As noted above, Knudson’s original proposal of the two-mutational concept of neoplastic devel- opment was based primarily on epidemiological data and appropriate models to explain the data. It was not until a number of years after the proposal was first enunciated that cytogenetic and molecular genetic evidence developed in its support. Both of these technologies are considered here, as well as much of the evidence that has developed in support of Knudson’s thesis.
The Cytogenetics of Germline, Dominantly Inherited Cancer
Each species of living organism has evolved in such a way that its genome is packaged into a number of structures termed chromosomes. Both the number and the structure of chromosomes are characteristic of the species, so that, for example, the human exhibits a haploid (one copy of each chromosome only) number of 23 chromosomes in its karyotype. However, most vertebrates exhibit two copies of the chromosome within each somatic cell in the female, while the male has one copy of the X and one copy of the Y chromosome. These last two are termed sex chromo- somes, while all others in the karyotype are termed autosomes. Thus, the human karyotype for all cells except certain germ cells and cells of some glandular organs is 46 chromosomes. This number differs for other species; e.g., the rat karyotype is 42 chromosomes, that of the mouse 40 chromosomes, the hamster 44, etc.
Appropriate methods have been developed to allow the microscopic examination of chro- mosomes and karyotypes of cells. In 1970, Caspersson and associates were among the first to develop techniques that allowed studies of the substructure of chromosomes with the light mi- croscope and assured definitive identification of individual autosomes. These techniques, which have now been modified and extended by a variety of workers, have allowed the identification of “bands” or regions of differential staining of the arms of individual chromatids in a variety of species. Specific patterns of banding, characteristic of the species as well as of each of the chro- mosomes within any one species, have now been described. Furthermore, the localization of in- dividual genes on specific chromosomes and in relation to specific bands has been described in a number of mammalian species as well as other species, both vertebrate and invertebrate. Figure 5.4 shows a diagram of chromosome 13 of the human karyotype, including its banding patterns and indicating the position of a number of genes in the two arms of the chromosome. As noted in the figure, the short arm is designated as the p arm, while the long arm is the q arm. The two are separated by the centromere. In chromosome 13 the short arm (p) is polymorphic in nature, and variations in size may occur in the heterochromatic satellite region (p13), the nucleolar organizer or secondary constriction (p12), and the short arm proper (p11) (Yunis et al., 1979). One should
Figure 5.4 Diagrammatic representation of human chromosome 13 in early metaphase, showing major and minor bands, with the designation of the band positions of a number of genes shown on the right. (Modified from Yunis et al., 1979; McKusick and Amberger, 1995.)
also note that each arm is separated into regions designated by the large numbers within the blocked spaces and bands designated by the smaller numbers adjacent to individual bands of the chromatid in the figure. This nomenclature was first standardized at an international conference held in Paris in 1971 (Paris Conference, 1972). Later refinements have been undertaken in this country for international use (ISCN, 1985). Thus, it is now possible to designate specific regions of the chromosome according to the specific banding pattern as standardized by this nomencla- ture. Although aberrations in karyotypes are not only characteristic of but perhaps ubiquitously associated with malignant neoplasia (see below), it is only in the last two decades that specific chromosomal alterations have been associated with neoplasms resulting from specific inherit- ance patterns.
As indicated earlier, the recessive conditions of Bloom syndrome, Fanconi anemia, and ataxia telangiectasia are associated with chromosomal instability, but no specific chromosomal alterations have been demonstrated in these and other conditions that exhibit a more generalized instability of karyotypes. However, in cancer-prone syndromes of a dominant type of inherit- ance, in a number of instances either specific chromosomal alterations or specific structural gene alterations have been demonstrated during the past 15 years, as evidenced by techniques of cyto- genetics or molecular biology, respectively. These findings have, in turn, led to the localization of specific gene(s) that appear to be responsible for the defect and the resulting neoplasm.
Table 5.3 lists some specific chromosomal changes associated with autosomal dominant inherited cancers. Best known is the association of hereditary retinoblastoma with a small dele- tion in the long arm of chromosome 13 (Francke and Kung, 1976; Wilson et al., 1973). Less than 5% of patients with retinoblastoma show such deletions (Horsthemke, 1992), but recent evi- dence indicates that even those patients with normal chromosomal karyotype express molecular biological and biochemical evidence of genetic abnormalities in this chromosomal structure. Benedict et al. (1983) reported that patients with the characteristic chromosomal deletion exhibit only half the normal activity of esterase D, the gene for which appears to be closely linked to the area of deletion. In one patient with bilateral retinoblastoma and a normal karyotype, only 50%
of the normal esterase D activity was found in the patient’s normal cells. Interestingly, in cells isolated from the neoplasm of this patient and exhibiting abnormalities of chromosome 13, no detectable esterase D activity was found. More recent studies have demonstrated that the esterase D gene is closely linked to that coding for retinoblastoma (van der Heiden et al., 1988; Table 5.3). In further support of the two-hit theory, the second retinoblastoma allele frequently is lost by chromosomal mechanisms (Cavenee et al., 1983).
Another example of chromosomal abnormalities in hereditary neoplasms is the demon- stration of a constitutional deletion on the short arm of chromosome 11 in some patients with Wilms tumor, specifically those with the WAGR syndrome, which includes a predisposition to Wilms tumor (W), aniridia (A), genitourinary abnormalities (G), and mental retardation (R). There is much variation in the size of these deletions, but chromosome band 11p13 is invariably involved in the WAGR syndrome (Riccardi et al., 1978). Other phenotypic features frequently associated with these deletions include congenital absence of the irises (aniridia), gonadal dyspla- sia, and mental retardation. However, only 50% of patients with the congenital defects actually develop Wilms tumor, indicative of the incomplete penetrance of the mutation predisposing to the neoplasm or linked genes with different functions being deleted. More recently, several authors have also demonstrated that hereditary renal cell carcinoma, an autosomal dominant condition, is associated with translocations between the short arm of chromosome 3 and other chromosomes, especially 11 and 8 (cf. Table 5.3). Although this condition is quite rare, when the translocation involves chromosome 8, it has also been reported to involve the c-myc proto-oncogene, reminis- cent of similar translocations seen in Burkitt’s lymphoma, resulting from somatic changes in the karyotype (Drabkin et al., 1985). However, no rearrangement or abnormality in the structure of the translocated proto-oncogene has yet been described. Finally, the balanced translocation be- tween chromosomes 17 and 22 seen in a female patient with von Recklinghausen neurofibroma- tosis 1 was actually an unusual manifestation, but, as shown below, it offered a clue to the localization of the gene for this condition. Not shown in the table is the fact that hereditary med- ullary carcinoma of the thyroid has also been found to be associated with a preponderance of chromosomal structural abnormalities, as noted in karyotype preparations (Hsu et al., 1991).
Obviously, chromosomal deletions visible in the light microscope represent major alter- ations in the genome, reflecting thousands of kilobases of DNA lost from that chromosome. Lo- calization of the gene involved (usually 15 to 40 kilobases in size) requires more refined technology, which has now been employed not only in the localization of genes for neoplasms exhibiting a Mendelian type of heredity but also for numerous other genetic diseases as well.