Tumor Progression and Homeostasis1

TUMOR PROGRESSION AND HOMEOSTASIS Richmond

T.

Prehn

The Institute for Cancer Research, The Fox Chore Cancer Center, Philadelphia, Pennsylvania

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I. Introduction . . . . . 11. Initiation: The First Step in Tumor Progression . . A. The Clonal Nature of Initiation . . . B. The Latent Tumor Cell or Clone . . C. Induction versus Selection; Genetic versus Epigenetic Change . 111. The Subsequent Steps in Tumor Progression . . . A. The Clonal Nature of Subsequent Steps . . . B. Latency during Progression . . . . . . C. Induction versus Selection in Progression; Genetic versus Epigenetic Change in Progression . , IV. Immunity as a Homeostatic Mechanism . . . A. Introduction . . . . . . B. Induced versus “Spontaneous” Tumors. . . C. Immunological Selection and Surveillance . D. Virally Induced Tumors and Lymphoreticular Neoplasms . . E. Is Subliminal Irnmunogenicity Adequate for Surveillance? . . F. Immunostimulation of Tumor Growth . . . G. Metastasis . . . . . . . H. Conclusions concerning Immunity. . V. Concluding Remarks . . . . . . References . . . . . . .

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1. Introduction

The idea that cancer evolves as a series of sequential heritable cellular changes is quite old. It probably had its genesis in the common cIinical observation that human neoplasia often appears to undergo change during its clinical course, such that what was originally a relatively benign tumor of low grade is transformed over a period of weeks or years into a highly malignant and now rapidly lethal disease. It must be emphasized that what I am discussing is not a change in the size or extent of the tumor, but a change in the biological properties of its cells. Transplantation studies have made it clear that the change resides primarily in the tumor cells themselves, usually not in an altered host. This process ‘This investigation was supported by Public Health Service Research Grant Nos. CA-08856, CA-06927, CA-05255, CA-13456, RR-05539 from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania. 203

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of change with time in the biological attributes of the tumor cells was extensively described in the mouse mammary tumor system (Foulds, 1954), and the term “neoplastic progression” was applied. It is widely believed, although still debated, that all tumors may pass through such a process of progression, perhaps in some cases much condensed, and that possibly no malignancy ever results from a single comprehensive change. The fact that tumors undergo progression over a time course that may extend over many years and numerous steps, implies that at each step of the process there are homeostatic mechanisms operating with considerable but imperfect success to limit or reverse the process. Unfortunately, these postulated mechanisms are mostly unknown while some that have been investigated extensively may prove to be of relatively minor importance. In this paper I will largely limit my discussion to those areas of tumor progression and homeostasis actually impinged upon by my own work or that of one or more of my students or postdoctoral associates, in particular, Dr. E. Andrews, now at Cornell; Dr. G. Bartlett, now at Hershey; Dr. M. Basombrio, now at Buenos Aires; Dr. M. LappC, now at Hastings on Hudson; Dr. G. Slemmer, now at Vancouver; and Dr. H. Outzen, still associated with me in Philadelphia. 11. Initiation: The First Step in Tumor Progression

A. THECLONAL NATUREOF INITIATION According to Willis ( 1960), cancer arises as the result of cellular changes over a large area or field. This conclusion was the result of clinical observation, and the accuracy of the observations is beyond question. There can be no doubt that clinical cancer often appears to arise over a wide area rather than focally, and if focal, may sometimes be at least highly multifocal. A striking example of this is the cancer that sometimes arises in an old burn scar. Sometimes the entire broad area of the scar is occupied, seemingly all at once, by the cancer tissue. However, despite this common clinical observation, there are many experimental data showing that, regardless of appearances, the essential nature of the process is clonal. Cancer is usually the result of the clonal amplification of a heritable alteration, which occurred in a single cell. The evidence for this statement is quite extensive and persuasive. Some of the best data concerning the clonal nature of neoplasia arises from a study of glucose-6-phosphate dehydrogenase ( G-6PD ) polymorphisms and their occurrence in neoplasms. This enzyme occurs in several isomeric forms determined by a structural gene on the X chromosome.

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It follows, since one X is suppressed in somatic cells, that in heterozygous females, approximately half of the somatic cells will express one form of the enzyme, the other half will express the other. Consequently, if such a female develops a tumor, all the tumor cells, if of monoclonal origin, should exhibit one and the same isozyme. If the tumor had originated from multiple normal ancestor cells, both isozymes would be expected to be present. A variety of tumors have been examined, and the answer has usually been clear; they are, with few exceptions, monoclonal ( Fialkow, 1972). The basically monoclonal nature of a malignant neoplasm is also supported by the fact that all the neoplastic cells can sometimes be shown to carry other markers indicating a common neoplastic precursor cell, for example, an atypical chromosome that is unlikely to have arisen in multiple ancestors. In chemically induced tumors, each tumor can be shown to be antigenically unique and non-cross-reactive, but all the cells of each individual tumor do cross react. Again, this seems to be strong evidence for the clonal origin of each tumor (Prehn, 1970). Neoplastic transformation in tissue culture can frequently be visualized directly as a very focal and almost certainly clonal process. Also, when “initiating carcinogen is painted on the skin of a susceptible mouse followed by a “promotor,” numerous neoplasms can be produced in each animal, each arising as a small punctate lesion with no apparent involvement of intervening areas of skin (Berenblum, 1954). A similar picture is seen in mouse breast oncogenesis. In response to chemical oncogen or virus, numerous small focal hyperplastic lesions are produced throughout the mammary gland with no apparent alteration of intervening tissue (Pullinger, 1952; Bern et aE., 1958). A similar pAttem is seen in chemically induced hepatoma formation (Farber et al., 1975; Kitagawa and Pitot, 1975). Although the evidence is strong that neoplasia is basically a clonal process, it must be recognized that if multiple clones of transformed cells were originally present in a field of tissue, the resulting gross tumor might still be derived largely from only one of them. The growth rates of the clones could hardly be identical, and therefore the most rapidly proliferating would soon predominate in the lesion. It is probable that tumors induced in high frequency by strong carcinogens may often begin as a multiclonal disease which rapidly, by selection, becomes essentially monoclonal (Prehn, 1970). Direct evidence of this was obtained by an analysis of mouse sarcomas induced by the subcutaneous implantation of paraffin pellets containing the carcinogen 3-methylcholanthrene ( Prehn, 1970). Samples from widely separated areas of primary tumors were propagated as separate

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sublines by transplantation in syngeneic mice. These separate sublines were tested as to whether or not they were of common origin by examining their antigenic specificities. It is known that chemically induced multiple primary tumors are very seldom cross-reactive ( Basombrio, 1970; Globerson and Feldman, 1964). The question was whether or not the tumor sublines would behave in the manner of independently derived tumors, and each be antigenically specific. It was found that thesublines isolated from the primary tumors did indeed occasionally show individuality in antigenic specificity in the manner of independently derived tumors. It can be inferred that, because of the vagaries of the sampling procedure, the percentage of primary tumors of multiclonal origin must actually have been quite high. In contrast, in no instance could non-cross-reactive subclones be isolated from subsequent tumor-passage generations ( Prehn, 1970 ) . If initiation, or the first heritable change in the cells, is essentially of the focal, clonal nature I have described, why did Willis and other observers arrive at the opposite conclusion? Several possible explanations can be offered, any one or more of which may have obtained in any given instance. The first explanation has already been discussed, namely that some tumors, under conditions of strong carcinogen application and/or high host susceptibility may indeed begin as multifocal lesions or among many cells in a field. This may be the case in some neoplasms of the breast in which multiple primary tumors in one susceptible individual are not rare. The second explanation of the apparent arisal of a tumor by change of many cells over a large area lies in the effect of neoplastic cells on normal cells. It was pointed out by Ewing (1940) that cancer cells could impose a neoplastic appearance on surrounding normal cells (collateral hypertrophy). Experimental verification of this conclusion was provided by Argyris and Argyris ( 1962), who inoculated a transplantable mouse tumor into the dermis and showed that the normal epithelium above the transplant, although not in direct contact with the tumor, became hyperplastic. Appropriate controls established that the effect was not due to mechanical stress, but to some diffusible influence. The third possible explanation for the seeming arisal of some tumors over a large area has recently been experimentally documented by Slemmer (1974). He demonstrated that, under some circumstances in mouse breast oncogenesis, already heritably abnormal cells could apparently migrate through the ductal tree while still maintaining a grossly normal appearance, and their subsequent growth would give the illusion of tumor arisal simultaneously over a wide area of the tree. One caution must be expressed concerning the conclusion that there

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is no continuous recruitment of normal cells into tumor cells in the manner proposed by Willis ( 1960). Transplantation studies in genetically controlled mice make it clear that, in a growing tumor, cancer cells are derived from cancer cells, not from the cells of the normal host, but this rule is violated in the case of some tumors of viral etiology in which the cells are productive, i.e., are actively shedding the oncogenic virus into the cellular environment ( Siegler, 1970).

B. THELATENT TUMOR CELLOR CLONE DeCossk has described a function of the immune reaction which holds target cells reversibly in a nondividing state (DeCossk and Gelfant, 1968). Other instances are common in which only a part of the neoplastic phenotype is unexpressed, the proliferative aspect, but the cell or clone remains recognizable morphologically as neoplastic. Perhaps the most dramatic of such examples is provided by mammary tumor development in the mouse. The literature on the mammary tumor of the mouse is very extensive, and its review here would simply detract from the points to be made. An entrk to this literature can be found in a recent publication by Slemmer (1974). The experimental technique that permitted the nature of the latency phenomenon in this system to be explored was provided by the transplantation methods developed by DeOme et al. (1959) and his colleagues. A brief description of this technique and its application is required to understand the conclusions that have been reached regarding latency. The mouse mammary gland develops by the growth of a ductal tree beginning at the nipple and extending into a well defined and circumscribed subcutaneous fat pad, the mammary fat pad. Prior to puberty, the gland consists of a rudimentary ductal tree that extends only a few millimeters from the nipple. At puberty, a rapid proliferation of the ducts begins by growth at terminal end buds, and the ducts rapidly extend into and through the fat pad, until the pad is well filled by the branching ductal tree. Attempts to transplant and obtain growth of ducts in other areas of the body fail, although some growth may be obtained in other fatty tissues, such as the “brown fat.” Transplantation of the entire gland-containing fat pad was achieved by Prehn (1953) and subsequently in the rat by Dao et al. (1964). Transplantation with subsequent growth is readily obtained if, prior to puberty, a fragment of duct is placed in a distal, and thus gland-free portion of the mammary fat pad of a syngeneic female. Transplantstion with growth can also be achieved when a ductal fragment is placed in the fat pad of an

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adult syngeneic female recipient if that fat pad had previously been “cleared of endogenous mammary gland. Clearing is accomplished by the excision of the nipple area and the adjacent mammary rudiment prior to puberty. A fragment of duct placed in an “uncleared” fat pad, i.e., a fat pad already filled with endogenous gland, fails to grow. When oncogenesis is produced in the mouse mammary glands by feeding chemical oncogens, by action of the mammary tumor virus, and/or by hormonal treatment, the initial lesion appears to be a small benign tumor, the so-called hyperplastic nodule. Numerous focal lesions of this type can often be visualized scattered throughout the ductal tree. They grow to a size of one to several millimeters, and then cease to enlarge. This state of dormancy or latency usually persists for long periods, perhaps throughout the entire life of the mouse. Whether or not they may sometimes regress is not known. It is only rarely that one of these lesions undergoes progression and acquires the capacity for renewed growth. The transplantation procedures of DeOme have permitted the nature of this dormant or latent period to be explored. If a hyperplastic nodule is transplanted to an uncleared fat pad, the transplant will persist indefinitely and usually not grow, just as it would have done had it been left in situ. However, when transplanted to a cleared fat pad, the hyperplastic nodule grows and eventually fills the fat pad with an abnormal hyperplastic grand (benign tumor). Such transplanted tumors usually grow slowly in the cleared fat pad and, like normal‘gland, will not grow beyond the confines of the pad. Such transplantation studies, showing that the nodule will grow only in cleared fat pads, make it apparent that the heritably abnormal cells of the hyperplastic nodule are, like normal gland cells, inhibited from growth by the surrounding normal ducts. They can thus remain latent for extended periods, until further progression endows them with the capacity to overcome the inhibition. The nature of the inhibitory principle, apparently elaborated by the normal ducts, is completely unknown. It does appear, however, that a major distinction between the normal and the hyperplastic nodule tissue is that the former both elaborates and responds to the inhibitory principle; the hyperplastic nodule usually responds to, but does not elaborate the principle. The recent work of Slemmer (1974)has added a new dimension to the study of the growth potential of the hyperplastic nodule. His analysis has suggested that the mammary gland is probably composed of three distinct lineages of epithelial cells: the ductal, the alveolar, and the myoepithelial. The initial neoplastic change may occur in any one of these three. The most interesting finding was that, in the early hyper-

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plastic benign lesion, the neoplastic cells were often associated with one or both of the other normal epithelial cell types. This association was obligatory, the neoplastic component being unable to grow in a cleared fat pad, except in association with the normal population. Since Young et al. (1971) have shown that normal mammary ducts grow poorly when transplanted to old, endocrine-deficient animals, it is possible, although still unproved, that the growth of an early breast neoplasm may sometimes be endocrine-dependent because of the endocrinedependency of the associated and necessary normal cell component. Latency in the breast tumor system is apparently not influenced by the immune mechanism-immunity in this system will be discussed at some length in Section IV. The problems connected with initiation, latency, and progression have been extensively examined in oncogenesis of mouse skin, particularly in the case of chemical etiological agents. The work of Rusch and Kline ( 1946), Rous and Kidd ( 1941), and subsequently of Berenblum and Haran ( 1955) established the so-called “two-stage hypothesis.” If a chemical oncogen, for example an oncogenic hydrocarbon, is applied to the skin of a mouse in a suboncogenic dosage, the skin will appear overtly normal after the initial inflammation, and consequent generalized hyperplasia, has subsided. However, despite the lack of overt changes, a profound and long lasting change has, in actuality, occurred. If, at some subsequent time, which may be as long as a year or more, a second similarly small dosage of the oncogenic chemical is applied, neoplasia may rapidly ensue. In other words, the effects of the chemical oncogen are cumulative even when the interval between applications is very long (Boutwell, 1964). Berenblum postulated that the invisible change represented by “initiation” was due to the presence in the oncogen-treated skin of “latent tumor cells” ( Berenblum and Haran, 1955). These could subsequently be made to proliferate and develop into gross neoplasms by the second application of the chemical, in this case, acting as a “promoter.” The reason for believing that the first application of oncogen produced latent, rather than some type of defective or incomplete tumor cells, was that promotion, i.e., the second step, did not apparently require a chemical oncogen. In the classical system, initiation was produced with a subthreshold dose of a hydrocarbon oncogen; promotion was accomplished by the repeated application of the highly irritating substance, croton oil. Croton oil alone, in any dosage, was only marginally oncogenic, and if the experimental procedure was reversed, i.e., if the croton oil preceded the hydrocarbon, few if any tumors were produced.

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The interpretation that initiation produced complete latent tumor cells was reinforced by the work of Lapp6 (1968), who showed that a very effective promoter was simple skin grafting. Apparently all that was required for promotion was the production of compensatory hyperplasia in the skin that was previously initiated. As in the case of Berenblum’s work, if the skin grafting preceded the oncogen by an interval sufficient that the hyperplasia had subsided to the normal resting state, no tumors were produced. A variety of other forms of irritation, such as simple wire brushing, will also serve as a promoter (Deelman, 1927). However, promotion is apparently not cumulative unless the applications of promoter are made at short intervals (Boutwell, 1964). It certainly does seem that initiation and promotion may be qualitatively distinct processes. It perhaps should be noted at this juncture that there is a difference between promotion with a complete oncogen, an agent that can serve as both initiator and promoter, and promotion with a pure promoter-an agent, such as skin grafting or croton oil, which has little or no capacity to initiate. In the latter case, the great majority of the neoplasms (SO-SO%) are benign papillomas which undergo spontaneous regression after a few days or weeks. If a potent oncogen is used for both steps, the total number of lesions may actually be less, but a higher percentage of them will be carcinomas that can grow progressively and kill the host (Shinozuka and Ritchie, 1967). Although many chemical oncogens may both “initiate” and then “promote” the growth of previously transformed cells, there is a class of agents, typified by urethane, the members of which have the power to “initiate” or transform, but little or no capacity to promote. Studies of urethane oncogenesis in the skin of the mouse have led to the conclusion that urethane is nearly, but not completely, a pure initiator (Salaman and Roe, 1953). Studies in my laboratory (R. T. Prehn, unpublished) have shown that a similar situation exists in the mouse lung. Lung adenomas can be induced by urethane, but the growth of already transformed cells or clones is not promoted. In contrast, a hydrocarbon oncogen, given by mouth, both initiates and promotes the adenomas (R. T. Prehn, unpublished). Lappe (1969) was able to show that at least some benign papillomas initiated by a hydrocarbon oncogen and promoted by skin grafting, were serially transplantable. The techniques are not yet available to permit analysis of the detailed type accomplished with the hyperplastic breast nodules of the mouse, but the possible role of immunity as a homeostatic mechanism has been explored. The available data suggest that, as in the breast, immunological surveillance does not operate in

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this system except under certain conditions which will be discussed in Section IV.

C. INDUCXION VERSUS SELECIION; GENETICVERSUS EPICENETIC CHANGE When a chemical oncogen is painted on the skin of a mouse, punctate focal lesions arise, surrounded by apparently normal intervening skin. Likewise, when an oncogenic virus is added to a culture of normal embryo target cells, scattered foci of transformation appear. The percentage of target cells so transformed is limited regardless of the multiples of viral units applied. One reasonable explanation of these data may be that, for one reason or another, only a minority of the cells exposed to the oncogenic agent have the capacity to give rise to a transformed clone or tumor (Stoker and MacPherson, 1961). In the case of chemical oncogens, I advanced the theory several years ago that the chemical might be not really a transforming agent, but merely a selector of transformed cells already present in the population prior to application of the chemical (Prehn, 1964). The theory was based upon the observation that normal cells are regularly much more susceptible to the toxic effects of chemical oncogens than are neoplastic cells (Alfred et al., 1964). Thus, application of an oncogen would provide neoplastic cells with a great competitive advantage and perhaps liberate them from the inhibition of surrounding normal cells, an inhibition discussed in a previous section. However, subsequent work by Huberman and Sachs (1966) and by DiPaolo et al. ( 1969, 1971) was, in some cases, able to separate oncogen toxicity from tumorigenicity and Heidelberger (1973) was able to show that under ideal conditions 100% of target tissue culture cells could be transformed. These experiments suggest that the chemical oncogen, like the oncogenic virus, when it produces transformation, does something more than merely select previously existing variants. Although the theory that a chemical oncogen selects only previously existing variants now seems untenable, the word “only” needs emphasis. That the chemical (and perhaps in some cases the virus also) exerts a selection pressure in favor of the transformed cells, cannot be doubted. Not only are the chemicals and viruses often lethal to normal cells, they may also depress the immunological defenses of the host and so favor the proliferation of transformed, but antigenic, clones ( Malmgren et d.,1952). The fact that under most circumstances only a small portion of a target population is susceptible to induced transformation still needs explanation. While other explanations are certainly possible, I venture

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to suggest that perhaps the cells preferentially capable of responding to some oncogenic agents may be those that have already undergone the first of the sequential steps in tumor progression. These variants may already be “primed” and have fewer steps remaining before they manifest transformation or tumorigenicity. Thus, for example in the experiments of Heidelberger ( 1973), so-called “initiation” by a chemical agent may not really be the true first step. If the oncogen preferentially works on already variant cells, the widespread correlation between an inbred mouse strain’s spontaneous incidence of a particular tumor type, and its susceptibility to carcinogeninduced tumors of the same type would be explained. For example, strain A mice are very susceptible to spontaneous lung adenomas; the incidence is vastly increased by a chemical oncogen. In such cases the oncogen seems to be merely accelerating a process that would have occurred without it, and it is easy to think of the oncogen as acting upon cells already “determined to develop into tumor. Also, it seems evident that the true initial step may, in the case of some congenital tumors, be present in the germ line (Knudson, 1974). Thus, in summary, it appears that the initiation of tumor by chemical oncogen or virus involves the induction of heritably stable variants, but that the “inducer” may often act preferentially on cells already predisposed to undergo the further changes leading to actual tumor formation. Actual examples of this have recently been described (Naha and Ashworth, 1974; DiPaolo et al., 1969,1971 ). Whether or not the variant clone that eventually gives rise to tumor, originates by genetic or epigenetic change, i.e., by mutation or abnormal differentiation, cannot yet be determined. The two mechanisms are not mutually exclusive. The arguments in favor of abnormal differentiation have been well summarized by Pierce (1974). They seem to depend upon whether or not those tumors that sometimes resolve by differentiation to a normal state (crown gall in plants, teratocarcinoma in mice, and neuroblastoma in children) are representative of tumors in general. In this connection it should be recalled that mouse skin papillomas usually regress. Lapp6 has occasionally been able to observe a thin monolayer of apparently normal epithelium around the residual keratin pearls that mark the site of such a regression (M. A. Lappk, unpublished). Farber et al. ( 1975) has also tentatively described the apparent resolution, by a return to a normal morphology, of the oncogen-induced hyperplastic foci of incipient hepatomata in rats. In my laboratory, Outzen et al. ( 1975b) has observed, in a chemically induced frog “sarcoma,” sequential changes from an undifferentiated malignant lesion to a benign neuroma.

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111. The Subsequent Steps in Tumor Progression

A. THECLONAL NATURE OF SUBSEQUENT STEPS

In the previous section, it was pointed out that whether or not the initial step in the oncogenic process was clonal was often not clear. The situation with regard to the subsequent steps in tumor progression is much less ambiguous. The mammary tumor system of the mouse is probably the system in which the events of progression can most easily be observed and analyzed. Progression in mouse tumors was extensively analyzed by Foulds (1956a-d) and the basic features were described in a classical series of papers. Most notable was the fact, pointed out by Foulds and others, that a mouse breast neoplasm often exhibited a variety of morphologies in various parts of the lesion. These variant areas were often homogeneous spheres of tissue which could hardly be considered as anything but proliferative clones. Foulds thus came to the conclusion that tumor progression was due to the selection of a large number of essentially independently assorting characters. That the variants observed in different parts of a tumor were indeed heritably stable subclones was shown, in a different tumor system, by Henderson and Rous (1962). These investigators fragmented tumors of mixed morphologies into small pieces. The pieces were transplanted individually and each was shown to possess a marked tendency to propagate the morphology of the particular parental fragment. The work in the mouse mammary tumor system makes the same points even more elegantly ( Slemmer, 1974). As was already described, hyperplastic nodde tissue transpIanted to “cleared fat pads propagates to fill the pad forming a so-called “hyperplastic outgrowth line.” Several different morphologies are seen. The striking observation is that further variants can be observed grossly to arise as punctate lesions in the hyperplastic outgrowth line. These variants can be isolated and similarly propagated and breed “true.” The frequency of arisal of such variants, which in some cases are malignant, is a characteristic of the particular hyperplastic outgrowth line, but their appearance is essentially a random process, both with regard to time and location.

B. LATENCY DURING PROGRESSION In Section I1 it was pointed out that the variant clone that initiates the neoplastic process may remain latent, as in the “latent tumor cells” of initiated skin, or as in the hyperplastic nodules of the mouse breast which remain in a stationary growth phase for very extended periods

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of time. It is also clear from the work of Slemmer (1974) that variant subclones, the product of tumor progression, may also enter similarly extended stationary periods. When a new variant arises in the breast, its growth is determined by its response to the “spacing factors” of the normal breast. To recapitulate, the normal ductal tree apparently elaborates some principle that inhibits normal ductal growth. This principle, which serves as a ductspacing factor during the growth of the normal ductal tree, also usually inhibits the growth of new variant clones of breast epithelial cells. The new variants seem to respond to the factor, but not to elaborate it themselves. They are therefore inhibited whenever they come too close to normal ducts, and their size is thus limited to a few millimeters. When this constraint is removed by transplantation to a cleared fat pad, growth ensues. New variant subclones which can be isolated from the hyperplastic outgrowth line are often malignant, i.e., they would not be constrained by the presence of normal ducts, were such present, and they will grow outside the fat pad. However, in other sublines, varying degrees of the capacity to override the inhibition of the spacing factor are encountered. In the untransplanted hyperplastic nodule, no further variants will be noted until one occurs that can override, at least to some extent, the inhibition of the spacing factor. Other variants that might arise will not grow and are probably irrelevant. It should be noted that variant formation in the growth-inhibited in situ hyperplastic nodules is probably low compared with that in an outgrowth line in a cleared fat pad, simply because the amount of tissue at risk is comparatively small. On the basis of what has been learned about progression in the mouse breast tumor, an explanation can be offered for the common observation of prolonged latency in human breast cancer after surgery. It is sometimes observed that after mastectomy, a patient may occasionally remain well for periods as long as 15 years, only to eventually develop a recurrence of tumor in the operative scar. It is usually thought that some peculiarity of the local environment changes at that time to permit the growth of the surgically seeded tumor cells. I think it perhaps more likely that the explanation lies in tumor progression. I have pointed out that the early hyperplastic lesions in the mouse cannot grow outside the environment of the fat pad, and for this reason we term them benign. I have also pointed out that malignancy arises as the result of further variation in these early lesions. Some of the hyperplastic outgrowth lines that cannot grow or grow poorly outside the fat pad would be diagnosed, with justification, as malignant by the pathologist-he has learned by experience that such lesions can

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be expected to metastasize. I suggest, however, that the true state of affairs may be that these lesions are in themselves often benign at the time of examination, but can be expected to give rise to variants that are indeed malignant and which are the elements that actually grow in a metastasis. The pathologist is thus making a forecast, rather than necessarily diagnosing the actual present state. The cells the surgeon accidentally seeds in the scar or the cells that seed naturally in distant organs to remain latent for many years may not be, and perhaps never were, endowed with the capacity to grow to any appreciable extent outside the fat pad. They therefore remain latent until further random progression occurs among them. This concept of the long latency of some metastatic human tumors, while I think quite reasonable in view of the lesions of the mouse, is entirely speculative. It is unfortunately a somewhat pessimistic theory inasmuch as it suggests that prolonged latency and subsequent metastatic recurrence may be innate functions of random changes in the tumor cells themselves, not in the host.

C. INDUCXION VERSUS SELECTION IN PROGRESSION; GENETICVERSUS EPIGENETIC CHANGE IN PROGRESSION I have pointed out earlier that the sporadic, random, clonal nature of the later steps of tumor progression seems very clear, at least in some systems, and is probably more firmly established than is true in the case of the initial event of the oncogenic process. In the initial event, the question of whether an oncogenic agent merely selected from among previously existing variants seems to have been answered in the negative. It will be recalled from the earlier sections, that the oncogen not only selects, but the evidence is good that it also can induce change or transform. This same question concerning induction versus selection has been asked in reference to the later steps of tumor progression. Some of the earliest and most definitive work was reported by Klein and Klein (1956). This work showed that the change in a mouse tumor from the solid to the ascitic form was due to a relatively stable, heritable, alteration in the tumor cells. If a number of tumor sublines were serially transplanted and exposed at intervals to the selective pressure of the intraperitoneal environment, conversion to the ascitic form was observed to occur at a very variable and unpredictable pace among the different sublines; in other words, it appeared to be a random process. The conclusion was thus made that the change must have depended upon the sporadic arisal of stable variants in the tumor cell population, and that these arose independently of the selective pressure of the peritoneal location. A similar conclusion was reached in another system by Law

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(1954), who applied a modified fluctuation test to another form of tumor progression. It seems clear from the preceding that tumor progression is due, at least in some cases, to the random appearance in the neoplastic cellular population of hereditarily stable variants with a selective advantage. This conclusion is reinforced by the observation of Lappk that a mouse skin papilloma possesses throughout its life a constant probability of progressing to malignancy. In the absence of further oncogen treatment, the number of malignancies is directly determined by the “papilloma days” that the mouse is at risk (Lapp6 and Prehn, 1969). The variants probably arise spontaneously and may be selected for by a carcinogen. There seems to be no critical evidence to show that an oncogen or other environmental change actually induces later steps in progression; an oncogen’s action in progression, in contrast to initiation, could be the pure selection of spontaneous variants. It should be noted that few systems have been critically examined by a form of fluctuation test (Luria and Delbruck, 1943), which is often difficult to perform in the context of tumor progression. However, there is some evidence to suggest that a potent oncogen can do more to influence progression than merely to select. The evidence suggesting induction, as well as selection, by an oncogen, of the successive steps in tumor progression is derived from both skin and breast. In the mouse skin, application of a carcinogen in low dosage followed by a nononcogenic promoter leads to the production of numerous benign papillomas. Larger dosages of the carcinogen result in more of them being malignant (Shubik, 1961). These facts could be nicely explained if one postulated that the carcinogen actually induces the successive steps in tumor progression (they can also occur spontaneously but more slowly) and that each successive step occurs with greater frequency or probability than the preceding one. There thus tends to be an induced cascade effect in transformed clones. Coupled with this; the carcinogen tends to be toxic to more normal cells (Alfred et al., 1964), and therefore some potential tumor clones might be eliminated by a high oncogen dose at a very early stage of progression, Thus, the net effect of these two processes, induction and selective toxicity, would be to favor, at high doses of oncogen, fewer lesions but those of higher malignancy. A similar situation may occur in the breast. If the oncogen is applied directly to the gland (high dose?) only malignant carcinomas, rather than hyperplastic nodules, are produced (Sinha and Dao, 1975). The situation is thus quite analogous to that in mouse skin and would be difEicult to explain if the subsequent steps in progression were due to mere selection of spontaneously occurring variants.

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I have already discussed the question of whether or not “initial” transformation by an oncogen is really the first step or whether the oncogen preferentially acts upon cells already abnormal. If the latter, then induction of the later steps of tumor progression seems established. Support for this idea comes from the observation by Basombrio and Prehn (1972a) that mildly tumorigenic 3T3 cells can be further transformed by a chemical oncogen. There is one example of neoplastic progression in which the mechanism of selection of previously existing variants is almost certainly not adequate to account for the observations. I refer to what is called the Barrett-Deringer phenomenon ( Barrett and Deringer, 1950). The phenomenon involves a change in the ability of a transplanted tumor to grow despite a moderate histocompatibility barrier. In the original experiment, a tumor that arose in an inbred mouse strain was transplanted to an F, hybrid formed by outcrossing that strain. The other parental strain forming the F, was quite resistant to the growth of the allografted tumor. The tumor, of course, grew well in the F, in accord with the “laws of transplantation.” After passage in the F,, the tumor was transplanted into resistant backcross mice, i.e., mice produced by crossing the F, with the tumor-resistant parental strain. Owing to Mendelian segregation, some backcross mice would be expected to grow the tumor, and others not. In fact, the extent of the growth of a tumor in resistant backcross mice has been used as a measure of the degree of histocompatibility difference between the tumor and the resistant allogeneic inbred strain (Prehn and Main, 1958). The Barrett-Deringer phenomenon consists of the observation that the percentage of resistant backcross mice growing the tumor was increased when the tumor was previously passaged through the F,, rather than transplanted directly from the syngeneic host. The alteration in the tumor was stable; once the change had occurred, passage of the tumor in the strain of origin did not restore the original transplantation characteristics. The observation has been repeated with a number of different tumors with essentially the same result, although with some tumors the change may be in the opposite direction, i.e., less growth in the back-cross animals (for review, see Klein and Klein, 1957). The Barrett-Deringer phenomenon seems to be an example of tumor change or progression that may not be explicable on the basis of the selection of preexisting, sporadically occurring, cellular variants. It has been shown that a very brief sojourn in the F, is all that is necessary for the full manifestation of the effect, and it is also independent of the number of tumor cells exposed to the F, environment ( a very small inoculum of tumor undergoes the change equally as well as a large

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inoculum, suggesting that the effect is not due to the existence in the inoculum of a small number of preexisting variant cells). The change is very constant and reproducible with no detectable element of sporadicity. It thus appears that the change is in the nature of a rapid adaptation of the tumor cell population, not a selection of randomly appearing cellular variants (Klein and Klein, 1957). It is still possible, however, that the adaptive change is induced in only a small portion of the tumor cells rather than in most of the population. The nature of the adaptation-inducing mechanism in the Barrett-Deringer phenomenon is not known. However, it has been shown by Klein that if the tumor cells are within the protective confines of a diffusion chamber ( Algire et al., 1954) while in the F, host, the adaptation still occurs (Klein and Klein, 1957 ). The change is thus not dependent upon cell-to-cell interaction within the F,. A further observation by Sanford (1965) was that the tumor change in the F, did not take place if those animals had been immunologically crippled by X-irradiation. In her work, the change observed, as a result of F, passage, was a decreased rate of tumor growth in the strain of origin; the effect on growth in backcross mice was not tested. As far as it has been possible to analyze progression in the autochthonous host, the observations favor the theory of the selection of random variants. However, the Barrett-Deringer phenomenon, produced by exposure of the tumor to a grossly foreign environment, seems to demonstrate that host-induced stable adaptations may occur in a tumor cell population. It may be well to remember that most of the changes associated with organ differentiation in normal ontogeny are presumably due to some form of induction rather than the selection of randomly occurring variants. IV. immunity as a Homeostatic Mechanism

A. INTRODUCTION The role of immunity as a defense against the growth of cancer cells has been the subject of intense investigation for nearly 20 years. During that period the prevalent opinion has varied from “it has no role” to “it is the major defense” and now perhaps back again to “it is a homeostatic mechanism of questionable importance.” Certain it is that 20 years of endeavor have left the role of the immune mechanism in cancer more controversial than ever. The possibility that immunity might play a major role in cancer stems

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from two observations. The first was that certain tumors in animals were caused by infectious agents, viruses; immunity certainly must play a role in controlling such agents. Second, there was the observation that most, perhaps all, tumor cells were at least potentially antigenic in the animal of origin (Main and Prehn, 1957). It is only this second observation that leads to hypotheses concerning the role of immunity as a homeostatic mechanism in relation to the cancer cells per se. It is only this role that has been of immediate concern to my laboratory and that will be examined in what follows. This is not intended in any way to minimize the possible importance of the immune mechanism in controlling cancer by controlling the spread of oncogenic viruses. There is little merit in reviewing the entire field of tumor immunology; this has been done by a number of authors (Haughton and Amos, 1968; Hellstrom and Hellstrom, 1969; Klein, 1973; Baldwin, 1973; Herberman, 1974). Rather, I shall confine myself to an examination of a few controversial issues, to which my laboratory has contributed, and discuss the implications of the findings in relation to the broad field of tumor homeostasis.

B. INDUCED VERSUS “SPONTANEOUS” TUMORS The first point to be made is that there is a profound immunological difference between so-called “spontaneous” tumors and those induced in the laboratory by chemical, viral, or physical means. It appears that, virtually without exception, those tumors that appear sporadically with low incidence and without known cause have little or no capacity to effectively immunize animals syngeneic to the animal of origin. Mice “immunized with such tumors usually grow a challenge inoculum of that same tumor as well or nearly as well as do the nonimmune controls. Immunization with these tumors produces little evidence of immunity effective against the growth of the challenge tumor cells; whatever immunogenicity they may exhibit is relatively weak (Main and Prehn, 1957; Baldwin, 1966; Hammond et al., 1967; Peters, 1975; RCvksz, 1960). The situation with regard to induced laboratory tumors is usually quite different. If tumors are induced, for example, with a standard oncogenic dose of 3-methylcholanthrene such that most of the animals will become tumorous within 3-6 months, most of the tumors can be shown to be immunogenic in mice syngeneic with the animal of origin (Main and Prehn, 1957). Such mice, immunized with tumor cells, are often highly resistant to the growth of a challenge inoculum. The immunity, in the case of chemical induction, is highly specific, being limited to the particular immunizing tumor and usually not cross-reactive with

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other tumors even of the same etiology, histology, organ, or animal of origin. The immunogenicity, as measured by the degree of resistance to the growth of the challenge inoculum, is very variable from tumor to tumor, ranging from nondetectable in some cases to nearly absolute immunity in others (Main and Prehn, 1957; Prehn, 1960; Old et d., 1962; Bartlett, 1972). I have recently been able to show that in the case of 3-methylcholanthrene (MCA) the average immunogenicity of the induced tumors is directly related to the concentration of the chemical (Prehn, 1975a). As the concentration was reduced, the tumor incidence declined, the average latency before tumor appearance lengthened, and the average immunogenicity decreased even when the latency was held constant. The more the concentration was reduced, the more these induced tumors modeled, as far as immunogenicity is concerned, the so-called spontaneous. The relationship of immunogenicity to dose of oncogen and to latent period suggest that the characters immunogenic and tumorigenic arise as independent variants. This idea is further supported by the fact, previously mentioned, that lines of 3T3 cells that are more tumorigenic in uiuo than are normal cells, can, nonetheless, be transformed by a chemical carcinogen. Each clone, separately transformed, produces a tumor of different antigenic specificity ( Basombrio and Prehn, 1972a). Thus, it is quite clear that the initial change which makes 3T3 cells more tumorigenic than are completely normal cells occurred prior to the antigenic transformation produced by the chemical.

C. IMMUNOLOGICAL SELECTIONAND SURVEILLANCE Since it is known that most oncogens, and in particular MCA, are iinmunodepressants ( Malmgren et al., 1952; Prehn, 1963; Stjernsward, 1967), one could entertain the hypothesis that the above facts support the concept of immunological surveillance ( Burnet, 1970). Thus, with a high concentration of chemical, immunodepression would permit the growth of highly immunogenic transformed clones. At lower concentrations, or at longer times after the MCA administration, when immunological recovery had occurred, surveillance would be more effective, fewer transformed clones would grow, and those that did grow would tend to be thosc of lesser immunogenicity. According to this formulation, the lack of immunogenicity of spontaneous tumors, as compared with those induced by higher concentrations of chemical, is exactly in accord with the expectations of the surveillance hypothesis. The idea that the inverse relationship that exists between latency

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(i.e., the period of time between initial exposure to MCA and the appearance of gross tumor) and immunogenicity (Prehn, 1962, 1969a; Old et al., 1962) might be due to imfhunoselection was challenged by Bartlett (1972), who tested whether or not the relationship would still be found if immunological selection were eliminated from the system. The method was to expose mouse cells to MCA within the confines of intraperitoneal diffusion chambers. The exposed cells were subsequently transplanted to the subcutaneous tissues of mice. Thus, at least the early stages of the oncogenic process took place within the immunologically isolated confines of diffusion chambers, a site presumably free from the possibility of immunological surveillance and selection (Prehn and Main, 1956). Tumors arose from the transplants after varying periods. It was found that under these conditions there was no detectable correlation between the latency and the immunogenicities of the tumors. Therefore, the conclusion seemed justified that the usually observed inverse relationship between the latency and the immunogenicity was indeed a function of immunological selection. Similar results were obtained by Parmiani et al. (1973). Concomitant with the above study, I examined the immunogenicities of tumors that arose among cells in tissue culture [immunogenicity again defined as the degree of immunity to challenge tumor growth that could be elicited in mice by prior immunization with tumor (Prehn, 1971c)l. Inasmuch as the tissue culture environment was presumably completely free of any possibility of immunological surveillance and immunological selection, it was anticipated that tumors originating in such an environment would be uniformly highly immunogenic. Such was not the case. It was found that whenever transfonnation took place “spontaneously” in the cultures, the resulting tumors were not detectably immunogenic in uiuo.? Likewise, “spontaneous transformation” in diffusion chambers was also shown to result in nonimmunogenic tumors (Prehn, 1971c; Bartlett, 1972; Parmiani et al., 1971). In contrast, when the cultures In the initial series of cultures not exposed to MCA, the tumors that were presumably the result of “spontaneous” in uitro transformation were all found to be immunogenic. [A similar finding has been reported by Kieler et al. (1972).] The tumors were also found to be cross-reactive, suggesting that the immunogenicity was the result of contamination with an unknown virus, possibly unrelated to the transformational event. Subsequent to that series of cultures, the laboratory was moved to another city, the substrains of mice were changed, and a new technical staff performed the work. Immunogenicity of “spontaneously transformed” cultures was never encountered again. The probable explanation that immunogenicity in the initial series was due to contaminating virus is strengthened by the observation that immunogenicity can be deliberately imparted to a tumor by infection with a passenger virus (SvetMoldovsky and Camburg, 1965; Sjogren, 1964).

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were exposed to MCA, and when transformation presumably resulted from the action of the MCA, the tumors were usually highly immunogenic. Thus, in tissue culture and diffusion chamber oncogenesis, the immunogenicity was apparently a function of the presence or the absence of MCA, not a function of immunological surveillance. At first glance the results obtained with MCA and with “spontaneous” transformation in immunologically protected environments seem paradoxical. However, the conclusions to be drawn from the two types of experiment are not mutually exclusive. The Bartlett work suggested that immunological selection plays a role in determining the immunogenicities of the MCA-induced tumors, and that immunological surveillance is a factor in chemically induced oncogenesis. The tissue culture work suggested that the oncogen is directly involved and necessary in producing immunogenicity. Without the MCA, there is apparently little or no immunogenicity for the immunological selection to act upon. The lack of detectable immunogenicity in “spontaneous tumors,” even in the absence of an immunological surveillance mechanism, raises questions about the possible role of immunity in human cancer. Judging from the mouse model, as I have thus far described it, the possible role is presumably dependent upon the etiology of the tumor. If the tumors arise in high frequency as the result of a high flux of an oncogen-as may be the case in, for example, bronchogenic squamous cell tumors of the lung or ultraviolet-induced skin tumors-then one would expect that immunological surveillance might play a significant role. On the other hand, tumors that arise infrequently in response to very low levels of environmental oncogens or which may be truly spontaneous, and which therefore probably have little or no immunogenicity, would presumably be little affected by immunological defenses. I will discuss this argument in greater depth after a short digression concerning those tumors overtly induced by oncogenic viruses.

D. VIRALLY INDUCEDTUMOFSAND LYMPHORETICULAR NEOPLASMS The evidence in favor of the hypothesis that immunity serves as a defense against the growth of cells neoplastically transformed by oncogenic viruses is even greater than that concerned with tumors chemically induced. It is not always easy to decide whether the surveillance is operating against the spread of the virus or against the growth of transformed cells. However, much evidence suggests that it is, at least in some cases, the latter, and thus quite analogous to the situation in MCA induced tumors, This evidence has been extensively reviewed by Klein ( 1973) and Hellstrom and Hellstrom (1969).

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The most persuasive work concerns the small oncogenic DNA virus tumors, such as those induced by polyoma, SV40, and the oncogenic adenoviruses. Tumors induced by these agents carry relatively strong tumor-specific transplantation antigens ( Klein, 1973; Hellstrom and Hellstrom, 1969). Antilymphocyte serum treatment or newborn thymectomy profoundly increases the tumor incidence. Curiously, the incidence of polyoma or SV40-induced tumors in neonatally inoculated animals can be reduced by inoculation of a second virus dose or of irradiated tumor cells during the oncogenic latent period (Deichman, 1969). The implication is clear that immunity to the transformed clones can apparently influence the tumor incidence. The resemblance of these systems to oncogenesis with MCA seems striking. The tumors are highly immunogenic; however, in the case of the viruses the tumors are of course cross-reactive, and because of viral multiplication in the host, dose response studies of the type done with MCA are not yet available. In my opinion, great caution should be exercised in interpreting work done with lymphoreticular tumors, however induced, as being either for or against the immunological surveillance hypothesis. This is because of the fact that these are tumors of the immune organ itself. It has been known from many other organ systems that excessive physiological demand can itself result in neoplasia. For example, there is the classical work of Biskind and Biskind (1949) on oncogenesis in the ovary. The ovary, transplanted in the spleen of the gonadectomized rodent, eventually becomes tumorous because the feedback inhibition to gonadotropin production is interrupted by the destruction of ovarian hormones in the liver. It is possible that increased lymphoma production in immunodepressed animals or man is due to an analogous physiological stress, in this case antigenic stimulation of an organ that is crippled and unable to respond adequately. This idea has recently been placed on a more sophisticated level by Schwartz, who suggests that lymphomas in kidney transplant recipients or in graft-versus-host disease are the result of antigenic stimulation under conditions in which feedback inhibititon by regulatory suppressor T cells has been blocked (Schwartz, 1975). Such tumors may therefore not be a manifestation of a failure of the surveillance mechanism. These considerations complicate the interpretation of Marek's disease in chickens. This virally induced lymphoma can be largely prevented by immunization of the birds with a related but nonpathogenic turkey virus ( Nazerian, 1973). Although the incidence of disease is drastically reduced, the amount of Marek's virus detected in the birds is only moderately altered (Nazerian, 1973). This fact might suggest that the turkey virus inoculation increases immune surveillance against incipient tumor

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cells per se, rather than against the virus. However, the lack of a dramatic effect on the Mareks virus is somewhat difficult to understand since the antigen on the tumor cell is either a part of, or determined by, the Marek‘s virus. Only in the former case would one expect a cross-reactive turkey virus to influence the growth of tumor cells, but in that event it should also markedly affect the virus. In explanation, it has been suggested that virus production is little affected by immunization because it occurs largely in a “sequestered site (Klein, 1973; Hellstrom and Hellstrom, 1969). If one were to speculate that the Marek‘s lymphoma is not a result of virally induced transformation, but is instead the result of an undampened but ineffectual immune reaction (perhaps due in part to a genetically determined lack of T-suppressor cells of the proper specificities) , the role of the turkey virus might be to activate T-suppressor cells-that could dampen the “run away” response to the Marek‘s virus. This is, of course, speculation, but it serves the purpose of showing that alternatives to the concept of a failure of immunological surveillance vis-8-vis tumor cells can be conceived even in Marek‘s disease, a malignant disease in which immunization is highly successful. If we disregard all evidence, for the reasons expounded above, derived from lymphoreticular neoplasms, the case for an effective immunological surveillance seems to depend almost entirely upon the fact that immunodepressive measures, such as neonatal thymectomy or antilymphocyte serum ( ALS ) , can sometimes increase the incidence of tumors in animals exposed to potent chemical oncogens or laboratory strains of oncogenic viruses (Klein, 1973; Hellstrom and Hellstrom, 1969; Prehn, 1974). In the case of some of the viruses, these data are much more persuasive than with the chemicals. In some of the chemically induced tumor systems, such as some hydrocarbon-induced tumors of the mouse, the available evidence suggests that the state of the immune mechanism influences neither the incidence nor the regression of the tumors to any appreciable degree (Outzen et d.,1975a; Andrews, 1971, 1974; Stutman, 1974). Even with the viruses, the situation is not clear. Virally induced mouse mammary tumors, for example, are much decreased following thymectomy or ALS, and this apparently is not a function of ovarian atrophy (Prehn, 1971a). On the other hand, there can be little doubt that regression in the Moloney sarcoma system is caused by a thymus-dependent immune reaction (Stutman, 1975). It is not yet known whether tumors induced by viruses, under conditions of natural infection, are highly immunogenic or are perhaps more analogous to those tumors produced by very low levels of chemical carcinogen. To summarize the discussion up to this point, there is good evidence

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that the immune mechanism apparently plays a role in the regulation of the growth of certain tumors, particularIy those induced in the laboratory by high concentrations of certain, but not all (Baldwin, 1973), chemicals and by infection with laboratory strains of oncogenic viruses. (Contrary evidence from studies with the “nude” mouse will be discussed later. ) On the other hand, spontaneous tumors and tumors induced with low concentrations of chemical have little or no immunogenicity (for reasons having nothing to do with immunological selection) and thus would presumabIy be difficult targets for immunological surveillance. Naturally occurring viral tumors remain to be investigated in this regard.

E. Is SUBLIMINAL IMMUNOCENICITY ADEQUATE FOR SURVEILLANCE? The fact that spontaneous tumors have little or no detectable immunogenicity does not necessarily mean that immunological surveillance could not operate against them. It is well established that the effectiveness of an anticellular immunity is dependent upon the size of the target population. A level of immune response adequate to deal with a tumor cell population of a certain size may be overwhelmed by a larger. Thus, it could be postulated that an immunogenicity too low to be recorded in the usual immunization-challenge type of in uiuo test, might still be adequate to cope with a very small nidus of transformed cells. In other words, a subliminal immunogenicity might be adequate for the purposes of effective immunological surveillance. However, several lines of evidence suggest that this is probably seldom the case. The first evidence against the concept that a subliminal level of tumor immunogenicity might be adequate for the purposes of surveillance arises from a consideration of the effects of a very small antigenic stimulus upon the immune reactivity of an animal. It has been shown that a very small exposure to even a strong transplantation antigen, such as might be the result of the growth of a small nidus of immunogenic tumor cells, tends to condition the immunological mechanism in such a way that the immune response to that antigen is very small; it tends to remain small throughout the remainder of life regardless of the size of subsequent antigenic stimuli (Stillstrom, 1974). Perhaps this is a form of partial immunological tolerance, but the mechanisms of the phenomenon are not known. Whatever the mechanism, it seems likely that it is because of exposure to a minimal antigenic stimulus during early tumor development that the animal in which an autochthonous MCA induced primary tumor has been excised is much less capable of resisting subsequent challenge inoculations of that tumor than are syngeneic controls that had been initially immunized by having the tumor implanted ( Stjernsward, 1968; Basombrio and Prehn, 197213). One can conclude

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that much of the immunogenicity observed during the syngeneic transplantation of laboratory tumors is in reality a laboratory artifact imposed by the transplantation method of initial antigen presentation ( Andrews, 1974). If the result of a very small initial exposure to a strong antigen results in partial tolerance, it is hard to see how exposure to the very weak antigens of a nidus of developing spontaneous tumor cells could result in an effective surveillance response. That transplantation can induce immunological artifacts was demonstrated dramatically in studies concerning MCA-induced papillomas in mouse skin, work already alluded to in Section II,B. Lapp6 devised a method of inducing papillomas that consisted of exposure of the mouse skin to a low dose of MCA followed by the syngeneic grafting of the exposed skin (Lap@, 1968; Prehn and Lapp6, 1971). The grafting provided a physiological stimulus ( “promotion”) that rapidly elicited a crop of papillomas in the previously MCA-treated epithelium. Lapp6 was able to show clearly that the papilloma incidence and the rate of regression were influenced by the immunological capacity of the host animal. Immunologically crippled hosts developed more papillomas, and these persisted longer than was the case in normal or in immunologieally potentiated hosts. Apparently this was clear evidence of immunological surveillance. Subsequent work by Andrews (1974) showed, however, that the immunological effect on tumor incidence was probably dependent upon an artifact introduced by transplantation of the skin containing the incipient papillomas. In the absence of such transplantation, the incidence was completely uninfluenced by the immunological status of the animal. This conclusion is consistent with the observation that life long immunodepression with the aid of ALS did not influence hydrocarbon-induced papillomas ( Haran-Ghera and Lurie, 1971) . Furthermore, Andrews showed that regression of the papillomas still occurred even when the possibility of host immunity was almost certainly excluded ( Andrews, 1971). He produced immunologically crippled animals by adult thymectomy followed by X-radiation. The crippled mice were then given a graft of already MCA-initiated skin from an allogeneic donor. The grafting served as a promoter, and papillomas appeared in the usual fashion; they also regressed despite the fact that the allograft in which they resided was surviving healthily. It thus seems most unlikely that host immunity plays a necessary role in ordinary papilloma regression. Immunity can contribute when, and only when, the immune response is aroused by some procedure such as skin grafting in immunocompetent hosts. The in situ papilloma does not apparently arouse an immune reaction. In the case of the skin papilloma system, the lack of effective immuni-

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zation by the in situ, untransplanted neoplasm could be accounted for by the conditioning effect of a low initial exposure to the pertinent antigens, as already described, and/or to a second phenomenon best illustrated in the work of Slemmer in the mammary tumor system. I have already described the system in Section II,B,l. Slemmer (1972) was able to show that an antigenic MCA-induced hyperplastic nodule could be transplanted into an uncleared fat pad in a syngeneic mouse and there persist without growth or regression for many months and perhaps indefinitely. At any time during this period, the nodule tissue could be rapidly destroyed by simply implanting another fragment of the same nodule subcutaneously, i.e., outside any mammary fat pad. Clearly, in this system the immune mechanism was capable of destroying the incipient tumor whenever immunization took place. However, the lesion inside the fat pad did not immunize, although it was kept from growing by the inhibiting influence of the normal epithelium. It seems probable that within many and perhaps all epithelial organs, such as skin and breast, in which the parenchymal stem cells are separated from mesodermal elements by a basement membrane, even transplanted, antigenic cells immunize poorly, if at all. It has definitely been shown that both the mammary fat pad and the superficial skin epithelium are immunologically privileged sites in which immunization to cellular antigens is very slight (Blair and Moretti, 1967; Billingham and Medawar, 1950). Thus, in these sites one could not expect a nidus of weakly immunogenic tumor cells to be eliminated by an immune reaction. Even when the tumor cells are potentially highly immunogenic, i.e., MCA-induced, such elimination does not occur in the natural course of events. In the breast, immunization does not occur until the tumor mass is large and/or has escaped the fat pad. Under these conditions, surveillance could not be effective against weakly immunogenic nascent tumors. Further evidence suggesting that a subliminal immunogenicity in tumor cells may not be adequate for the purposes of surveillance derives from a consideration of two distinct but possibly related phenomena: “sneaking through and “immunostimulation.” The “sneaking through phenomenon was first described in relation to the growth of an allogeneic tumor, but has subsequently been confirmed by a number of laboratories with respect to completely syngeneic systems (Humphreys et al., 1962; Old et d.,1962; Potter et al., 1969; Marchant, 1969). In brief, the phenomenon consists of the observation of an anomalous behavior in the growth of transplanted tumor cells when these are implanted in varying dosages. With at least some immunogenic tumors it can be shown that a very small inoculum may grow better than a larger. Recent evidence suggests that the phenomenon is due to what can be called a specific, immunological, partial tolerance

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produced by the small inoculum ( Bonmassar et al., 1974). The phenomenon is thus closely related to the effect of an initial small antigen dosage already discussed; it can be demonstrated even in highly immunogenic systems. Again, a very low dosage of inoculated tumor cells probably mimics in situ tumor formation. It seems unlikely that a weakly immunogenic, in situ, untransplanted tumor can produce an effective immune resistance to its growth when this is unattainable by the inoculation of a small dose of highly immunogenic tumor cells.

F. IMMUNOSTIMULATION OF TUMORGROWTH Thus far the argument against the efficacy of immunity in surveillance against newly formed, weakly immunogenic, tumors has related to the ineffectiveness of a very weak antigenic stimulus, such as a newly developing tumor cell clone would provide. Immunostimulation is a phenomenon which suggests that a weak immune response may not only be ineffective in controlling tumor growth; it actually may make the tumor cells grow better. The original idea of immunostimulation was obtained by consideration of the rather extensive literature suggesting that a weak immunological reaction by the mother against the conceptus could result in larger placentas and larger and more numerous offspring (Prehn and Lapp6, 1971 ) . Although this literature remains controversial, it did suggest, by analogy, that the immune reaction might also be able to favor tumor growth. The initial tests of this hypothesis were done with the aid of Winn tests in immunologically crippled host mice (Prehn, 1972). It was found that small numbers of specifically immune spleen cells, when implanted together with tumor cells, produced better tumor growth than did similar numbers of normal or nonspecifically immune spleen cells. Subsequently, similar phenomena have been recorded in a variety of systems both in uitro and in uiuo (hledina and Heppner, 1973; Fidler, 1973; Jeejeebhoy, 1974; Kall and Hellstrom, 1975; Bray and Keast, 1975; Ilfeld et al., 1973; Bartholomaeus et al., 1974). Shearer has shown clearly that heterologous antibody can be directly stimulatory to target cells (Shearer, 1973). At present, the mechanisms are not known, and it appears that several different effector systems, i.e., T cells and/or antibody, may stimulate under different conditions or in different systems. It now appears that some of the work subsumed under the title of “enhancement” may be synonymous with immunostimulation rather than with “blocking,” a position not unlike that long championed by Kaliss (1965). If immunostimulation is a general phenomenon, and this is not yet established, it probably plays a crucial role in tumor biology. Any tumor, regardless of its immunogenic potential, should produce a weak immune

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response early in its development; the immune response must pass though a weak phase before it gets strong. The immune reaction would thus give tumors an initial impetus; the immunity might or might not, depending upon the circumstances, mature into an inhibitory type of reaction. That this actually occurs is supported by the report by Jeejeebhoy (1974). If nothing else, the existence of the phenomenon of immunostimulation suggests that the weak immune response, probably elicited by an in situ nidus of spontaneously transformed, weakly immunogenic, tumor cells, would not be likely to result in surveillance. On the contrary, immunostimulation might be more likely. Immunity might result in more tumors than would occur if there were no immune reaction at all. The “nude” mouse would seem to offer a definitive means of assessing the role of immunity in oncogenesis, and of determining the relative roles of surveillance and immunostimulation. The congenitally athymic “nude,” despite the fact that some of its lymphoid cells are theta-antigen positive, has no detectable ability to reject allografts or xenografts. This immunological deficit results in a short life-span in most conventional colonies; however, longevity can be extended and approach normal under pathogen-free or germ-free conditions ( Outzen et al., 1975a). Inasmuch as this mouse has no resistance to allografts, it would have no resistance to the growth of antigenic tumors unless the surveillance function and allograft immunity are completely different types of reactions. Although data have been accumulating slowly, there appears, thus far, to be no increase in tumors induced in nude mice by a hydrocarbon oncogen (Outzen et al., 1975a; Stutman, 1974). With the exception of lymphoreticular tumors, there has been, as yet, no increment of spontaneous tumors (Outzen et aZ., 1975a). This result is identical to that obtained by the life-long administration of antilymphocyte serum to normal mice (Nehlsen, 1971; Sanford et al., 1973). On the other hand, neither has there been a tumor deficit; the nude mice seem to be about as susceptible to tumor formation as are their heterozygous littermates. The data from the nude mouse seem to suggest that neither immunological surveillance nor immunostimulation plays a significant role in chemical oncogenesis. On the other hand, Moloney sarcoma virus-induced tumors do not regress in nudes as they usually do in conventional nonnude controls (Stutman, 1975). Although the published accounts of oncogenesis in nudes seem to denegrate the role of the immune response in hydrocarbon oncogenesis (and thus seem contrary to the implications of the Bartlett ( 1972) work previously discussed), there exist several as yet unpublished observations that suggest the possibility of a different conclusion. Reed has data to suggest that although the nude mouse is resistant to skin oncogenesis,

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produced by painting a hydrocarbon, it becomes susceptible when restored by thymus grafting ( N. Reed, personal communication, 1975). Although nonimmunological explanations have not been excluded, this result is consistent with the immunostimulation hypothesis, since it suggests that thymic function may facilitate oncogenesis. Also consistent with the immunostimulation interpretation is the general observation in many laboratories that transplanted allogeneic tumors do not grow as well as expected in nude mice (Skov et al., 1975; Maguire et al., 1975). Furthermore, tumors that metastasize in the strain of origin seldom do so in nudes ( Maguire et al., 1975). Nonimmunological explanations cannot yet be excluded; for example, macrophages have been reported to nonspecifically inhibit tumor growth in nudes (0.Stutman, personal communication, 1975). An as yet unpublished account, with a bearing on the role of the immune response in oncogenesis was not done in nudes, but in mice rendered immunoincompetent by thymectomy and whole-body radiation ( Prehn, 1975a). Partial restoration of immunocompetence by injection of normal syngeneic spleen cells resulted in accelerated oncogenesis with MCA as compared with controls that were either unrestored or completely restored. The result was thus exactly in accord with the immunostimulation hypothesis, but again nonimmunological explanations must be considered. If the increased tumor production in partially restored immunocrippled mice is indeed immunological, how is it that, apart from the observation of Reed, there has been no indication of a difference in induced-tumor susceptibility between nude mice and their heterozygous littermate controls? The only explanation I can presently advance is to point out that there was no significant difference between the tumor incidence in unrestored immunocrippled mice and the maximally restored group. Perhaps these groups were comparable to the nudes and the heterozygotes, respectively. If so, partial restoration experiments in nude mice will eventually give similar results. The immunostimulation hypothesis leads to a further prediction-one that in fact seems to have been realized by observations made before the theory was conceived. According to the theory, immunostimulation occurs only when the immune response is relatively “weak” (Prehn and LappC, 1971) . Consequently, the theory predicts that in tumor systems in which the immunogenicity of the tumors is low, measures that lower the immunological responsiveness of the animal still further will decrease the growth of transplanted tumors and the tumor incidence. This is in contrast to the usual laboratory tumor systems in which immunogenicity is high and immunodepression tends, if anything, to increase the tumor incidence, and certainly the “take” of transplanted tumors. In

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other words, if the immune reactivity of the system is already in the low or irnmunostimulatory range, further reduction will decrease tumor growth-if it is in the high or tumor-inhibitory range, a decrease will lead to decreased inhibition and perhaps to actual stimulation. This consequence of the immunostimulation theory, namely that immunocrippling procedures will result in decreased tumor growth in weakly immunogenic tumor systems, is actually seen among mammary tumors in mice. In this system much of the immunogenic potential of the tumors is due to viral antigens-but this potential cannot be appreciably realized in animals that are infected from birth by natural transmission of the virus from the mother. In such animals, although antiviral antibodies are formed, the virus persists throughout life. In these mice, transplantation studies reveal very little evidence of immunity capable of inhibiting tumor growth (Vaage, 1968; Morton et al., 1969; Prehn, 1969b). If the general immunological capacity of such mice is interfered with by any of a variety of means (X-rays, ALS, thymectomy, etc.), tumor growth and incidence are usually reduced ( Prehn and Lapp6,1971) . In contrast, these same tumors are markedly accelerated in their growth by similar measures when they are transplanted to syngeneic mice lacking the virus, mice in which they are markedly more immunogenic (Prehn and Lapp6,1971) .

G. METASTASIS

It has long been bruited that an immune response to tumor antigens might play a role in the prolonged latency of some neoplasms or in their slowness to metastasize. The fact that in the breast tumor system the immune mechanism can be aroused by tumor cells only when they leave the protective confines of the fat pad mighst support such a hypothesis. However, firm support of the hypothesis has not yet been achieved, although there are several reports that are highly suggestive. Perhaps outstanding among these is the report of Lewis et al. (1969), which we have recently been able to confirm (Bodurtha et d.,1975), that patient serum is uniformly inhibiting in culture to the autochthonous melanoma when, and only when, the tumor is regional in extent. The patient's serum loses this property in association with, and perhaps prior to, dissemination of the tumor. Whether or not this is a cause and effect relationship has not been determined, but the observation is highly suggestive.

H. CONCLUS~ONS CONCERNING IMMUNITY Throughout this discussion of tumor immunity I have made virtually no reference to the interesting work being pursued in so many labora-

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tories concerning the results of varied in uitro tests of tumor antigenicity and the immune status of patients or experimental animals. This is not because of lack of interest or familiarity-much work of this type is being pursued in my laboratory. However, I find the results to date confusing, and it is difEcult to draw firm conclusions from them. Most irritating is the fact that there has sometimes been a striking lack of correlation between the results of in uitro and in uiuo testing in mouse systems ( Baldwin, 1973). Lymphoid cells can be immunized against and can develop cytotoxicity against normal syngeneic targets ( Wekerle et al., 1973). Normal lymphoid cells tend to be as cytotoxic as are patient cells to target tumors (Takasugi et al., 1973; Berkelhammer et al., 1975; Jeejeebhoy, 1975). Also, I am distressed by the lack of tumor or even organ specificity so often encountered (Berkelhammer et d.,1975). I have no doubt that all the vaned phenomena described by the in vitro methods are real-blocking, unblocking, arming, etc. ( Herberman, 1974). However, I am uncertain of what role these phenomena play in the gesta1.t of the reaction in uiuo. Despite these problems, it is ultimately only by dissecting these processes in uitro that progress will be made. The present confusion will no doubt gradually resolve. Perhaps I can summarize this section on the immune reaction and cancer by stating my own tentative conclusions. 1. A cytotoxic or inhibiting immune reaction usually plays little or no role in determining the occurrence or nonoccurrence of tumors except under exceptional laboratory conditions, i.e., large doses of chemical oncogens, laboratory-selected viruses, etc. ( Naturally occurring viral tumors require much further investigation in this regard.) In other words, immunological surveillance is, at best, probably a very limited phenomenon, 2. In many systems immunity, especially in low titer, may directly stimulate rather than inhibit the growth of tumor cells. (When immunodepression increases the incidence of tumor, it is because of decreased surveillance or increased stimulation?) 3. Some human cancers that occur in high frequency as a result of potent environmental oncogens may, like the analogous laboratory tumors, be influenced by an immune reaction, i.e., ultraviolet-induced skin cancers and bronchogenic squamous cell tumors might fall in this category. However, the probability of immunostimulation rather than surveillance must be remembered. 4. Immunity may sometimes function late in the course of disease to limit blood-borne metastases. 5. The usual lack of effectiveness of immunity as a defense mechanism increases the chance that effective methods of improving the immune

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response (immunotherapy ) may be found. These may already be present in adjuvant immunotherapy with agents such as Bacillus Calmette-GuCrin ( BCG) (Berkelhammer et ul., 1975), but the possible hazards of immunostimulation must constantly be considered. V. Concluding Remarks

This survey of tumor initiation, progression, and homeostasis has omitted many topics and references that would belong in any comprehensive review. Indeed, a comprehensive review of such a broad subject would cover most of what is known of tumor biology. This survey has been limited to those topics of particular interest to me and thus reveals my biases. What I have presented is a personal view. However, even in the limited areas that I have chosen to discuss, this brief survey makes it apparent that knowledge has been accumulating at a rapid pace; it makes it even more apparent that our ignorance remains profound. Perhaps most critical is the lack of knowledge concerning homeostatic mechanisms other than the immune response. I have discussed one such in the regulation of growth in the mammary tumor system, but nothing is known concerning its mechanism of action. Perhaps one area for fruitful investigation is the role of nerves and regenerative capacity in oncogenesis (Prehn, 1971b). Recently, my colleagues and I have been able to show that oncogenesis in the frog by a hydrocarbon is markedly potentiated by nerve section-possibly via the critical effect of nerves on regenerative phenomena (Outzen et al., 1975b). Although the future of such studies is hard to predict, it is my feeling that, in the long run, important as immunological studies may be, they will be counterproductive if they so monopolize attention that other mechanisms of tumor homeostasis are thereby neglected.

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