of the affected individual, their widespread use in the characterization of different tumors has revealed that MSI is another relevant process in carcinogenesis. In simplified terms, the difference between LOH and MSI resides in the qualitative difference of the DNA migration patterns one can find when the constitutional (nucleated blood cells) and the tumor genotypes of an affected individual are compared. LOH is the loss of one or more bands in the tumors with respect to the related constitutional genotype. MSI is not a loss but a band shift, as shown in Figure 1.3, which presents

FIGURE 1.3. Three examples of MSI in embryonal rhabdomyosarcoma of the orbit. MSI, like LOH, is a modification, in comparison to the normal constitutional cells (blood) of the affected individual, of the DNA migration pattern of tumor cells as revealed on agarose or polyacrylamide gel. Unlike LOH, which is evidenced as the loss of one or more bands in the tumor DNA, MSI is a qualitative difference that is evidenced as a “band shift.”

three cases of MSI in embryonal rhabdomyosarcoma of the orbit.

Cells with a defective mismatch repair system are said to carry a mutator phenotype, a phenotype in which many genes carry mutations because of uncorrected errors in DNA replication. These cells are also said to carry genome instability, the process leading, through accelerated somatic evolution, to a genomically heterogeneous population of cells naturally selected for their ability to proliferate and invade, while simultaneously evading host defenses.31 This picture leads to at least two different considerations.

1.It is likely that several mutations in different mismatch repair genes, rather than a single mutation in one gene, represent the most relevant early event in carcinogenesis.

2.The genome instability theory brings us back to Boveri’s observations, since an unstable genome is more likely to produce unbalanced distribution of chromosomes between the daughter cells during mitosis, as observed by Boveri in his experiments on double-fertilized sea urchin eggs.

RELEVANCE OF MSI

TO ORBITAL TUMORS

Several reasons could be suggested for the extreme rarity of reports on molecular analysis of orbital tumors. For example, these tumors are relatively rare and in many countries a consistent gap exists between oph-