J Cancer Res Clin Oncol (1996) 122: 135-140 �9 Springer-Verlag 1996

Alfred G. Knudson

Hereditary cancer: two hits revisited

Received: 29 August 1995 / Accepted: 14 September 1995

Abstract According to a "two-hit" model, dominantly inherited predisposition to cancer entails a germline muta- tion, while tumorigenesis requires a second, somatic, muta- tion. Non-hereditary cancer of the same type requires the same two hits, but both are somatic. The original tumor used in this model, retinoblastoma, involves mutation or loss of both copies of the RB1 tumor-suppressor gene in both hereditary and non-hereditary forms. In fact, most dominantly inherited cancers show this relationship. New questions have arisen, however. When a tumor-suppressor gene is ubiquitously expressed, why is there any specificity of tumor predilection? In some instances, it is clear that two hits produce only a benign precursor lesion and that other genetic events are necessary. As the number of necessary events increases, the impact of the germline mutation diminishes. The number of events is least for embryonal tumors, and relatively small for certain sarcomas. Stem-cell proliferation evidently plays a key role early in carcino- genesis. In some tissues it is physiological, as in embryonic development and in certain tissues in adolescence. In adult renewal tissues, the sites of the common carcinomas, mutation may be necessary to impair the control of switch- ing between renewal and replicative cell divisions; the APC gene may be the target of such a mutation.

Key words Heredity �9 Somatic mutations �9 Suppressor genes

Work dedicated to Dr. Haruo Sugano on the occasion of his 70th birthday. The material of this paper was essentially presented at the 60th Anniversary Symposium of the Cancer Institute and the Cancer Institute Hospital, Tokyo, held in September 1994

A.G. Knudson Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 USA Fax: (215) 728 3574

Introduction

Although there are recessively inherited conditions, such as xeroderma pigmentosum and ataxia telangiectasia, that predispose to cancer, the term "hereditary cancer" is here applied to a dominantly inherited predisposition to cancer. Virtually all types of human cancer can occur in genetically susceptible individuals. Sometimes there are non-neoplastic phenotypic features that identify such persons, as in neu- rofibromatosis, but more often there are none. A mutant gene's penetrance for neoplasia can be very high, but some obligate carders of a mutation never develop a cancer. The tumor specificity of the mutation may be extremely narrow (e.g., hereditary chemodectoma) or may be quite broad (e.g., Li-Franmeni syndrome), but no known mutation predisposes to all forms of cancer. It has been estimated that there may be 50 or so different genes in which mutations can impart high susceptibility to heterozygous carriers.

Dominant transmission of a trait indicates its passage to 50% of the offspring of an affected person, possibly for numerous generations. However, some persons will suc- cumb before the end of the age of reproduction or will be infertile, thereby causing the gene's disappearance from that line. Yet the disease does not disappear from the population, because new mutations occur at some back- ground rate and replace them. For a condition that fre- quently causes death in childhood these new mutants can constitute a large fraction of the total germline cases, about 50% for neurofibromatosis type 1 and 80% for hereditary retinoblastoma, for example. The loss of mutations by selection and their gain by mutation reach a mutational equilibrium. The new mutants are, of course, important in genetic counseling, since their offspring carry a 50% risk for receiving the mutation from an affected parent.

The fact that penetrance is incomplete for cancer muta- tions in the germline indicates that heterozygosity is not a sufficient condition for the development of cancer; some- thing else must occur. For hereditary retinoblastoma I supposed that this may be a second mutation, occur r ing

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post-zygotically (Knudson 1971). Compatible with this notion was the calculation that it would occur at a rate that was reasonable for a somatic mutation. The mean number o f such events was estimated to be three, which would also explain the high incidence of bilaterality among hereditary cases. The distribution of numbers of tumors among cases approximated to a Poisson distribution, sug- gesting that it was determined by chance. The 60% or so of non-hereditary (no germline mutation) cases were then attributed to two somatic mutations occurring at the same sites as in the hereditary ones. The only difference between hereditary and non-hereditary cases was the timing of the first event as prezygotic or postzygotic, respectively; the two forms were viewed as involving the same two events. The two mutations could be viewed as affecting the two copies of a single gene, or one copy of each of two different genes. In the former instance oncogenesis would be reces- sive, with the implication that the normal allele of a retinoblastoma gene (RB1) is protective against cancer, by being a negative regulator of cell division (Comings 1973; Knudson 1973).

Retinoblastoma and Wilms tumor

Louise Strong and I extended the two-hit hypothesis to Wilms tumor (Knudson and Strong 1972a), to neuroblas- toma and pheochromocytoma (Knudson and Strong 1972b), and, with David Anderson, to all hereditary cancers (Knud- son et al. 1973). For Wilms tumor we noted that the association of some cases with aniridia might be attributed to a deletion of adjacent genes and that knowledge of the site of such a deletion could aid in the isolation of a Wilms tumor gene. Indeed such deletions were subsequently de- scribed (Ladda et al. 1974; Riccardi et al. 1978), and a Wilms tumor gene (WT1) was localized to chromosomal band l lp13 (Francke et al. 1979). Deletions were known for a minority of patients with retinoblastoma, and were localized to chromosomal band 13q14 (Francke and Kung 1976; Knudson et al, 1976). In addition, some deletions were found in tumors but not in normal cells, supporting the idea of homogeneity of hereditary and non-hereditary cases (Hashem and Khalifa 1975). A similar" observation was made for Wilms tumor (Kaneko et al. 1981).

For retinoblastoma Sparkes et al. (1983) found a bio- chemical marker, esterase D, that was closely linked to RB 1 and was reduced to 50% of normal activity in the normal cells of germline deletion cases (Sparkes et al. 1980). The examination of the tumor from one such individual revealed the complete absence of the enzyme's activity; both events affected the same chromosomal site, and provided the first direct evidence for the recessive hypothesis (Benedict et al. 1983). Using an electrophoretic variant that Sparkes had found, Godbout et al. (1983) were able to show that the tumors arising in persons heterozygous for the variant often lost one of the two alleles present in normal cells. This finding was confirmed in the first application of restriction- fragment-length-polymorphisms (RFLP) to the study of

cancer (Cavenee et al. 1983). In addition, multiple RFLP on chromosome 13 permitted the demonstration that the second event could be a local one, a deletion, loss of a chromosome 13, or somatic genetic recombination, as predicted (Knudson 1978). Retention of both alleles sug- gested a local second event, whereas loss of all hetero- zygous alleles on a chromosome indicated whole chromo- some loss, and loss of heterozygosity for some alleles was compatible with deletion or recombination. These two possibilities could be distinguished by a consideration of the familial segregation of linked alleles. The powerful technology could be generalized for all cancer, and was quickly extended to Wilms tumor (Fearon et al. 1984; Koufos et al. 1984; Orkin et al. 1984; Reeve et al. 1984).

With the precise localization of RB1 and WT1 and use of the technique of positional cloning, the RB1 gene was subsequently cloned (Friend et al. 1986; Lee et al. 1987; Fung et al. 1987), and, still later, so was WT1 (Call et al. 1990; Gessler et al. 1990). We now perceive that all retinoblastomas, whether hereditary or non-hereditary, con- tain no normal copy of RB1, whereas this is true for at most 20% of Wilms tumors, the remaining cases involving one or more other genes than WT1. Still, RB1 and WT1 qualify as recessive cancer genes, also known as tumor-suppressor genes (Klein 1987), or antioncogenes (Knudson 1985).

The marked tissue-specificity of Wilms tumors that are abnormal for WT1 is readily explained by the tissue-limited expression of the WT1 gene. However, RB1 is expressed in all tissues, creating a new puzzle.

The problem of tissue specificity

The problem of tissue specificity arose not only for RB1, but also for other antioncogenes that were cloned. One such gene is TP53, which was not discovered by positional cloning, but rather by its protein product, p53, which interacted with certain DNA tumor virus proteins (Linzer and Levine 1979; Lane and Crawford 1979). This antion- cogene is the most frequently mutated gene in human cancers, and is particularly so in carcinomas. This observa- tion appears to be compatible with its ubiquitous expres- sion. The puzzle derives from the fact that germline muta- tion of TP53 does not most characteristically predispose to these carcinomas. Patients with the Li-Fraumeni syndrome that harbor germline TP53 mutations are chiefly susceptible to carcinoma of the breast, a tumor which, in its sporadic form, shows one of the lower frequencies of TP53 mutation among all carcinomas (Malkin et al. 1990). The other principal tumors are not even carcinomas, but rather sarco- mas, leukemia, and brain tumors (Li mad Fraumeni 1975), A possible explanation is that the characteristic tumors typi- cally occur in childhood or early adulthood in the syn- drome, and that patients die before reaching the age at which most carcinomas become common. This then shifts the question to the reason for these typical age-specific incidences.

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Patients with hereditary retinoblastoma are susceptible to certain other tumors too, notably osteosarcoma and soft- tissue sarcomas (Draper et al. 1986), categories also ob- served with Li-Fraumeni syndrome. As expected, sporadic tumors of these histologies often have mutations of RB1 or TP53 or both (Friend et al. 1986; Masuda et al. 1987; Diller et al. 1990). Just as with the Li-Fraumeni syndrome, hereditary retinoblastoma patients are not characteristically susceptible to some tumors that are known for mutations in sporadic cases, such as small-cell carcinoma of the lung. The sporadic cases of this tumor are almost invariably mutant for both RB1 and TP53, yet small-cell carcinoma of the lung is not a typical feature of either hereditary condition. However, as more patients with these conditions survive to later age, this tumor may be found in excess in both. More events may be critical to the development of small-cell carcinoma; in fact, deletion in chromosome 3p, the site of a putative tumor-suppressor gene, is found in most cases, but not in sarcomas (Whang-Peng et al. 1982; Naylor et al. 1987). It seems that, as the number of genetic aberrations increases, the impact of the inherited mutation on the frequency of a particular tumor diminishes. Thus, for persons who carry a germline mutation of RB1 the relative risk for one retinoblastoma is approximately 105, for osteosarcoma 103, and for small-cell carcinoma of the lung, at most 10 (Knudson 1992).

Genetic events in carcinogenesis

The two-hit hypothesis explains most tumors satisfactorily in that the predicted relationship exists between the heredi- tary and non-hereditary forms of the same tumor, where the gene has been cloned. An exception is the gene BRCA1, having mutations in the germline that account for a signif- icant fraction of hereditary breast cancer, but for none of the non-hereditary examples of this tumor (Futreal et al. 1994). Another exception is provided by the REToncogene, which is heterozygously mutated in the germline in multiple endocrine neoplasia type 2 (Mulligan et al. 1993; Donis- Keller et al. 1993). This is the only 'example so far of a dominantly inherited condition due to mutation in an oncogene. A third exception is hereditary non-polyposis colon cancer (HNPCC), which is caused by mutation in one of several DNA mismatch repair genes, usually MSH2 or MLH1 (Fishel et al. 1993; Leach et al. 1993; Bronner et al. 1994; Papadopoulos et al. 1994). Except for MEN2 and HNPCC, all dominantly inherited cancers for which the genes have been cloned arise in persons heterozygous for antioncogene mutations.

However, the idea that the two events that lead to mutation or loss of both copies of one antioncogene are sufficient to cause cancer is not generally true. The carci- nomas are clear exceptions, as illustrated by colon carci- noma. Familial polyposis coli is caused by germline muta- tion of the adenomatous polyposis coli (APC) gene (Groden et al. 1991; Nishisho et al. 1991). In adenomatous polyps the second copy of APC is mutant or lost, as befits an

antioncogene (Ichii et al. 1992; Smith et al. 1993); this is also true for adenomas in mice that carry a germline mutation of the homologous apc gene (Oshima et al. 1995). There may also be a mutant ras oncogene, but this is apparently not necessary. Adenomatous polyps may fairly be considered as two-hit tumors. However, their transformation to carcinomas requires additional, rate-lim- iting, events. Mutation or loss of both copies of TP53 may constitute such a change (Fearon and Vogelstein 1990), especially since this gene is mutated in few adenomas but in many carcinomas, even intramucosal ones (Kikuchi-Ya- noshita et al. 1992). One possibility is that one TP53 gene may mutate at the adenomatous stage, but progression to carcinoma requires loss or mutation of the second copy of the gene. The rate at which subsequent genetic events occur may be rapid enough for the time to growth of clinically detectable cancer to be very small compared to the time between the appearance of polyps and the trans- formation to carcinoma. In such a circumstance the sub- sequent events are not rate-limiting, even if biologically important. The appearance of carcinoma could be consid- ered as occurring in two broad stages, normal to adenoma- tous polyp, and polyp to carcinoma, with each stage requiring two hits that result in loss of an antioncogene. If the second stage occurs immediately after the first has been accomplished, then it would not be rate-limiting and could be viewed as a two-hit tumor. Such could be the case for retinoblastoma and other apparently two-hit tumors. On the other hand, if hosts regularly died without malignant transformation of the intermediate tumor, then it would be considered a two-hit benign neoplasm.

Proliferation of stem cells: a critical phenomenon in carcinogenesis

Colon carcinoma is enigmatic in that both the APC and the TP53 genes are mutant at a very high frequency, yet germline mutation is dramatically predisposing for the former, but not for the latter. A related question concerns their places in the sequence; why does mutation of APC always precede mutation of TP53? The loss of normal copies of APC obviously results in a tumor, even if benign. Normally the colon behaves as a typical renewal tissue, in which mitosis in a stem cell gives rise to one stem cell and one differentiating cell. The stem-cell population remains constant under normal conditions. During tissue repair this control is evidently relaxed temporarily so that stem-cell mitoses increase in number over time. If such relaxation were continued, even with ultimate differentiation of cells, the total population of cells would increase considerably, as happens in the adenomatous polyps. Mutation or loss of both normal copies of the APC gene leads to such a relaxation of control, producing a benign tumor. This carries an important implication for multi-step carcinogen- esis. As long as the stem-cell population remains constant, there is a low probability that one of them can acquire

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multiple mutations, but a replicating stem cell can accu- mulate mutations much more readily.

The idea that stem-cell proliferation can play an impor- tant role in carcinogenesis is supported by a consideration o f embryonal tumors. For example, retinoblasts proliferate during histogenesis of the retina. A single mutation in the RB1 gene in such a cell would produce 103 mutant daughter cells in 10 doublings, and the first retinoblasts normally undergo more than 20 doublings (Hethcote and Knudson 1978). This situation greatly increases the probability that some cell will sustain a second hit that will render it tumorigenic. Such a tumor may initially be benign, but continued proliferation virtually ensures that other critical, but not rate-limiting events will occur. Mutation of the APC gene may partially convert a colonic renewal stem cell to something resembling an embryonal stem cell. Elucidation of the mechanism of such a change could obviously advance our understanding of carcinogenesis.

The precise role of the TP53 gene in the process is not clear, but it has been shown that this gene can regulate passage from the G1 phase of the cell cycle to the S phase, in a conditional manner (Diller et al. 1990; Kastan et al. 1992). If the growing polyp elicits such a response, its growth will be at least partially regulated by TP53. Muta- tion of TP53 could then be associated with progression. However, in a renewal tissue TP53 loss might not be critical in the presence of an intact APC gene. On the other hand, if stem-cell proliferation is disturbed by chronic ulcerative colitis, the APC mutation might not be important. This disease is associated with a 30% incidence of colon cancer by the age of 60 years (Ekbom et al. 1990), but the tumors have a low incidence (less than 10%) of APC mutations (Tarmin et al. 1995). Mutations in TP53 are still important, since they occur in a high frequency (Harpaz et al. 1994). As with small-cell carcinoma of the lung yet another gene, perhaps alike in some way to that on chromosome 3p, must be mutated. The deleted in colon cancer gene (DCC) is a candidate (Fearon et al. 1990).

It is interesting that the tumors associated with TP53 mutations in the germline in the Li-Fraumeni syndrome often occur in childhood, adolescence, or early adulthood. Breast cancer is the major carcinoma and occurs quite early. Perhaps the normal physiological growth of breast tissue involves stem-cell proliferation that can also be regulated by TP53; if so, loss of such regulation could be tumori- genic. Germline mutation would put this target tissue one step closer to carcinoma. The physiological proliferation of stem cells could render a mutation in a gene like APC unnecessary. Certain embryonal tumors, such as retinoblas- toma, are not a significant feature of the Li-Fraumeni syndrome, and TP53 mutation is seldom if ever, found in these tumors. This suggests that TP53 is not a normal regulator of stem-cell proliferation in this tumor, thereby reducing the number of required events.

Another way in which stem-cell proliferation can be produced is illustrated by multiple endocrine neoplasia type 2, which is caused by mutation of the RET oncogene. The target tissues in this disease, the thyroid medulla and the adrenal medulla, both show hyperplasia of the target cells

(Knudson 1993). This hyperplasia is not neoplastic, but in effect it increases the number of stem cells that can accumulate critical transforming mutations.

Conclusions

I conclude with the thought that the two-hit hypothesis for the origin of some cancers has been useful in that it provided a framework for comparing hereditary and non- hereditary forms of the same cancer, and for considering a mechanism of negative control of growth. Retinoblastoma is a rare tumor of limited importance, but it has served as a useful model in thinking about carcinogenesis. On one hand its relative simplicity was useful for the construction of a model; on the other hand, it is too simple a tumor to equate with the common carcinomas. Yet it provides a foundation for the elaborations necessary to understand the complex cancers. Its origin in an embryonic tissue underscores the importance of stem-cell proliferation in carcinogenesis, helping to explain the specificities of target tissues in the hereditary cancers.

Acknowledgement This manuscript is dedicated to Dr. Haruo Sugano on the occasion of his 70th birthday and celebrates the 60th anniversary of The Cancer Institute, whose reputation for important contributions to cancer research Professor Sugano has done so much to enhance. The author was supported by grants from the Lucille R Markey Charitable Trust, the USPHS (CA-06927), and the Commonwealth of Pennsyl- vania

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