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Appendix A
Workshop Summary
TWO-STAGE MODEL OF CARCINOGENESIS
The goals of this workshop were (1) to assess the scientific basis for the two-stage model of carcinogenesis and (2) to evaluate the possible applications of the two-stage model to the health risk assessment process. Two-stage models are based on the assumptions that carcinogenesis is a multistage process, and that in its simplest form, two critical events are sufficient to convert normal cells to cancer cells (e.g., retinoblastoma in children).
The workshop was opened by the vice-chair, D. Mattison, who welcomed the participants and provided perspective on the relation of this workshop to the overall activities of the Committee on Risk Assessment Methodology (CRAM). The workshop chair, R. Griesemer, emphasized that the workshop is one mechanism through which CRAM obtains information and urged the participants to share additional ideas or information with CRAM after the workshop.
BIOLOGICAL FACTORS IN TWO-STAGE MODELS
A.J. Knudson, who first proposed the concept of two-stage models, presented a keynote address on the evidence from studies of heritable cancers in humans that supports the concept of two-stage models.
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If cancer is related to somatic mutations, there should be some background incidence for all cancers. One would anticipate that there would be an increase in incidence upon exposure to agents that affect this process and that there would be specific targets for those mutations with tissue specificity.
At present we know of two classes of targets, proto-oncogenes and anti-oncogenes (suppressor genes). Where oncogene mutations are found in human tumors, the evidence indicates they may not be the initial events; in some instances, specific translocations seem to be the only identifiable event in the origin of a cancer. The translocations seem to be dominant in the sense that activation of one copy of an oncogene confers malignancy on a cell. In the case of suppressor genes, with release of control of cell growth, two copies must become inactivated and the events can be hereditary or nonhereditary.
Hereditary cancers have provided useful information about the genetic events in carcinogenesis. Virtually every cancer type has a dominantly inherited subgroup. The hereditary fraction for retinoblastoma is rather large (about 40%). The probability of hereditary retinoblastoma in children with an inherited abnormal rb gene is 100,000 times greater than that for the nonhereditary form. The relation of incidence to age suggests that one event is necessary in somatic cells of carriers and two events in nonhereditary cases. Now that the gene has been isolated and mapped on chromosome 13, this suggestion has been supported by genetic analyses of cells from affected children. Somatic mutations depend on the mutation rate per cell division and the number of cell divisions per unit time; retinoblastomas do not develop in adults because the retinal cells have differentiated and no longer divide.
In embryonal tissues such as retina, there is no conditional cell division; a mutation results in a clone of cells carrying the mutation. One can imagine initiation as a loss of one retinoblastoma suppressor gene and promotion as the proliferation that normally occurs in retinoblasts.
Survivors of the hereditary form of retinoblastoma are at risk for other cancers. About 15% of gene carriers develop osteosarcoma. About 95% of patients with osteosarcoma have mutations in both the rb and p53 genes. These two genes are involved in virtually all small cell lung tumors but a third presumed gene on chromosome 3p is also involved in 90% of those tumor cases. A comparison of these three tumors that appear to involve a different number of genes (one for retinoblastoma, two for osteosarcoma, three for small cell lung tumors) sug-
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gests that as the number of genes involved goes up, the relative risk goes down because the inherited gene is a smaller and smaller fraction of the total number of important events.
For adult carcinomas, the picture is less clear. Renewal tissues in which stem cells replicate may have some properties like embryonal tissues and a number of gene changes are being found in a variety of human tumor types (e.g., colon). Still unknown, however, is how many genetic events are required for a particular cancer and what is the meaning of the various events.
R. Maronpot's discussion dealt with oncogenes and cell proliferation from the perspective of an experimentalist. He suggested that the one-hit model may be more appropriate for cancers that arise from exposure to ionizing radiation, potent alkylating agents, or in transgenic mice developing lung cancer, for example, but he admitted that the multiclonal nature of the response indicated that a second event would have to be postulated. The mouse skin tumor model is a well known example of two-stage tumors induced by xenobiotics. He cautioned that the data to which models may be applied may have implied but unwarranted precision.
Illustrations of the importance of the ras-oncogene in the B6C3F1 mouse followed. Liver tumors from mice exposed to vinyl chloride have a high frequency of ras-oncogene activation. Those associated with methylene chloride, trichloroethylene, and dichloroacetic acid are similar in ras activation patterns to nonexposed controls. Ras-oncogene activation is not detected in liver tumors associated with the administration of tetrachloroethylene, chloroform, or phenobarbital. Furan produces some novel ras mutations. Maronpot suggests if specific types of oncogene mutation and activation are found in both animal and human tumors that those findings would be important for risk assessment.
Characterization of the cell proliferative response has a number of pitfalls and limitations. Examples were given where cellular proliferation appears to be an important consideration such as in the kidney with respect to d-limonene and unleaded gas and in the bladder for saccharin, but the hepatic-cell proliferation after methylene chloride increased only slightly at the 12-month interval and not at all at the 3-,6-, and 18-month intervals. There is an important temporal relationship between cancer and cellular proliferation but that by itself is not evidence of a causal association.
During the ensuing discussion, S.H. Moolgavkar assured the audience
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that previous representation of hereditary cancers was consistent with his concept of the two-stage model. Knudson suggested that childhood cancers such as retinoblastoma may represent genetically altered embryonic cells with unconditional cell proliferation. Cancer of the mid-years (osteosarcoma, small cell cancer of the lung, breast cancer) may represent genetic hits—embryonal or not—but with conditional control of cellular proliferation. This control could, in the case of breast cancer for example, be hormonal. Then, it might be that late-age cancers arise from normally dividing tissue in which genetic injury occurs and cell proliferation is enhanced.
TWO-STAGE MODEL OF CLONAL EXPANSION
The next speaker, S.H. Moolgavkar, described the two-stage model of clonal expansion. He noted that from the data presented by Knudson, it is fairly well established that there are two rate-limiting events for retinoblastoma, loss of two antio-ncogenes. For other tumors, particularly adult cancers, the process described by Knudson is more complex, but Moolgavkar stated those data are consistent with their being two rate-limiting and necessary events on the pathway to malignancy. Further, observations of the appearance of tumors in populations of people or animals, providing cell division kinetics are taken into account, are consistent with two necessary steps.
For risk assessment, we need models that relate exposures to the agents of interest to the concentration of the active metabolite in tissue of interest. Secondly, we need models that relate the microdosimetry (interaction of metabolites with macromolecules, for example) with macrodosimetry (tumor formation). Because risk assessment involves extrapolation outside the range of data, the model needs to be at least approximately correct for accurate extrapolation.
Models may have biological or mathematical misspecifications. In describing the Armitage-Doll model and its limitations, Moolgavkar concluded that this model as currently used ignores the fact that cell division and differentiation are likely to be important in carcinogenesis. Also, the waiting time from stage to stage may differ from exponential distributions (mathematical misspecifications likely). Moreover, the
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approximations may be useful for epidemiologic studies but do not hold when the probability of tumor is high as in animal studies.
The Moolgavkar two-mutation model postulates two rate-limiting steps called initiation and conversion, represented by irreversible hereditary transitions from normal cells to intermediate cells to cancer cells. In addition to the conversion rates, each cell population has birth and death processes which affect the clonal expansion rates. The ratio of the death rate to the birth rate is the probability that a fraction of initiated cells does not give rise to foci. Moolgavkar postulates that when more than two events are described, as for skin carcinogenesis in mice or for colon tumors in people, only two events may be necessary for the occurrence of the malignant cell and that the other events simply provide a growth advantage (increasing the probability of transformation by increasing the number of target cells for the second event or increasing progression and metastasis after transformation has occurred). The model has been used for human breast and lung cancers and for retinoblastoma.
In presenting examples of applications of the model (radon and lung cancer in rats; N-nitrosomorpholine and liver foci in rats), Moolgavkar emphasized that with this model the shape of the incidence curve is determined by tissue growth and differentiation in contrast with the Armitage-Doll model where the age-specific incidence curve is determined by the number of stages required for malignant transformation. Both examples provided estimates of initiating and converting (promoting) potencies that can be expressed as the proportionate increase per unit dose over background.
The data needs for application of the model include labeling indices for putative intermediate cells at several time points (serial sacrifice studies). Also needed are better models for the cell-cycles. The model assumes, for example, that cells divide and die with exponential waiting times and that all cells in the intermediate foci are in the active dividing stage.
The planned formal discussion ensued. J. Wilson noted that Drs. Knudson and Moolgavkar had brought together two competing theories of carcinogenesis—that mutations lead to cancer and cancer is an adaptive response. He suggested that the inability of current assays to identify initiated cells and to approximate the increased cell number with sufficient sensitivity would be a continual problem. R.J. Sielken reminded us that the components of exposure assessments are probability
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distributions. For risk assessment he advocated considering the variety of estimates that are generated from the use of several forms of two-stage models. T. Thorslund thought that the two-stage model is a desirable start on a new way of estimating risks in the regulatory process. He indicated that the data required by Moolgavkar are rarely available and that considerable training is required to use the model.
APPLICATION OF THE TWO-STAGE MODEL TO ANIMAL DATA
S.M. Cohen presented the third major address of the workshop based largely on his own research. Cohen initially indicated that he feels that the current bioassay procedure was a good way to screen for carcinogens (he doesn't know a better way), but that it does not provide sufficient information on the mechanism of action to be useful in biological-based cancer risk assessment. He and his colleagues undertook the problem from the engineering simulation approach.
Cohen agreed fundamentally with the two-stage model described by Moolgavkar. The dividing cell has the greatest susceptibility of a genetic mistake and if cancer arises from genetic mistakes the two factors that influence tumorigenesis are increasing the rate at which genetic events occur or increasing the number of times a given cell divides. The initiated and transformed populations of cells can be augmented in size or in proliferative rates by genetic or nongenetic factors.
Noteworthy aspects of the mouse ED01 study of 2-acetylaminofluorene (2-AAF) were outlined. In the liver, the development of cancer is linear, whereas it is not linear in the bladder, but there appears to be a linear dose-response relationship with respect to DNA adduct formation in both the liver and bladder. This occurs because of a difference in the pharmacokinetics related to the development of tumors at the two different sites. In the liver, 2-AAF is metabolized to the N-hydroxyl intermediate and then to the reactive sulfur-containing metabolite. Because the initiated cells or the ''cells in the foci" apparently do not metabolize 2-AAF, only the first genetic event occurs in the liver. There is no compound-induced increase in cell proliferation. In contrast, in the bladder the N-hydroxylation occurs as in the liver, but then an N-glucuronide is formed. This glucuronide is excreted into the urine where it is hydrolyzed and can lead to DNA adduct formation in the urothelium. Thus
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in the bladder, the reactive metabolite causes both initiation and proliferation. Proliferation is only observed above 60 ppm, although DNA adduct formation occurs at dietary concentrations as low as 5ppm. Proliferation in the bladder appears to be essential for tumor formation, however. In conclusion, a two-stage model may be used for either case, but understanding the pharmacodynamics or oncodynamics of the particular tumor-target site is a prerequisite.
Saccharin, which causes bladder cancer in the rat but not in the mouse, hamster, or nonhuman primate, served as an example of a nongenotoxic agent. The male rat is affected to a greater degree than the female. Several types of information were offered to support the importance of proliferation in the development of bladder tumors with saccharin. First, bladder tumors develop principally if the material is given early in life, when cell division in the bladder is normally high. The labeling index (indicator of cell proliferation) is 10 percent at birth, 1.5 percent at seven days, and 0.1 percent at 21 days of age in the rat. Saccharin, co-administered with a cell-proliferating agent (promoter), increases tumor production. Thus, an increase in cell division in the bladder, by irritation as from saccharin or by a promoter, can result in bladder cancer.
The formation of crystals in the urine is the second factor in saccharin induced tumors. The solubility of the saccharin salt is an important aspect in tumor development and can be dependent on the acidity (pKa) of the salt. If the urine is made acid, crystals are not formed in the urine and tumors do not develop. If alkaline, saccharin leads to the formation of silicate crystals in the urine. These crystals irritate the bladder epithelium to cause an increase of cell turnover rate. This increased proliferative rate is considered responsible for increasing the rate of spontaneous mutation and thus for the induction of bladder cancer.
This sequence of events does not occur with significant frequency in the female rat, nor at all in the mouse. In the mouse, the pH of the urine is not changed. Presumably, in nonhuman primates and humans at doses that are not otherwise toxic, the increase in cellular division would not occur. Several other materials, melamine and uracil, that cause an increase in bladder epithelial cell division by forming crystals (although the crystals are of a different nature) were cited additionally to support the apparent relationship between bladder cancer and urinary crystals.
With other nongenotoxic carcinogens, such as dioxin, it is important
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to determine whether they mediate cancer through a receptor mechanism. Such a mechanism can induce cancer by increasing cell proliferation as in the case of a hormonal-related receptor, or it may play a role in affecting the immune system via a receptor site on the human-leukocyte antigen (HLA).
In conclusion, just as an indication of mutagenicity does not necessarily indicate that a material is carcinogenic, neither does the ability of an agent to produce cell proliferation at a high dose indicate that it will be carcinogenic (or carcinogenic at lower doses). Therefore, the determination of the dose-relationship to cellular proliferation is important when considering the likelihood of risk.
A regulator's point of view was presented by W. Farland of the USEPA. He said that risk assessment is not merely the estimation of the risk of cancer. The models presented are useful in considering both the quantitative and qualitative assessment of risk and may lead to the establishment of situational and conditional carcinogenic risks. Conditional carcinogens are those that could cause cancer at some dose, whereas situational carcinogens are those that cause cancer only under certain circumstances. Thus, situational carcinogens are important only if the situations under which they cause cancer are likely to occur as a result of conditions. The issues of benign tumors, target specificity, or other mechanisms of action (e.g., decreased immune surveillance) and anticarcinogenesis may have a place in consideration of the two-stage model. Finally, he indicated that the EPA has considered biologically-based modeling in those few cases where sufficient information is available—particularly in the area of characterization of risk.
C. Barrett followed and commented from the viewpoint of a molecular oncologist on the multiple causes of cancer, suggesting that many mechanisms may be involved. A chemical might cause cancer by inducing a heritable mutation on one critical gene, by inducing heritable epigenetic changes in critical genes, or by clonal expansion of one heritable alteration. Additionally, he suggested that one compound might cause cancer through several pathways.
To emphasize the complexities, Barrett said that if the same processes are operating in humans and rodents but rodent tumors have shorter latent periods, then either there are fewer steps or the rates of transitions from step to step are faster in rodents than in humans. Individual tumors in patients may have anywhere from zero to 10 chromosomes
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showing loss of heterozygosity (loss of suppressor genes); that is, tumors develop individually.
Three ways by which a substance can influence a multistep carcinogenic process are (1) it can induce heritable mutations in the critical genes (directly or indirectly), (2) it can cause heritable epigenetic changes in critical genes, and (3) it can cause one heritable alteration that increases clonal expansion. Substances acting late in the process may be producing secondary mutational events rather than clonal expansion. Adaptation and potentiation must also be taken into account.
Barrett also cautioned us of the difficulties of generating dose-response curves for mitogenesis and of defining mutagenesis. Cell-cycle control genes and genetic instability are as yet little understood but potentially important, as is also transcriptional control. He concluded that cancer is multicausal, multistep, multigenic, and probably multimechanistic.
In the subsequent general discussion, W. North remarked on the richness of possible modeling approaches and Moolgavkar agreed that we should continue to use the old models until we have more experience with the new ones. K. Crump pointed out that the models would be more helpful if we had comparable data in humans.
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Representative terms from entire chapter:
cell proliferation
