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[CANCER RESEARCH 44, 2259-2265, June 1984] Perspectives in Cancer Research Tumor Heterogeneity GlorÃ-aH. Heppner1 Michigan Cancer Foundation, Detroit, Michigan 48201 In 1977, Dan Dexter and I, with colleagues at Roger Williams General Hospital, submitted a manuscript to Cancer Research in which we reported the isolation of four distinct tumor subpopu lations from a single, spontaneously arising mouse mammary cancer. We speculated that these subpopulations were evidence for intraneoplastic heterogeneity and that heterogeneity was a general phenomenon, knowledge of which was important in the treatment of cancer. Editorial review of this manuscript, if not swift, was unambiguous: it was unworthy of publication in Can cer Research. Two reasons were given: our results were not novel; and anyway "everyone knew" that cancers are mono clonal. The first was certainly true; previous investigators, most notably Levan and Hauschka (48), Klein and Klein (47), Makino (50), Henderson and Rous (34), Gray and Pierce (28), Prehn (76), Mitelman (59), and Foulds (23, 24), had reported evidence of multiple tumor subpopulations within single cancers. With regard to the second objection, it seemed that the reviewers were implying that single-cell origin somehow ruled out subsequent variation as the population grew. I could only reflect that I too was originally monoclonal but became undeniably heteroge neous. Ultimately, the skeptical reviewers gave in to the force of this simple logic (15). At about the same time, Fidler and Kripke (22) published an analysis of tumor heterogeneity as exemplified by metastasis. These reports fell on fertile ground; witness that in the first 9 months of 1983, Cancer Research contained more than 20 papers dealing directly with some aspect of tumor heterogeneity. Symposia have been held on the topic (62, 68), and a new periodical has appeared (Invasion and Metastasis) subtitled A Journal of Cancer Dissemination and Tumor Cell Heterogeneity. It might seem then that our early speculations were well founded. However, now finding myself more in demand as a reviewer than as an author, I see a new merit in skepticism. Furthermore, I am becoming increasingly distressed by what I see as a focus on the difference between tumor cell subpopulations, without the necessary parallel understanding that tumor cells share a fun damental unity. The populations that we observe and therapeuticaiiy attack are not only contiguous in space and origin but are also complex ecosystems with characteristics that transcend those of their individual members. To understand how these systems develop, it is necessary to fuse the concepts and methods of developmental and population biology with those of cell biology. In short, this is my perspective on research in tumor heterogeneity. What I Mean by "Tumor Heterogeneity" An inevitable result of the increased use of any term that 1Supported by NIH Grants CA-27437 and CA-27419, the Concern Foundation, the E. Walter Albachten bequest, and the United Foundation of Metropolitan Detroit. To whom requests for reprints should be addressed, at Michigan Cancer Founda tion, 110 East Warren Avenue, Detroit, Ml 48201. Received December 19,1983; accepted March 2,1984. encompasses a popular concept is a gradual blurring of its meaning until it becomes an umbrella that covers, and perhaps shields from close examination, phenomena that are only periph erally related. This has been the fate of the "stem cell," and it is happening to the term "tumor heterogeneity." Tumors are vari able in several ways. Their characteristics change with organ site and cell origin. Numerous host variables, such as age and hormonal status, also introduce differences. The same cancer can even vary in putatively similar hosts. However, this intertumor variation is not what I, and I think most investigators, mean by tumor heterogeneity. The types of variability usually meant by the term tumor heterogeneity are cellular differences within a single neoplasm. Even here, however, there is room for confusion. Tumors are architecturally complex, differing regionally in vasculature, host infiltrates, connective tissue components, and other character istics which can alter the phenotype of otherwise identical cells. Marked differences in the proliferation behavior of tumor cells within a single cancer are commonplace. Some cells, perhaps most, are reproductively dead; others are out of cycle; and still others are cycling but are, at a given time, at different stages in the process. Furthermore, many cellular phenotypes, such as antigen expression (12, 17), membrane composition (7, 69), response to chemotherapy (87), metastatic proclivity (84, 89), to name a few, are themselves functions of the cell cycle. These differences, which we might call "secular" (as distinguished from those transmittable genetic and epigenetic differences which determine cell lineages), may be of biological and clinical signifi cance, but their analysis is only confused by lumping them with lineage differences under the single term, tumor heterogeneity. In this essay, "tumor heterogeneity" will be reserved for those cases in which tumor cell differences are believed to be due to differences in cell lineage, i.e., due to the presence of distinctly different subpopulations capable of breeding true. (The extent of temporal stability of subpopulations will be discussed below.) This definition is not intended to discriminate between variability that arises from genetic, as distinguished from nongenetic or epigenetic, processes. Cells within clones are certainly not iden tical; they too are subject to secular variation. However, this usage allows a focused experimental analysis of at least one type of neoplastia variability. Evidence for Tumor Heterogeneity Numerous reviews have appeared recently citing evidence of subpopulations within tumors (14, 21, 36, 54, 74). Subpopulations have been isolated from cancers of every major histological type and organ site and from both experimental and human cancers. They have been isolated from tumors induced by chem ical, physical, or viral agents; from long-term cell lines; and from tumors of recent origin. The list of characteristics by which subpopulations differ is 2259 JUNE 1984 Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. G. H. Heppner also extensive: cellular morphology; tumor histology; karyotype and other cytogenetic markers; growth rate; Å“il products; re ceptors; enzymes; immunological characteristics; metastatic abil ity; and sensitivity to therapeutic agents. In addition, since sister cells usually remain contiguous in solid tumors, sublines tend to be localized regionally or zoned (20, 31, 76). There might seem to be overwhelming evidence for tumor heterogeneity. It is important to recognize, however, that much of the evidence is similar in kind. In the first place, cultured, tumor-derived subpopulations are by definition obtained through isolation; consequently, the finding that they differ is subject to the interpretation that the differences are isolation induced. Dur ing isolation, cells are released from variability-inhibiting in vivo controls, and the special circumstances of cloning, i.e., separa tion of cells and their subsequent growth, can be stimuli for production of new phenotypes (32, 73). Secondly, the finding of heterogeneity in situ, as by immunohistochemical methods, is compatible with clonal heterogeneity but does not prove it, since "secular" explanations are also possible. A third argument, that tumors are heterogeneous because metastatic and primary tu mors differ in cellular composition, is also not definitive but, rather, circular, since this interpretation depends on the prior conviction that métastasesarise from a nonrepresentative sub set of parental cells to begin with. It seems to me that convincing evidence for tumor heteroge neity in nature consists of the demonstration of lineage variation in situ, coupled with the experimental dissection of the same variation in cloned, true-breeding subpopulations. This type of evidence is not abundant. The usual evidence is more ambigu ous; results from my laboratory are typical. While we have shown that different mammary tumor karyotypes (and marker chromo somes) can be found in subpopulations of clonal origin, we have never demonstrated the direct descent of these phenotypically distinct isolates from their putative parental cells. One would like to see studies in which lineage heterogeneity is demonstrated in fresh uncultured tumor specimens and the lineage markers are then used as a basis for isolating and characterizing the different cell populations. This ideal has been approached in the Shapiros' laboratory (82) where karyotype heterogeneity was first used as a marker for subpopulations in fresh primary human gliomas and then was used as a reference to identify these subpopulations in clones isolated from the same tumors. The subpopulations differed in several parameters, including sensitivity to drugs (91) and genetic stability (81). Karyotype differences have been de scribed in many other kinds of tumors by conventional techniques (59) and by flow cytometry (2), but to my knowledge the Shapi ros' work is unique in being a prospective study, one in which subpopulations are first identified in fresh tumors and their existence is subsequently confirmed in culture. Changes in kar yotype have been used to monitor tumor progression in situ (40). Insofar as progression reflects differential reproduction of subpopulations (63), karyotype frequency changes are proof of tumor heterogeneity. On the other hand, if differences in subpopulation generation are only assumed, this argument is also circular. These comments should not be taken as reflecting serious doubt on my part that tumor heterogeneity exists (Reviewers: take note), but I do argue that every claim for tumor heteroge neity, or homogeneity for that matter, should be subjected to skeptical examination and that the quality of evidence be evalu 2260 ated. Weakness in an experimental proof remains a weakness whether the experiment is done once or 100 times. Origin of Tumor Heterogeneity As mentioned, tumor heterogeneity can be viewed as suspect because superficially it appears at variance with the idea that cancers arise by the transformation of a single cell (19). Setting aside the evidence that not all tumors arise as single cells (77), the suspicion seems unjustified when one considers that most eukaryotic organisms begin as single cells but soon become heterogeneous. Tumors also appear to undergo developmental and differentiative changes, at least some of which result from altered gene expression. Heterogeneity is not a property unique to tumors, but one they share with other organs. Indeed, Griffen ef al. (29) found that nonneoplastic skin fibroblasts from normal carriers of 5<*-reductase deficiency can be cloned into subpopulations with a wide range of enzyme activity. Heterogeneity in lineages derived from a common stem cell has been documented in many normal cells, most thoroughly in those of the hemopoietic system (85). Heterogeneity is a feature of neoplastia development that can precede the tumor itself. Normal cells differ in susceptibility to carcinogens (6, 43), and the heterogeneity in characteristics of different SV40-transformed clones has been shown to reflect the heterogeneity of the normal cells from which they are derived (67). Evidence of cellular heterogeneity has been reported in preneoplastic hepatocytes (65) and mammary epithelial cells (1). In addition, even cells that are not transformed by a carcinogen are often altered physiologically by the exposure. Such carcino gen-altered, nontransformed cells constitute an abnormal envi ronment for the initiated cells and, as Rubin (79) has discussed, can be shown to influence the expression of transformation. Cellular heterogeneity must be viewed then as a feature of both normal and precancerous tissues. It seems not unlikely that the mechanisms that are responsible for variability under these circumstances could also be responsible for generating tumor heterogeneity. Studies on teratocarcinoma (71), on small cell carcinoma of the lung (3), and on mammary carcinoma (4, 30, 80) all attest that differentiation of neoplastic "stem cells," quite analogous to that in normal tissues, results in tumor heteroge neity. Pierce and Cox (71) described the development of heter ogeneity within teratocarcinoma as a "caricature of embryogenesis." Besides "normal" mechanisms, heterogeneity may arise by tumor specific mechanisms. Increased genetic instability is a case in point (63). This instability leads to more errors in tumor cell DNA (point mutations, genomic rearrangements, chromo some losses, gene amplification, etc.) and is reflected in in creased phenotypic variability. Evidence comes from observa tions of karyotypic abnormalities that accompany neoplastic progression (40) and from the work of Cifone and Fidler (11) who found in fibrosarcoma cells that rates of spontaneous mutation are greater in metastatic than in nonmetastatic subpopulations. However, considerable caution is necessary when one compares mutation rates in different cell populations. Observed mutation frequencies depend on several factors that are difficult to control; these include gene copy number, parental and mutant cloning efficiencies, cell size, and cell-cell interactions. Elmore et al. (16) used stringent criteria and could not measure a difference in CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. VOL. 44 Tumor Heterogeneity mutation frequency between normal and transformed diploid human skin fibroblasts. Furthermore, although one might antici pate that aneuploidy itself would result in variant formation, and indeed some aneuploid tumor lines are unstable (81), in our experience aneuploid mammary lines in vivo can be highly vari able or relatively stable (30, 57). Recent determinations of cell variant production rates have tended to direct attention away from mutation to extra- or epigenetic mechanisms as the chief source of variability in tu mors. For example, Bosslet and Schirrmacher (8), Harris et al. (32), and Peterson et al. (70) have reported rates of 10"2 to10~5 variations/generation, which exceed by several orders of mag nitude the rates of 10"6 to 10~8 mutations/generation usually considered to be consistent with genetic mutation. Of course, the high rates could reflect a specific carcinogen-induced hypermutability (25). Nor are the differences in the rates of phenotypic variant appearance and mutation in normal versus neoplastia cells as large, on close examination, as the above frequencies imply. The data of Peterson ef al. (70) indicate mutation rates in cultures of normal breast epithelial cells that are nearly as high as those of their malignant counterparts. This suggests that some as yet unrecognized variable (genetic or epigenetic), found in normal and neoplastic cells, causes the high rates of pheno typic variation. In any case, the cognoscenti have, for better or for worse, shifted their attention in recent times to epigenetic mechanisms as an important factor in the generation of tumor heterogeneity. A hypothesis that narrows the apparent differences between genetic and epigenetic mechanisms was provided by Frost and Kerbel (26), who suggested that DNA hypomethylation, and the activation of otherwise repressed genes, may be the cause of tumor variant production. In support of this view are the follow ing: (a) hypomethylation accounts for the parallel between neo plastic and normal tissue development since alteration in DNA methylation plays a role in the latter (10,42); (b) hypomethylation is consistent with the cases of inappropriate gene expression in neoplastic progression (39); (c) methylation patterns are herita ble, passed on from parent to daughter cells (5); (d) methylation patterns change with high frequencies and rates reminiscent of variant formation (26). The evidence provided by these obser vations is circumstantial; direct evidence for the hypomethylation hypothesis is more limited. However, Feinberg and Vogelstein (18) have reported differential methylation of specific genes in different cells of a colon carcinoma and an instance of progres sive hypomethylation in a primary lung carcinoma and its (liver) métastases. In addition, the hypomethylating agent, 5-azacytidine, has been shown to induce cell variants in several tumor lines (26). Although suggestive, each of these studies is still incomplete proof. Missing is convincing evidence, in the differ ential methylation studies, that the changes observed are actually intraclonal and, in the 5-azacytidine studies, adequate measure ment of concentration and kinetic parameters to establish that the action of the agent is actually attributable to hypomethylation (specifically, hypomethylation of those genes for which putative expression is altered). Somatic cell fusion, another epigenetic process, has also been suggested as a mechanism of variant production (13,44). What ever the mechanism(s), it is subject to influences from outside the tumor cell. For example, in some tumor systems, the rates at which variants appear in a cell lineage are greater in vivo than in vitro (9, 30). Furthermore, variant production is not always a random event. Kiang ef al. (46) reported the periodic appearance and disappearance of several characteristics during the serial transplantation of GR mouse mammary tumors. Similarly, Vaage (86) found that, on repeated testing, serially passaged pieces of the same C3H mammary tumor underwent characteristic changes at about the same time. The histories of the several tumors passaged by Vaage were different, but among the lin eages derived from a given tumor, the temporal relations were identical. These examples of tumor progression, which is thought to result from the successive production and selection of new variants (63), suggest a degree of orderliness unexpected from a stochastic process. Some Complications Reports of tumor heterogeneity have in general been well received because they seem to explain troubling observations and experiences. The weakness of experimental cancer research has always been variability. Generalizations are hard to pin down. Experiments are often difficult to reproduce. By definition, vari ability is difficult to duplicate. In a way, the study of tumor heterogeneity answers a prayer; ¡(reproducible results are no longer a problem. Indeed, they become evidence, another ex ample of tumor heterogeneity! To the clinician, heterogeneity can be a serviceable explanation for the failure of a therapy: "What is lacking is a drug to which the few invasive and metastatic cells are sensitive! The remainder of the cancer is irrelevant." I have described this approach as a horse opera scenerio: the good tumor cells are in white hats, the bad ones in black. We "target" our magic bullets and await the hero's (heroine's, for a very few) reward (35). These simple notions ignore important complications. One is that the data which distinguish particular subpopulations as, for example, highly metastatic or nonmetastatic, fast or slow grow ing, drug resistant or sensitive are always obtained by studying isolated subpopulations or clones. Cloning is a way of multiplying almost identical components and is useful for magnifying the individual properties of tumor cells. However, it is now clear that tumors have a "societal" aspect which is changed or lost when the numbers of one component are varied at the expense of another. Study of isolated tumor subpopulations shows the behavior of cells when cloistered like monks, not when in their relevant tumor society. My associates and I have studied the ability of mammary tumor subpopulations to influence each other's growth and behavior. We found that subpopulations behave very differently when isolated and when growing and interacting together. Cell interactions alter properties as diverse as growth rate (37, 52), immunogenicity (56), sensitivity to drugs (51), and ability to metastasize (55). Parallel findings have been reported by Hauschka (33), Klein and Klein (47), Nowotny and Grobsman (64), Janssen and Revesz (41), and recently by Keyner eÃ-al. (45), Newcomb ef al. (61 ), Olsson and Ebbesen (66), Wang ef al. (88), and Woodruff (90). Especially interesting is the effect of subpopulation interaction on metastatic behavior described by Poste ef al. (73) and Miner ef al. (58). Cloned tumor cells may be unstable, changing from highly to poorly metastatic (and vice versa) from generation to generation. By contrast, the behavior of tumor lines formed 2261 JUNE 1984 Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. G. H. Heppner without cloning (and consequently possessing some of the di versity of the parental tumor) is far more stable. These mixed lines faithfully transmit a characteristic metastatic behavior through successive generations. This stability implies either (a) the presence in mixed populations of several lines, each mutable, but with mutability masked by averaging or because the meta static conversion in one line signals the reverse conversion of another or (b) the interaction of the unlike lineages in the mixed population in ways that suppress their intrinsic instabilities. Poste ef al. (73) and Miner ef al. (58) provided convincing evidence for the latter explanation by showing that donai lines, each of which is unstable when grown in isolation, are stabilized when grown together (either in vitro or in vivo). Not all clonal lines can be stabilized or can stabilize, and these abilities exist only among clones from the same tumor type, not among types of different histological origin. A general mechanism to explain tumor subpopulation interac tion has not been described (38). Our group has uncovered numerous examples of the ways that subpopulations can inter act. Some interactions require host participation (51, 52); others occur in vitro (37, 51). Some interactions involve a diffusable factor (37); others require cell contact (53). What is certain, however, is that tumor cells are similar to members of other societies. The ways in which they interact depend upon their potential and the circumstances in which they find themselves. We have recently extended this picture of a tumor ecosystem to include host cell components, lymphocytes, and macrophages. These emigres, like the autochthonic components, influence and are influenced by their tumor neighbors (49, 78). Interactions complicate the study of tumor heterogeneity because, while heterogeneity is detected by isolating cell subpopulations, the properties of the parental tumor cannot be deduced by the simple addition of its component parts; a purely reductionist analysis is bound to fail. A second, related complication is cell line instability. Several recent studies have raised doubts about the signifi cance of tumor heterogeneity by showing that daughter lines subcultured from variant populations behave differently than does the parental line; that is, that tumor subpopulations are reproductively unstable. At the outset, it should be recognized that this is not a generalization that holds for all tumor subpopulations and clones. Phenotype stability is yet another charac teristic by which different subpopulations vary, that is, are het erogeneous (57, 60). The systems used to demonstrate highfrequency instability are, in general, long-term cell lines. If genetic instability increases as a function of tumor progression (63), one would expect such lines to represent the furthest end of the spectrum. Nevertheless, as Stackpole (83) thoroughly docu mented, B16 melanoma clones have unstable characteristics with regard to metastasis, so that new lines derived from estab lished metastatic lines are themselves not necessarily metastatic, and nonmetastatic lines can give rise to metastatic clones. Poste ef al. (75) have shown that growing metastatic foci, of proven clonal origin, become heterogeneous with time. In other words, events in the primary tumor are recapitulated in the metastasis. Similar results using the KHT sarcoma line have already been cited (32). The group responsible for the latter work, under the leadership of Victor Ling, has proposed a model to explain their observations. Their "dynamic heterogeneity" model contrasts with models in which tumor subpopulations are thought of as stable units, subject only to selection by the host environment. In the dynamic heterogeneity model, although tumors are also 2262 considered to consist of multiple clones, the genotype of these clones is changeable. At a given time, a particular clone may give rise to metastatic cells, but, having done so, its progeny may back-mutate, producing a nonmetastatic phenotype. In this view, a tumor is a collection of unstable cells, the instability of which is masked in the whole by mutually canceling "forward" and "backward" mutations in the individual clones. To my mind, the "dynamic heterogeneity" model, white ingen ious, fails to explain critical experimental data. The model is in fact one of the alternative hypotheses tested by Poste et al. (73) and Miner ef al. (58) (see above). The dynamic heterogeneity model predicts that, if several unstable lines are grown in polyclonal mixtures, each will maintain a high rate of mutation. Poste ef al. (73) and Miner ef al. (58) found that clonal instability is suppressed, not just masked, in polyclonal cultures. While Ling's group (32) worked with tumor systems different from those of Poste ef al. (73) and Miner ef al. (58), the stabilization phenom enon has been demonstrated in several different tumor types, so there is no reason to believe that it is restricted to a particular system. The observation that clonal instability is suppressed by clonal interactions does not explain the origin of the instability. At a certain level, instability can be seen as a continuation of the processes discussed under "The Origin of Tumor Heterogeneity" as well as evidence of their dynamic nature. At issue in both cases is not the fact of change, however, but the frequency with which it occurs. Most investigations of clonal instability have been focused on the ability to metastasize. We may assume that metastasis, like virtually every complex phenotypic property that has been thoroughly studied, is not determined uniquely by a single gene but is an "emergent" property, resulting from both the direct and pleiotropic actions of many genes. Furthermore, there is more to the cell than genes and chromosomes. In somatic cell duplication, much information is presumably carried by cytoplasmic and membrane components, and because of the parallel binary nature of replication and cell duplication, extranuclear transfer can resemble the segregational information transfer of the genotype. Depending upon the nature of the informationbearing material (e.g., mRNA, organelles), extranuclear informa tion may remain an influence for varying periods of time and through varying numbers of cell divisions. Insofar as such infor mation influences gene penetrance, the variation in its transfer properties may suggest an instability in the genome itself. The problem that confronts us is, "What fills the enormous gulf that exists between the genotype and the phenotype of a cell?" Rather than implying that tumor heterogeneity, as defined here, does not exist, the apparent instability of the metastatic phenotype may bear witness to its extent and significance. Metastasis is considered to be a process in which only some of the cells in the primary tumor can engage and, as a complex process, it depends on a constellation of characteristics each of which can be transmitted to daughter cells and is required for maintenance of the metastatic phenotype. Change in any of the determinants of the process will lead to a change in metastatic potential. If the determinants are genes (by no means a necessity, as emphasized above), mutation at any of the several loci (per haps dozens) will appear phenotypically as a change in meta static potential. Obviously, under these circumstances, mutation rate deduced from phenotypic change would appear far higher than the true rate experienced by each gene. When pleiotropy is present, one cannot expect to obtain meaningful mutation rates CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. VOL. 44 Tumor Heterogeneity from observation of phenotypic change. Without detailed knowl edge of both the genetic and extragenetic mechanisms that underlie a particular change in phenoty pe, no rational basis exists for choosing a class of mechanism with a frequency of change that can be taken as a norm. Certainly, when the complexity of change in cellular systems is considered, it is logically inappro priate to adopt point mutation frequency as a standard of com parison, as is conventional, especially since point mutation may yet prove to be among the rarest sources of cell variability. A multigenic basis for the metastatic phenotype has other experimental consequences. Consider a single cell populating a new metastatic locus and duplicating (the argument does not change in cases when a few cells are involved). Early in the colonization process, the population will be homogeneous with respect to the metastatic phenotype. However, because several "determining" genes are at risk of mutation, there will be, in a short time, an accrual of nonmetastatic cells which cloning will divulge. Thus, it should be no wonder that clones of cells from metastatic foci may be no more capable of metastasis than cells from their parent tumor. Both are mixed cell populations. The development of a tumor is a complex process, characterized by the early appearance of cell line heterogeneity, constant produc tion of new cell lines, and evolving cell line interdependence. Although these processes might be modified by the experimen talist, none of them stops when he enters the picture. In addition to the problem of pleiotropy, there are other inherent difficulties to a meaningful assessment of cellular variability. Definition of the extent of variability depends upon the number of measurable characteristics and the precision with which the measurements can be made. It is likely that the number of different subpopulations in a tumor exceeds the number of measurable phenotypes. Furthermore, some characteristics, such as metastatic ability, do not lend themselves to stringent quantitative analysis. Under these circumstances, the distinction between experimental variability and phenotypic instability is clouded. Other characteristics, such as sensitivity to drugs or expression of a particular marker protein, may be capable of precise measurement in any particular experiment, but investi gators have come to accept a degree of variability between experiments as the norm. Tumor heterogeneity, then, is always going to be a "working definition," and a minimal one at that. The reality may be that the mosaic of characteristics isolated cell is different from that of every other. of every tem in which evolution through natural (and artificial) selection might take place and, at least superficially, tumor progression resembles evolution. However, change is not always evolution. Paradoxically, even though variability is necessary to evolution, the great heterogeneity within tumors can be interpreted as suggesting that selection is not a major determinant in the population biology of cancer. Presence of strong selective forces leads to homogeneous, not heterogeneous, populations, and on this basis one may be led to discount the role of selection in tumor progression. Before doing so, however, it may be valuable to look to organismal evolution for arguments that, by analogy, may be applicable to cancer biology. Instrumental in my own thinking on these problems is the work of Wright (92), especially his shifting balance theory of the evolution of species. A feature of this theory is its emphasis on the multifaceted processes that stand between genotype and phenotype, such that each phenotype is determined by many genes and each gene has numerous pleiotropic effects. The result is that evolution is determined by "natural selection among interactive systems." Wright argues that, in complex organisms which have thousands of genetic loci, among which there are numerous opportunities for interactions that favor survival, the observed phenotypes will be variable and will tend "to wander continually" because of accidents of sampling, selec tive migration, changes in the environment, etc. This "microevolution" allows for relatively rapid population adjustment as com pared to the rates to be expected were selection operating on a simple one gene-one phenotype organism. More rapid "macroevolution" occurs when a population encounters the opportunity for expansion offered by new niches in previously uninhabited territory, after surviving a catastrophe that has eliminated other species or when reaching, through microevolution, a new adap tive level that opens up previously nonexistent niches. The pleiotropy of the "malignant" genotype, the inherent vari ability of neoplastic populations, the interactions among tumor subpopulations, and the opportunity for niches in the cancer cell environment all suggest that a Wright-like "shifting balance" approach may be useful in understanding the temporal devel opment of tumors. As examples, one might begin by considering the dynamics of cell cloning or of tumor repopulation after chemotherapy as finding their analogues in the macroevolutionary thrust provided by expansion into new territory or survival of a catastrophe. Tumor Heterogeneity and Population Biology Conclusions The importance of multigenic determinants and pleiotropy, discussed above, is in the implication that tumor heterogeneity is not only extensive but also the "natural" condition of these cell Understanding of the nature of tumor heterogeneity has under gone change in the last 5 years. From early attempts to dem onstrate tumor subpopulations, we have progressed to studying their biological significance. It is clear that our first ideas about tumor heterogeneity were too simple. As isolation of tumor subpopulations became more routine, the phenotypic differences between them have come to seem less important. We began as tumor taxonomists seeking to classify the characteristics and origin of different neoplastic populations and now are develop mental biologists studying their ontogeny, structure, interactions, and collective behavior. In this maturing view, a particular isolated tumor subpopulation is unimportant except as a reminder of the diversity of the cell society from which it came. Recognition of tumor heterogeneity is essential to any theory of neoplastic development, as well as to experimental design and clinical populations. The variable cancer cell population appears to be a likely object upon which selective forces might act. The environ ment of a cancer is one in which selective forces are present. There are, for example, regional differences in oxygen supply, acidity, nutrient supply, and the presence or absence of immunocompetent infiltrates which give growth advantage to some, but not other, cells within their spheres of influence. Likewise, different organs also offer different microenvironments, and these will be advantageous to certain metastatic tumor variants. There are also powerful negative selection forces provided by the therapeutic efforts of the clinician. The combination of transmittable variability and environmental diversity suggests a sys 2263 JUNE 1984 Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. G. H. Heppner treatment. Tumor societies are highly adapted for survival. They survive natural and artificial (therapeutic) selection through het erogeneity by producing new variants to "outflank" it and by utilizing subpopulation interactions to counteract its destructive influence (52). Goldie and Goldman (27) have analyzed the ther apeutic implications of variant production, showing that early and combined therapy is required to defeat its protective effects. Cellular interaction also offers opportunity for intervention (36). Having recognized their complexity, we must now learn to anni hilate tumor societies. Acknowledgments I gratefully acknowledge the influence of all my colleagues, particularly Drs. 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The shifting balance theory and macroevolution. Annu. Rev. Genet., 16:1-19,1982. 2265 1984 Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research. Tumor Heterogeneity Gloria H. Heppner Cancer Res 1984;44:2259-2265. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/44/6/2259.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on January 27, 2016. © 1984 American Association for Cancer Research.