In Chapter 9 were discussed the basic biological and molecular mechanisms of the stage of pro- gression in the development of neoplasia. Unlike the stages of initiation and promotion, the stage of progression encompasses, for the most part, the clinical disease resulting from the pres- ence and growth of neoplastic cells within the host. While the control or elimination of the stages of initiation and promotion are considered as methods of cancer prevention, the control of neoplastic disease during the stage of progression is almost entirely in the realm of therapy. Even early diagnosis of the disease in the stage of progression is not a form of prevention but rather a form of efficient therapy. The longer the stage of progression is allowed to develop, the less efficient become the therapeutic modalities used to treat this disease. It is the changes that occur during this stage of progression that dictate the way that neoplasia is diagnosed and treated.
Clonality of Neoplasms
Previous chapters discussed alterations in single cells (initiation) as well as in cellular popula- tions (promotion), leading to the development of the stage of progression. Stem cells (Chapters 9 and 14), each of which may give rise to a clone of genetically identical cells, occur in both nor- mal and neoplastic cellular growth. However, a question that has always intrigued investigators in the field of oncology is whether a neoplasm arises from a single cell or from multiple cells (a field) that have been transformed to neoplasia almost simultaneously, e.g., by viruses or germ- line genetic alterations. The answer to this question is by no means simple (Tanooka, 1988). In considering clonality, one must determine at what stage in the development of neoplasia the “founder” cell (not identical to a stem cell as defined in Chapter 14) occurs. Such a founder cell may be a normal cell that becomes initiated, and its progeny develop through the stage of pro- motion, with subsequent transition of a single cell in the clone to the stage of progression. Alter- natively, the founder cell may be in the stage of promotion, which then gives rise to a clone in the stage of progression. Finally, since genetic abnormalities occur commonly during the stage of progression, subclones may appear, suggesting that the neoplasm is polyclonal in origin (Fey and Tobler, 1996; Woodruff, 1988). Usually the founder cells of neoplasms are those resulting from the transition of a cell in the stage of promotion to the stage of progression. However, pre- neoplastic lesions produced experimentally in the liver (Weinberg and Iannaccone, 1988) and the skin (Deamant and Iannaccone, 1987) are reportedly clonal.
Methods for Determining Clonality
The first indications that neoplasms were clonal came from cytogenetic and biochemical analy- ses. In chronic myelogenous leukemia, in which the leukemic cells all possess the Philadelphia chromosome, a monoclonal origin of the neoplasm would be expected. Myelomas and several types of lymphomas almost always produce only a single type of immunoglobulin. From our knowledge of immunobiology (Chapter 16), myelomas and lymphomas thus were felt to arise from a single cell or a very few cells. However, the most widely used method for the determina- tion of a monoclonal or polyclonal origin of neoplasms depended on investigations of the iso- zymic forms of X-linked enzymes in cells.
In the normal mammalian female, early during embryonic development but after meiosis, one of the two X chromosomes in each cell is repressed (Figure 10.1). The mechanism of this repression is unknown, but it culminates, in the fully developed organism, in a mosaic cellular pattern consisting of a number of populations of cells expressing the genes on one X chromo- some, while the remaining cellular populations of the organism express the genes on the other X chromosome. If an individual is heterozygous (different alleles or copies of the gene in each of the two chromosomes), then some cells will express one form of the gene and other cells the other form. In relation to the monoclonal derivation of neoplasms, some females are mosaic for two isozymic forms of the enzyme glucose-6-phosphate dehydrogenase, resulting from the re- pression of one or the other X chromosome in individual cells during early embryonic life, as stated by the Lyon hypothesis. Thus, if a neoplasm arising in such a mosaic individual contains only a single form of this enzyme, it is likely to have resulted from a single cell. On the other hand, it is difficult to rule out the possibility that the early neoplastic transformation occurred in many cells, but that then one or very few cells attained a growth advantage and overgrew the vast majority of the population. In studies with chimeric mice produced by the amalgamation of two
Figure 10.1 Diagrammatic representation of the Lyon hypothesis. The zygote (fertilized egg) is de- picted as that of a female inheriting one X chromosome from the mother (Xm) and the other from the father (Xp). These chromosomes are passed to daughter cells, but at some early time in embryogenesis a differ- ence occurs in the behavior of each of the two X chromosomes in each somatic cell, so that only one X chromosome remains active in each cell and its subsequent daughter cells. The other X chromosome is inactive and becomes the Barr body, as noted in the figure. Thus the adult female becomes a mosaic with a number of cells expressing the genes of Xp, while others express those of Xm. (After Fialkow, 1974, with permission of the author and publisher.)
embryos (allophenic mice), neoplasms of the skin induced in such animals exhibited an apparent clonal growth in that genetic markers characteristic of only one of the two parent embryos ap- peared in the neoplasms (Condamine et al., 1971). However, hepatomas produced in such ani- mals were mosaic; that is, they possessed genetic characteristics of each of the parent embryos.
With the advent of newer methods of molecular biology, newer methods for the determina- tion of clonality in cells have been developed. Table 10.1 lists more traditional methods, as de- scribed above, and a number that utilize techniques of molecular biology to analyze genetic alterations. The restriction fragment length polymorphism (RFLP) analyses have been discussed previously (Chapter 5). One may also combine the polymerase chain reaction with RFLP deter- mination to increase the sensitivity of the analysis, such that the clonality of as few as 100 cells may be determined (Gilliland et al., 1991). While cells of a myeloma or lymphoma may be shown to be clonal by measuring the genetic structure of their immunoglobulin or T-cell recep- tor, owing to the production of only a single molecular species of these proteins by a lympho- cyte, studies have demonstrated an apparent polyclonality of some lymphomas when the structure of the T-cell receptor gene is measured in these lesions (Ohno et al., 1997; Bignon et al., 1990). This phenomenon may be explained by arguing that the founder cell of each clone was produced during the stage of progression rather than in the transition from promotion to progression or earlier (Collins, 1997). DNA fingerprint analysis may also be somewhat variable, possibly owing to the appearance of subclones. Analysis of breakpoint cluster regions, as in the Philadelphia chromosome, is another method giving results similar to cytogenetic analysis but taking advantage of the unique, stable genetic alteration in the breakpoint region. Finally, the structures of the genomic termini of the Epstein-Barr virus (EBV) in episomes of infected cells may indicate whether the neoplasm arose from a very low multiplicity of infection (very few viral particles per target cell) or from a high multiplicity, with many cells being simultaneously infected and giving rise to a lymphoma (Chapter 12). Table 10.2 list data indicating the mono- or polyclonality of a variety of neoplasms of the human. As noted from the table, only two heredi- tary neoplasms, neurofibroma and trichoepithelioma, have been shown to be consistently poly- clonal in origin, although recent studies have suggested polyclonality for several other neoplasms, both hereditary and spontaneous. These studies, which are now more than 20 years
old for the most part, were carried out on relatively large amounts of neoplastic tissue. More recent techniques have allowed the analysis of microdissected samples from morphologically distinct microscopic lesions. Such studies have in general confirmed the monoclonal origin of each distinctive lesion (Cheng et al., 1998).
Studies in experimental animals have indicated a clonal nature for some neoplasms (Collins and Fialkow, 1982); in other cases, however, chemicals have induced neoplasms of multicellular origin (Reddy and Fialkow, 1979). A follow-up analysis of the latter studies dem- onstrated that sarcomas induced by the subcutaneous injection of methylcholanthrene may de- velop as monoclonal or polyclonal neoplasms, depending on whether benzene or olive oil is the solvent vehicle in which the carcinogen is administered (Reddy and Fialkow, 1981). The same authors (Reddy and Fialkow, 1983) later showed that papillomas induced by a single painting of dimethylbenzanthracene, followed by promotion with TPA, are predominantly monoclonal in origin, whereas those neoplasms resulting from the repeated applications of the carcinogen itself exhibit a much higher incidence of polyclonal origins. A suggested explanation of these apparent discrepancies is that a relatively small dose of initiating agent may tend to induce more mono- clonal neoplasms simply because fewer cells will be initiated, whereas relatively higher doses of an initiating agent will ultimately result in polyclonal neoplasms because of a greater chance of adjacent cells being initiated and developing together. However, Deamant and Iannaccone (1987) argued that the different observations of monoclonal or polyclonal neoplasms were the result of confounding by the presence of varying amounts of nonneoplastic dermal tissue. On the other hand, differences in growth rates and other factors such as nutrition (blood supply) and interaction among clones of neoplastic cells may result in the apparent monoclonality of a neoplasm even if the process of initiation was not clonal (cf. Woodruff, 1988; Chow and Rubin, 2000).
An alternative view of the clonality of neoplasms is that taken by Nowell (1976) and oth- ers on the clonal evolution of a neoplastic cell population, with neoplasms considered to evolve by the selection of one or more clones of cells during their natural developmental history. By this concept, the clonality of a neoplastic cell population in the stage of progression may simply be the result of the selective growth advantage of a clone of cells appearing during the natural his- tory of development of the neoplasm, resulting in the overgrowth by this population. This has been recognized over the years by the demonstration of “stem” cell populations in neoplasms with a characteristic karyotype. Such clonal evolution can also account to a great degree for the progression of many neoplasms. It has also been suggested that diseases heretofore not classified as neoplastic in origin may exhibit clonal development of their basic pathological lesion and thus could possibly be related to the neoplastic transformation. This appears to be the case in atherosclerosis, as suggested by Benditt (1974), who demonstrated that the fibrous plaque seen in blood vessels in the early development of this disease is clonal, as shown by the use of X- linked markers.
Thus, the data (Table 10.2) in human neoplasia argue that most neoplasms are clonal in origin. However, this does not imply that their clonality arose at the stage of initiation but rather at the stage of progression. Preneoplastic lesions in the stage of promotion both in skin (Reddy and Fialkow, 1983) and in rat liver (Weinberg and Iannaccone, 1988) are clonal, presumably arising from single initiated cells. However, as discussed in Chapter 9, neoplastic lesions arise within preneoplastic lesions, presumably initiating a new clone, as evidenced both from different karyotypes and other markers of progression (Giaretti, 1997; Scherer et al., 1984). Apparently polyclonality may, in turn, result from high doses of the carcinogenic agent (Reddy and Fialkow,
1983), resulting in cells transiting into the stage of progression within a short time period. Therefore the clonal nature of both preneoplasia and neoplasia is an important aspect of our con- sideration of the natural history of neoplastic development.