The Natural History of Neoplasia Newer Insights Into an Old Problem Henry C. Pitot, MD, PhD
The stages of initiation and promotion in the natural history of epidermal carcinogenesis have been known for many years. Recently, experimental systems other than skin have been shown to exhibit similar, if not completely analogous, stages in the natural history of neoplasia. In particular, the demonstration by Peraino and his associates that phenobarbital may enhance the production of hepatomas by a relatively subcarcinogenic dose of acetylaminofluorene was one of the first demonstrations of stages occurring in an extraepidermal neoplasm. Studies reported in this paper have demonstrated that administration of phenobarbital (0.05% in the diet) for 6 months following a single dose of diethylnitrosamine (5 to 10 mg/kg) given within 24 hours after partial hepatectomy resulted in a marked increase in the number of enzyme-altered foci in the liver as well as in the production of hepatocellular carcinomas. This was compared to animals receiving only a single dose of diethylnitrosamine following partial hepatectomy with no further treatment, in which only a relatively small number of foci were evident in the absence of phenobarbital feeding. Using three different enzyme markers, a distinct degree of phenotypic heterogeneity of the enzyme-altered foci in liver was demonstrated. These studies have shown that liver carcinogensis can be readily divided into two stages: a) initiation by a single dose of diethylnitrosamine following partial hepatectomy and b) promotion by the continuous feeding of phenobarbital. Furthermore, the immediate progeny of the initiated cells, the enzyme-altered focus, may be recognized by suitable microscopic means prior to the formation of gross lesions as required in the skin system. These initiated cell populations exhibit a degree of biochemical heterogeneity which reflects that seen in fully developed hepatic neoplasms, suggesting that promotion and progression in this system does not significantly alter the basic biochemical characteristics of the initiated cell. (Am J Pathol 89:401-412, 1977)
WHILE NUMEROUS OBSERVATIONS on the clinicopathologic characteristics of neoplasia in both men and animals have been described since the middle of the past century, the systematic study of the natural history of the neoplastic process in vivo dates principally from the pioneering studies of Berenblum and Shubik who reported, in the latter half of the fifth decade of this century, that the induction of carcinomas in the skin by hydrocarbons could be separated into two distinct stages. 1,2 These classic experiments demonstrated that the first stage of epidermal carcinogenesis, termed initiation, was brought about by the single direct application to the skin of mice of a carcinogenic hydrocarbon or, as later studies From the Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706. Presented at the Seventy-fourth Annual Meeting of the American Association of Pathologists, Toronto, Ontario, Canada, March 15, 1977. Address reprint requests to Dr. Henry C. Pitot, Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI
63706. 402
Vol. 89, No. 2 November 1977
NATURAL HISTORY OF NEOPLASIA
403
have shown, alkylating agents including ,8-propiolactone ' or by the oral administration of other compounds such as urethane.4 The stage of initiation appeared to occur relatively rapidly after the administration of the carcinogenic stimulus, and the change brought about was not characterized by a specific or even detectable permanent morphologic change in the structure of the epidermis or dermis. Subsequent to the administration of the initiating agent, if a second material were repeatedly applied to the initiated area of skin, neoplasms ultimately arose in response to both treatments. This phenomenon is seen schematically in Text-figure 1. Promoting agents in most instances induced a significant if not massive hyperplasia of the skin, although other studies clearly demonstrated that the hyperplasia itself without the initiator was insufficient to provoke the neoplastic transformation.5 Late studies by Tannenbaum and others'7 demonstrated that the phase of promotion could be modulated by dietary manipulation. On the other hand, the process of initiation appeared to be irreversible, although recent reports by Roe and his associatesI suggested that, if the time between initiation of the neoplastic transformation and the beginning of the process of promotion is extensively prolonged, many fewer neoplasms result. However, as has been pointed out by Van Duuren and his associates,' this effect may be primarily a reflection of the age of the animal. In any event, the present generally accepted concept of the characteristics of the stages of initiation and promotion in mouse skin is that the process of initiation is essentially an irreversible process, while that of promotion is capable of modulation, if not total reversibility, prior to the appearance of unqualified neoplasms. 1.
x
2.
x
3.
x
4.
l 4f 4 4 ff 4 4 4f 4f x > 144444444 X~~~~~~~~~~~~~~~~~~~~~~~0 4 4 4 4 4 4444444>
5.
4
No Tumors
4 s4s 4 44
>
l 4 4 4 4f 4f 4 4
Time X=Application of Initiator
Tumors
-> Tumors No Tumors No Tumors
>
4 =Application of Promoter
TEXT-FGURE 1 -Outline of the dassic experiments demonstrating the two-stage natural historv of carcinogenesis as exemplified in mouse skin.
404
PITOT
American Journal of Pathology
An extension of the two-stage concept was formulated by Foulds to include the progression of neoplasms from their earliest discernible form, usually a relatively benign lesion, to the highly malignant, rapidly growing malignant neoplasm.10 Characteristics of the phase of tumor progression identified by Foulds included a) the rule of independent progression of tumors, which maintains that progression occurs independently in different primary neoplasms within the same host, and b) the rule of independent progression of characters, which means that a variety of characteristics of a particular neoplasm progress independently of one another. The characteristics mentioned by Foulds included growth rate, invasiveness, metastatic capability, hormone responsiveness, and morphology. Unfortunately, it is not always possible to distinguish between the phase of tumor promotion and tumor progression, since these two blend with each other in the natural history of many neoplasms. Furthermore, a number of neoplasms, upon induction, may already represent a fully progressed population of neoplastic cells. On the other hand, in the vast majority of so-called spontaneous neoplasms of man and animals, the natural history of the neoplasm involves all of the stages of initiation, promotion, and progression. However, it has only been recently that these stages have been readily demonstrable in tissues other than skin. Such investigations in other organ and tissue systems, one of which will be considered in some detail here, have required a reevaluation and rethinking of some of the basic mechanisms thought to be involved in the natural history of neoplasia. Stages in Neoplastic Development in Nonepidermal Tissues Some of the earliest demonstrations of stages in the natural history of neoplasia in extraepidermal tissues were related to the effects of irradiation on tissues. Cole and Nowell demonstrated that irradiation of the liver by itself resulted in few if any neoplasms of this organ, whereas, subsequent to the irradiation, a stimulation to proliferate induced by the administration of carbon tetrachloride resulted in numerous hepatocellular carcinomas.11 Another example of a two-stage phenomenon was reported by Hicks et al.,12 who showed that the induction of bladder carcinoma following a single intravesicular administration of methyl-Nnitrosourea required the subsequent administration of more of the carcinogen or the feeding of saccharin. One of the more interesting and rapidly advancing areas of study of the natural history of neoplasia is that which is characteristic of hepatocarcinogenesis.
Vol. 89, No. 2 Novernber 1977
NATURAL HISTORY OF NEOPLASIA
405
Evience for Stages in the Development of Hepatoceilular Carcnomas in Rodents Some of the earliest indications that hepatocarcinogenesis in the rat was the result of several stages in its development were reported by Farber
and his associates,13 who demonstrated both morphologic and biochemical evidence that nodules of hepatocytes, termed regenerating or hyperplastic nodules, which resulted from feeding various chemical hepatocarcinogens, were in all likelihood the prenoplastic lesion from which developed the malignant hepatocellular carcinoma. The demonstration of carcinoma in cells that appeared to arise within the nodules was also reported,13 suggesting a precursor relationship of the nodule to the malignant neoplasm. On the other hand, as was shown by Farber and others, the majority of the regenerating or hyperplastic nodules disappeared on cessation of the feeding of the carcinogenic diet. Thus, despite the more recent suggestion that these nodules should be termed neoplastic nodules,14 it is difficult to understand how the precursor relationship of the nodule to carcinomas can be maintained if the existence of the putative precursor is so transient. More recent studies by Farber have suggested that the cells of the hyperplastic nodule do not disappear when the nodule vanishes from livers of rats on a normal diet, but rather that the cells are "remodeled" and again become part of the normal liver structure.15 Using a marker antigen, the PN antigen, these investigators have been able to demonstrate that many of the cells of the nodule remain in the remodeled liver and may under appropriate stimuli give rise to neoplasms. However, the direct staging phenomenon as seen in the skin is not so clear in the evolution of the hyperplastic nodule in the liver. In the late 1960s, Friedrich-Freksa and his associates 16 demonstrated that in animals given diethylnitrosamine, there appear in the liver, within 6 weeks of initiation of the diet, small foci of cells which by standard histopathologic techniques cannot be distinguished from normal liver cells but which exhibit a marked deficiency of the enzyme glucose-6phosphatase and therefore show as enzyme-deficient islands of hepatic tissue when a histochemical stain for this enzyme is applied to the section. These investigators theorized at this time that these cells were the precursors of hepatocellular carcinomas. Later studies by Rabes and his associates 17 demonstrated that islands of tissue deficient in canalicular ATPase, as well as glucose-6-phosphatase, possess a greater rate of DNA synthesis than the normal hepatocytes. In a number of such islands, foci of cells develop having an extremely high rate of DNA synthesis and an altered morphology quite comparable if not identical to that of hepatocellular carcinomas.1i
406
PITOT
American Journal of Pathology
In their studies, Scherer and Emmelot 18 reported that a linear relationship existed on a log-log scale between the number of enzyme-deficient islands produced in the liver after a single dose of diethylnitrosamine immediately after a partial hepatectomy and the dose of diethylnitrosamine administered, up to a dose of 30 mg/kg. Beyond this dose no such relationship was maintained, but more significantly, carcinomas developed, while at doses of 30 mg or less only enzyme-deficient islands appeared during the period of study of the animal. Furthermore, at the lower doses as the animal aged, the size of the islands increased, but their number did not. These studies also strongly supported the precursor relationship of the enzyme-deficient islands to hepatocellular carcinomas and furthermore suggested that diethylnitrosamine in sufficient amounts could "promote" the enzyme-deficient islands to hepatocellular carcinomas. The Two-Stage Hypothesis in Hepatocarcinogenesis The first distinct evidence that hepatocarcinogenesis was the result of at least two stages in its natural history was reported by Peraino and his
associates."9 These investigators demonstrated that upon feeding acetylaminofluorene to animals for 3 weeks followed by 1 week of basal diet and subsequently 0.05% phenobarbital in the diet, the number of hepatomas resulting was more than tenfold greater in the phenobarbital-treated animals as compared to those receiving no phenobarbital. The duration of the experiment extended for somewhat more than 5 months after the administration of the carcinogen, during which time the animal was constantly maintained on a diet containing phenobarbital. In addition, Peraino and his associates demonstrated that the feeding of phenobarbital to mice having a relatively high incidence of "spontaneous" hepatomas resulted in a much more rapid appearance of these hepatomas than seen in untreated animals.20 While phenobarbital has been suggested to be a carcinogen both for the. rat and the mouse, in view of its ability to bring out spontaneously occurring lesions, careful controls extending for the life-span of the animal must be carried out to insure that phenobarbital is not merely increasing a specific base line of neoplasms when it is tested as a carcinogen. Morphologic and Functional Characteristics of Initiated and Promoted Hepatocytes In attempting to relate the experiments of Peraino with those of Scherer, Friedrich-Freksa, Farber, and others, one may hypothesize that
the enzyme-deficient islands do represent the immediate progeny of the initiated cell(s). To demonstrate this it would be necessary to determine whether populations of enzyme-deficient islands may be promoted to
Vol. 89, No. 2 Novernber 1977
NATURAL HISTORY OF NEOPLASIA
407
hepatocellular carcinomas and/or whether their number may also be increased. Experiments in this laboratory were therefore devised utilizing the procedure described by Scherer and Emmelot,15 which involves a partial hepatectomy followed within 24 hours by a single gastric installation of diethylnitrosamine in water at doses between 1 and 30 mg/kg. This was carried out in our laboratory, and the animals were then allowed to remain on a normal basal diet for 2 months. At the end of this time, one set of animals was placed on a diet containing 0.05% phenobarbital while the other was fed the same diet but with no phenobarbital. At the end of 6 to 8 months the animals were sacrificed, and their livers examined grossly and morphologically as well as for the histochemical presence of three different enzymes. The enzymes utilized as markers of the altered hepatic cells are listed in Table 1. The rationale for the use of these enzymes as markers has already been discussed in the case of glucose-6-phosphatase, while the data of Rabes has demonstrated that the absence of canalicular ATPase is as good a marker of altered foci of hepatic cells or better than the former. Y-Glutamyl transpeptidase has recently been shown by Fiala and his associates 21 to increase markedly during hepatocarcinogenesis and also to be at high levels in many transplanted hepatomas. All of these enzymes occur within membranes of the cell, as can be seen from the table. Investigations by Goldfarb and Pugh22 have demonstrated that enzymedeficient islands contain i -glutamyltranspeptidase, whereas the remainder of adult liver do not. Utilizing serial frozen sections, it thus became possible to determine the number of enzyme-deficient islands in liver sections using three different marker enzymes: two of these, glucose-6-phosphatase and ATPase, would be deficient in the altered foci of liver cells, and the other,-, glutamyltranspeptidase, would be present in these foci. On this basis we were able to demonstrate two significant characteristics of animals undergoing the regimen indicated above. First, the number of enzyme-altered Table 1-Enzyme Markers Used to Identify Altered Foci of Hepatic CelIs Enzyme
Subcellular localization
Glucose-6-phosphatase
Endoplasmic reticulum membrane Bile canalicular membrane Bile canalicular and/or plasma membrane
Canalicular ATPase
Y-Glutamyl transpeptidase
Reaction catalyzed Glucose-6-P - glucose + PI ATP -. ADP + Y
Pi
-Glutamyl methoxynaphthylamine R-glutamyl + methoxynaphthylamine
408
PITOT
American Journal of Pathology
islands in animals fed phenobarbital was approximately seven times higher than those in the controls (Table 2). In phenobarbital-treated animals, large lesions histologically compatible with hepatocellular carcinomas were found, whereas no such tumors were seen in animals not receiving phenobarbital. Second, by utilizing three different enzyme stains and monitoring the same enzyme-altered island by means of serial sections, it was clear that not all islands exhibited the same phenotype. The results of overlaying tracings of the individual enzyme-altered foci employed to determine the biochemical phenotype with respect to the three enzymes in Table 1 of each focus is seen in Text-figure 2. It is apparent from such tracings that the enzyme-altered foci exhibit all possible enzyme patterns, with the seven possible biochemical phenotypes evident. As we have previously reported, using glucose-6-phosphatase and serine dehydratase,23 there is significant biochemical heterogeneity in the population of islands in both sets of animals. This phenotypic heterogeneity is quite reminiscent of that which has been reported from our laboratory to be present in both primary and transplanted hepatocellular carcinomas in the rat.24 In addition, hepatocellular carcinomas have been shown to be heterogeneous for glucose-6-phosphatase and, to a lesser extent, -' -glutamyltranspeptidase.2126 Conclusions That specific stages exist during the genesis of hepatocellular carcinomas seems no longer to be a question but a reality. The principal questions remaining are concerned with the nature, morphology, and biochemistry of the initiated cell population and its immediate progeny, and the modulation of tumor promotion and progression as has been described in the case of the skin. The experiments reported in this discussion do not completely answer these questions but propose interesting Table 2-Effect of Phenobarbital on the Production of Altered Foci or Islands of Hepatocytes in Livers of Rats Given Diethylnitrosamine After Partial Hepatectomy
Treatment
No. of islands/sq cm liver at 18 mo
No. of hepatomas
0.05% Phenobarbital DEN + PH DEN + PH + 0.05% phenobarbital (6 mo)
0 18±5 132 ± 50
0 0 7
Six to ten animals were sacrificed in each group 8 to 10 months after partial hepatectomy (PH) and administration of diethylnitrosamine (DEN). See text for further details of the experiment.
\'- J~ ~ _-.6 "E NATURAL HISTORY OF NEOPLASIA
Vol. 89, No. 2 November 1977
409
\~~~~~~~~
VrA~~~~~~~~~~~~~m
-
0
The stages of initiation and promotion in the natural history of epidermal carcinogenesis have been known for many years. Recently, experimental systems other than skin have been shown to exhibit similar, if not completely analogous, stages in the natural history of neoplasia. In particular, the demonstration by Peraino and his associates that phenobarbital may enhance the production of hepatomas by a relatively subcarcinogenic dose of acetylaminofluorene was one of the first demonstrations of stages occurring in an extraepidermal neoplasm. Studies reported in this paper have demonstrated that administration of phenobarbital (0.05% in the diet) for 6 months following a single dose of diethylnitrosamine (5 to 10 mg/kg) given within 24 hours after partial hepatectomy resulted in a marked increase in the number of enzyme-altered foci in the liver as well as in the production of hepatocellular carcinomas. This was compared to animals receiving only a single dose of diethylnitrosamine following partial hepatectomy with no further treatment, in which only a relatively small number of foci were evident in the absence of phenobarbital feeding. Using three different enzyme markers, a distinct degree of phenotypic heterogeneity of the enzyme-altered foci in liver was demonstrated. These studies have shown that liver carcinogensis can be readily divided into two stages: a) initiation by a single dose of diethylnitrosamine following partial hepatectomy and b) promotion by the continuous feeding of phenobarbital. Furthermore, the immediate progeny of the initiated cells, the enzyme-altered focus, may be recognized by suitable microscopic means prior to the formation of gross lesions as required in the skin system. These initiated cell populations exhibit a degree of biochemical heterogeneity which reflects that seen in fully developed hepatic neoplasms, suggesting that promotion and progression in this system does not significantly alter the basic biochemical characteristics of the initiated cell. (Am J Pathol 89:401-412, 1977)
WHILE NUMEROUS OBSERVATIONS on the clinicopathologic characteristics of neoplasia in both men and animals have been described since the middle of the past century, the systematic study of the natural history of the neoplastic process in vivo dates principally from the pioneering studies of Berenblum and Shubik who reported, in the latter half of the fifth decade of this century, that the induction of carcinomas in the skin by hydrocarbons could be separated into two distinct stages. 1,2 These classic experiments demonstrated that the first stage of epidermal carcinogenesis, termed initiation, was brought about by the single direct application to the skin of mice of a carcinogenic hydrocarbon or, as later studies From the Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706. Presented at the Seventy-fourth Annual Meeting of the American Association of Pathologists, Toronto, Ontario, Canada, March 15, 1977. Address reprint requests to Dr. Henry C. Pitot, Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI
63706. 402
Vol. 89, No. 2 November 1977
NATURAL HISTORY OF NEOPLASIA
403
have shown, alkylating agents including ,8-propiolactone ' or by the oral administration of other compounds such as urethane.4 The stage of initiation appeared to occur relatively rapidly after the administration of the carcinogenic stimulus, and the change brought about was not characterized by a specific or even detectable permanent morphologic change in the structure of the epidermis or dermis. Subsequent to the administration of the initiating agent, if a second material were repeatedly applied to the initiated area of skin, neoplasms ultimately arose in response to both treatments. This phenomenon is seen schematically in Text-figure 1. Promoting agents in most instances induced a significant if not massive hyperplasia of the skin, although other studies clearly demonstrated that the hyperplasia itself without the initiator was insufficient to provoke the neoplastic transformation.5 Late studies by Tannenbaum and others'7 demonstrated that the phase of promotion could be modulated by dietary manipulation. On the other hand, the process of initiation appeared to be irreversible, although recent reports by Roe and his associatesI suggested that, if the time between initiation of the neoplastic transformation and the beginning of the process of promotion is extensively prolonged, many fewer neoplasms result. However, as has been pointed out by Van Duuren and his associates,' this effect may be primarily a reflection of the age of the animal. In any event, the present generally accepted concept of the characteristics of the stages of initiation and promotion in mouse skin is that the process of initiation is essentially an irreversible process, while that of promotion is capable of modulation, if not total reversibility, prior to the appearance of unqualified neoplasms. 1.
x
2.
x
3.
x
4.
l 4f 4 4 ff 4 4 4f 4f x > 144444444 X~~~~~~~~~~~~~~~~~~~~~~~0 4 4 4 4 4 4444444>
5.
4
No Tumors
4 s4s 4 44
>
l 4 4 4 4f 4f 4 4
Time X=Application of Initiator
Tumors
-> Tumors No Tumors No Tumors
>
4 =Application of Promoter
TEXT-FGURE 1 -Outline of the dassic experiments demonstrating the two-stage natural historv of carcinogenesis as exemplified in mouse skin.
404
PITOT
American Journal of Pathology
An extension of the two-stage concept was formulated by Foulds to include the progression of neoplasms from their earliest discernible form, usually a relatively benign lesion, to the highly malignant, rapidly growing malignant neoplasm.10 Characteristics of the phase of tumor progression identified by Foulds included a) the rule of independent progression of tumors, which maintains that progression occurs independently in different primary neoplasms within the same host, and b) the rule of independent progression of characters, which means that a variety of characteristics of a particular neoplasm progress independently of one another. The characteristics mentioned by Foulds included growth rate, invasiveness, metastatic capability, hormone responsiveness, and morphology. Unfortunately, it is not always possible to distinguish between the phase of tumor promotion and tumor progression, since these two blend with each other in the natural history of many neoplasms. Furthermore, a number of neoplasms, upon induction, may already represent a fully progressed population of neoplastic cells. On the other hand, in the vast majority of so-called spontaneous neoplasms of man and animals, the natural history of the neoplasm involves all of the stages of initiation, promotion, and progression. However, it has only been recently that these stages have been readily demonstrable in tissues other than skin. Such investigations in other organ and tissue systems, one of which will be considered in some detail here, have required a reevaluation and rethinking of some of the basic mechanisms thought to be involved in the natural history of neoplasia. Stages in Neoplastic Development in Nonepidermal Tissues Some of the earliest demonstrations of stages in the natural history of neoplasia in extraepidermal tissues were related to the effects of irradiation on tissues. Cole and Nowell demonstrated that irradiation of the liver by itself resulted in few if any neoplasms of this organ, whereas, subsequent to the irradiation, a stimulation to proliferate induced by the administration of carbon tetrachloride resulted in numerous hepatocellular carcinomas.11 Another example of a two-stage phenomenon was reported by Hicks et al.,12 who showed that the induction of bladder carcinoma following a single intravesicular administration of methyl-Nnitrosourea required the subsequent administration of more of the carcinogen or the feeding of saccharin. One of the more interesting and rapidly advancing areas of study of the natural history of neoplasia is that which is characteristic of hepatocarcinogenesis.
Vol. 89, No. 2 Novernber 1977
NATURAL HISTORY OF NEOPLASIA
405
Evience for Stages in the Development of Hepatoceilular Carcnomas in Rodents Some of the earliest indications that hepatocarcinogenesis in the rat was the result of several stages in its development were reported by Farber
and his associates,13 who demonstrated both morphologic and biochemical evidence that nodules of hepatocytes, termed regenerating or hyperplastic nodules, which resulted from feeding various chemical hepatocarcinogens, were in all likelihood the prenoplastic lesion from which developed the malignant hepatocellular carcinoma. The demonstration of carcinoma in cells that appeared to arise within the nodules was also reported,13 suggesting a precursor relationship of the nodule to the malignant neoplasm. On the other hand, as was shown by Farber and others, the majority of the regenerating or hyperplastic nodules disappeared on cessation of the feeding of the carcinogenic diet. Thus, despite the more recent suggestion that these nodules should be termed neoplastic nodules,14 it is difficult to understand how the precursor relationship of the nodule to carcinomas can be maintained if the existence of the putative precursor is so transient. More recent studies by Farber have suggested that the cells of the hyperplastic nodule do not disappear when the nodule vanishes from livers of rats on a normal diet, but rather that the cells are "remodeled" and again become part of the normal liver structure.15 Using a marker antigen, the PN antigen, these investigators have been able to demonstrate that many of the cells of the nodule remain in the remodeled liver and may under appropriate stimuli give rise to neoplasms. However, the direct staging phenomenon as seen in the skin is not so clear in the evolution of the hyperplastic nodule in the liver. In the late 1960s, Friedrich-Freksa and his associates 16 demonstrated that in animals given diethylnitrosamine, there appear in the liver, within 6 weeks of initiation of the diet, small foci of cells which by standard histopathologic techniques cannot be distinguished from normal liver cells but which exhibit a marked deficiency of the enzyme glucose-6phosphatase and therefore show as enzyme-deficient islands of hepatic tissue when a histochemical stain for this enzyme is applied to the section. These investigators theorized at this time that these cells were the precursors of hepatocellular carcinomas. Later studies by Rabes and his associates 17 demonstrated that islands of tissue deficient in canalicular ATPase, as well as glucose-6-phosphatase, possess a greater rate of DNA synthesis than the normal hepatocytes. In a number of such islands, foci of cells develop having an extremely high rate of DNA synthesis and an altered morphology quite comparable if not identical to that of hepatocellular carcinomas.1i
406
PITOT
American Journal of Pathology
In their studies, Scherer and Emmelot 18 reported that a linear relationship existed on a log-log scale between the number of enzyme-deficient islands produced in the liver after a single dose of diethylnitrosamine immediately after a partial hepatectomy and the dose of diethylnitrosamine administered, up to a dose of 30 mg/kg. Beyond this dose no such relationship was maintained, but more significantly, carcinomas developed, while at doses of 30 mg or less only enzyme-deficient islands appeared during the period of study of the animal. Furthermore, at the lower doses as the animal aged, the size of the islands increased, but their number did not. These studies also strongly supported the precursor relationship of the enzyme-deficient islands to hepatocellular carcinomas and furthermore suggested that diethylnitrosamine in sufficient amounts could "promote" the enzyme-deficient islands to hepatocellular carcinomas. The Two-Stage Hypothesis in Hepatocarcinogenesis The first distinct evidence that hepatocarcinogenesis was the result of at least two stages in its natural history was reported by Peraino and his
associates."9 These investigators demonstrated that upon feeding acetylaminofluorene to animals for 3 weeks followed by 1 week of basal diet and subsequently 0.05% phenobarbital in the diet, the number of hepatomas resulting was more than tenfold greater in the phenobarbital-treated animals as compared to those receiving no phenobarbital. The duration of the experiment extended for somewhat more than 5 months after the administration of the carcinogen, during which time the animal was constantly maintained on a diet containing phenobarbital. In addition, Peraino and his associates demonstrated that the feeding of phenobarbital to mice having a relatively high incidence of "spontaneous" hepatomas resulted in a much more rapid appearance of these hepatomas than seen in untreated animals.20 While phenobarbital has been suggested to be a carcinogen both for the. rat and the mouse, in view of its ability to bring out spontaneously occurring lesions, careful controls extending for the life-span of the animal must be carried out to insure that phenobarbital is not merely increasing a specific base line of neoplasms when it is tested as a carcinogen. Morphologic and Functional Characteristics of Initiated and Promoted Hepatocytes In attempting to relate the experiments of Peraino with those of Scherer, Friedrich-Freksa, Farber, and others, one may hypothesize that
the enzyme-deficient islands do represent the immediate progeny of the initiated cell(s). To demonstrate this it would be necessary to determine whether populations of enzyme-deficient islands may be promoted to
Vol. 89, No. 2 Novernber 1977
NATURAL HISTORY OF NEOPLASIA
407
hepatocellular carcinomas and/or whether their number may also be increased. Experiments in this laboratory were therefore devised utilizing the procedure described by Scherer and Emmelot,15 which involves a partial hepatectomy followed within 24 hours by a single gastric installation of diethylnitrosamine in water at doses between 1 and 30 mg/kg. This was carried out in our laboratory, and the animals were then allowed to remain on a normal basal diet for 2 months. At the end of this time, one set of animals was placed on a diet containing 0.05% phenobarbital while the other was fed the same diet but with no phenobarbital. At the end of 6 to 8 months the animals were sacrificed, and their livers examined grossly and morphologically as well as for the histochemical presence of three different enzymes. The enzymes utilized as markers of the altered hepatic cells are listed in Table 1. The rationale for the use of these enzymes as markers has already been discussed in the case of glucose-6-phosphatase, while the data of Rabes has demonstrated that the absence of canalicular ATPase is as good a marker of altered foci of hepatic cells or better than the former. Y-Glutamyl transpeptidase has recently been shown by Fiala and his associates 21 to increase markedly during hepatocarcinogenesis and also to be at high levels in many transplanted hepatomas. All of these enzymes occur within membranes of the cell, as can be seen from the table. Investigations by Goldfarb and Pugh22 have demonstrated that enzymedeficient islands contain i -glutamyltranspeptidase, whereas the remainder of adult liver do not. Utilizing serial frozen sections, it thus became possible to determine the number of enzyme-deficient islands in liver sections using three different marker enzymes: two of these, glucose-6-phosphatase and ATPase, would be deficient in the altered foci of liver cells, and the other,-, glutamyltranspeptidase, would be present in these foci. On this basis we were able to demonstrate two significant characteristics of animals undergoing the regimen indicated above. First, the number of enzyme-altered Table 1-Enzyme Markers Used to Identify Altered Foci of Hepatic CelIs Enzyme
Subcellular localization
Glucose-6-phosphatase
Endoplasmic reticulum membrane Bile canalicular membrane Bile canalicular and/or plasma membrane
Canalicular ATPase
Y-Glutamyl transpeptidase
Reaction catalyzed Glucose-6-P - glucose + PI ATP -. ADP + Y
Pi
-Glutamyl methoxynaphthylamine R-glutamyl + methoxynaphthylamine
408
PITOT
American Journal of Pathology
islands in animals fed phenobarbital was approximately seven times higher than those in the controls (Table 2). In phenobarbital-treated animals, large lesions histologically compatible with hepatocellular carcinomas were found, whereas no such tumors were seen in animals not receiving phenobarbital. Second, by utilizing three different enzyme stains and monitoring the same enzyme-altered island by means of serial sections, it was clear that not all islands exhibited the same phenotype. The results of overlaying tracings of the individual enzyme-altered foci employed to determine the biochemical phenotype with respect to the three enzymes in Table 1 of each focus is seen in Text-figure 2. It is apparent from such tracings that the enzyme-altered foci exhibit all possible enzyme patterns, with the seven possible biochemical phenotypes evident. As we have previously reported, using glucose-6-phosphatase and serine dehydratase,23 there is significant biochemical heterogeneity in the population of islands in both sets of animals. This phenotypic heterogeneity is quite reminiscent of that which has been reported from our laboratory to be present in both primary and transplanted hepatocellular carcinomas in the rat.24 In addition, hepatocellular carcinomas have been shown to be heterogeneous for glucose-6-phosphatase and, to a lesser extent, -' -glutamyltranspeptidase.2126 Conclusions That specific stages exist during the genesis of hepatocellular carcinomas seems no longer to be a question but a reality. The principal questions remaining are concerned with the nature, morphology, and biochemistry of the initiated cell population and its immediate progeny, and the modulation of tumor promotion and progression as has been described in the case of the skin. The experiments reported in this discussion do not completely answer these questions but propose interesting Table 2-Effect of Phenobarbital on the Production of Altered Foci or Islands of Hepatocytes in Livers of Rats Given Diethylnitrosamine After Partial Hepatectomy
Treatment
No. of islands/sq cm liver at 18 mo
No. of hepatomas
0.05% Phenobarbital DEN + PH DEN + PH + 0.05% phenobarbital (6 mo)
0 18±5 132 ± 50
0 0 7
Six to ten animals were sacrificed in each group 8 to 10 months after partial hepatectomy (PH) and administration of diethylnitrosamine (DEN). See text for further details of the experiment.
\'- J~ ~ _-.6 "E NATURAL HISTORY OF NEOPLASIA
Vol. 89, No. 2 November 1977
409
\~~~~~~~~
VrA~~~~~~~~~~~~~m
-
0
