cooperative action has been analyzed most thoroughly inmammary neoplasia in mice, which will be discussed firstto illustrate some general principles of wider applicability.

MAI\'I VIARY NEOPLASIA IN NIICE

The detailed analysis of mammary neoplasia in inbredmice has disclosed 3 important etiologic factors, comprising, as stated in Bittner's 3-factor hypothesis, a geneticfactor, a hormonal factor, and a milk-borne tumor agentwith the properties of a virus and now usually called themilk agent or the Bittner virus. Within certain limits,an excess of one factor can compensate for deficiency inanother, but ordinarily, to ensure a high and early mcidence of mammary tumors, the 3 factors must act cooperatively. i\Iammary neoplasia develops spontaneouslyand can be induced experimentally by administering car

This paper is concerned primarily with the multiplicityof factors, acting simultaneously or consecutively, that areimplicated in the development of individual tumors. Themost conspicuous primary inductive or causative mechanisms of carcinogenesis, as revealed by experimentalanalysis, do not necessarily provide the best clues topracticable measures of prophylaxis and therapy in man.The present discussion therefore is not restricted to theactions of proved carcinogens but extended to the variedfactors both carcinogenic and noncarcinogenic that to asignificant degree influence the initiation of neoplasiaand its subsequent course. The title applies most directlyto the multiple factors that act cooperatively to induce aparticular kind of neoplasia in a single individual. The

The abbreviations used are: DNA, deoxyribonucleic acid;RNA, rihonucleic acid.

1339

Multiple Etiologic Factors in Neoplastic Development

LESLIE FOULDS

Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fuiharn Road, Londan S.W. 8, England

SUMMARY

This discussion is concerned with the multiplicity of factors that influence the initiation of neoplasia and its subsequent course. Factors that are not strictly “carcinogenie― may, nevertheless, affect the ultimate result decisively. Both genetic andnongenetic factors are implicated in the delivery of a carcinogen to the target tissuein an effective form arid amount, and others govern the responsiveness of the tissue.Several factors may operate concurrently or consecutively. The cooperative actionof genetic and hormonal factors together with a milk-borne virus is ordinarily mosteffective in producing a high yield of mammary tumors in mice. Epidermal carcinogenesis provides evidence of successive stages differently controlled. The first stageof initiation establishes a persistent region of incipient neoplasia whence tumors ofvaried kinds emerge at a later time. When the carcinogenic stimulus is optimal andthe genetic sensitivity high, the early lesions are multiple, benign, or “imperfect―neoplasms of the kind usually described as “precancerous― ; they are important because progression to malignant neoplasia may take place in one or more of them andbecause they disclose a region of incipient neoplasia within which malignant tumorsmay emerge later without spatial relationship to the early lesions.

Effective management of neoplastic disease is likely to depend on better knowledgethan is yet available of the successive stages of neoplastic development and of thediverse factors operating at each of them. Different forms of neoplasia will probablycall for different kinds of management. Initiation by known carcinogenic agentsshould be minimized. If the initiating agent is unknown, progression n-maystill becontrolled effectively, as in neoplasia of the uterine cervix, by treatment at the “precancer― stage of carcinoma in situ. Even at later stages, some control is practicable,as evidenced by the substantial, if limited, benefits of endocrine therapy for carcinoma of the breast and prostate.

Initiation, the crucial event in experimental carcinogenesis, probably entails persistent replicable change in the affected cells, not necessarily of the kind described asmutation. Recent developments in the theory of genetic structure and genetic act.ion will predictably become increasingly important in the study of neoplasia and arediscussed here in a preliminary way.

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cinogenic hydrocarbons or by irradiation in certain mice inwhich no milk agent is demonstrable. The Bittner virusis an important etiologic factor in mice but not an indispensable one, and miocomparable agent has been demonstrated in animals other than mice. The genetic factoris complex. Multiple genetic factors are implicated, andthey affect several different mechanisms, including thepropagation and transmission of the milk agent, thespecific reactivity of mammary tissues to particular hormones, and the production of those hormones. It remainsto be decided whether or not genetic factors determine in amore direct way the liability of mammary epithelium toneoplastic change.

The hormonal factor also is complex. Several or manyhormones contribute to the end result. Formerly theimportance of estrogens was exaggerated ; if forced to pickone hormone in preference to the others, many investigators would now choose prolactin. In inbred mice thatcarry the milk agent, the hormonal factor is doubtfullycausative or inductive. Its action seems to be mainlypermissive or supportive, being restricted to eliciting andmaintaining a sufficiency of mammary tissue upon whichother factors can operate to initiate neoplasia (4). Inother circumstances, when hormones evoke mammary tumors in mice lacking the milk agent or in rats, whichrarely develop mammary carcinomas spontaneously, theiraction is probably inductive and, surely, more thanpermissive.

Genetic factors severely restrict the inductive action ofhormones. Prolonged administration of hormones, especially estrogens, to innumerable guinea pigs and hamstershas not yet induced mammary neoplasia, nor has it doneso in the much fewer experiments on monkeys. Only 1mammary carcinoma seems to have been induced experimentally in guinea pigs and that by radioactive thorium(Thorotrast) injected below a nipple (17). The fewmammary tumors induced in hamsters have resulted fromintragastric administration of a carcinogenic hydrocarbon(14). In general, the 2 most consistentlysuccessfulmethods for inducing mammary neoplasia in laboratoryanimals are intragastric administration of chemical carcinogens and irradiation. Nevertheless, the effectivenessof the chemical carcinogens is considerably affected byhormones, which are important at least permissively(36, 37, 43, 51). Moreover, as first shown by Shay et at.(52), hormonesinfluencethe typeof tumorinducedby thecarcinogens ; when estrogenic stimulation preponderatesmost of the tumors are carcinomas, whereas a balancedstimulation by androgens and estrogens evokes fibroadenomas. This action of hormones may be described asdirective; it does not initiate neoplasia, but it directs itscourse.

Mammary neoplasia, broadly surveyed in animals ofvarious species, confirms other evidence to the effect thattumors of one general type can be elicited in severaldifferent ways ; it illustrates how multiple factors can actcooperatively to produce a maximum effect and showshow one factor, such as the hormonal factor, can act in atleast 3 different ways, namely, inductively, directively or,merely, permissively. Genetic factors are decisive in someof the inductive mechanisms and are important, at least

permissively, in all. In certain circumstances, even withother factors operating optimally, the permissive factorsmay be the limiting or decisive ones.

Mammary neoplasia in mice serves also to introduceanother matter that needs to be taken into account in ageneral discussion of multiple etiologic factors, namely,the possibility that varied factors operate at differentstages of neoplastic development.. Over 50 years agoApolant and Haaland described what are now calledhyperplastic nodules in the breasts of mice, and Haaland(33) believed that they were precursors of mammarycarcinomas. About a quarter of a century later Gardner,followed by others, confirmed Haaland's opinion by observations on inbred mice. It has become recognizedthat most mammary carcinomas in inbred mice developeither in hyperplastic nodules or in bigger lesions calledplaques, which have been described more recently (21).These precursor lesions and in particular the plaques,which can be observed clinically, have yielded illuminatinginformation about the development of tumors throughqualitatively different stages as a result of irreversiblechanges in tumor cells. This mode of development hasbeen called progression (19, 20).

Histologic examinations indicate that. most mouse mammary carcinomas develop by focal or multifocal progression in nodules or plaques. The miodules are often verynumerous, but carcinoma develops in only one or a few ofthem whereas the others persist indolently or regress.Plaques are usually multiple but not numerous, and progression to carcinoma takes place in only one of them at atime. Progression may be multifocal, but it is restrictedto a small'fraction of the plaque tissue (20, 21). Clinicalobservations disclose a remarkable responsiveness ofplaques to pregnancy ; the plaques grow during the 2ndhalf of pregnancy and regress quickly after part.urition.Plaques are conditional neoplasms that grow only in certam appropriate environments. During intermissions ofbreeding they remain in abeyance, and in mice killedduring this period, fibrotic remnants of plaques containingatrophic tubules lined by ragged epithelium are sometimespresent. Often traces of the regressed plaques are hardto find ; yet, in mice that live, regressed plaques alwaysgrow again, at the same place, during the 2nd half of thenext pregnancy, however long it is delayed. Some residualneoplasia (24) with a capacity for neoplastic growth mustpersist throughout the period of regression, even though itis hard to find until it is stimulated to grow by anotherpregnancy. Cycles of growth and regression may recurin several successive pregnancies, but often one plaquechanges its behavior abruptly and continues to grow afterparturition whereas other plaques in the same mouseregress, as all of them had done after former pregnancies.A pregnancy-independenttumor is thus established byprogression in a pregnancy-dependent plaque. It is emphasized that progression is not a mere extension of apre-existing lesion in space and time but a revolutionarychange in a portion of the old lesion establishing a tumorwith new properties not formerly present.

It is not known whether or not extrinsic factors directlyincite progression through nodule or plaque to carcinoma.Hormones are essential for progression through nodules to

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carcinoma but only, it seems, in a permissive sense, andthe hormonal requirements vary from one stage of development to another (4). Although not demonstrated inmammary neoplasia in mice, there is evidence for the important possibility that the hormonal environment prevailing during the early stages of neoplastic developmentacts directively to determine the hormone responsivenessof the definitive tumor and, in women, the reaction toendocrine therapy (15, 42).

The hormone responsiveness of mammary plaques inmice has proved convenient as a “marker―for the study ofsome general principles of tumor progression applicable tovaried types of neoplastic development in animals andin man.

TUMOR PROGRESSION

Some general principles or rules of progression wereproposed in 1949 and have been re-evaluated recently(25). Two of them are especially relevant to the presentdiscussion. The 1st is the rule of independent progressionof tumors, which maintains that progression occurs independently in different primary tumors in the same animal.It is valid for the neoplastic diseases of animals and of manthat are characterized by multiple primary tumors or“precancerous―or “precursor―lesions. As a rule progression to carcinoma takes place at one time in only oneof the considerable number of lesions that may be presenton the skin of men exposed to carcinogenic chemicals or ofchildren suffering from xeroderma pigmentosum. Similarly only one or a few of the abundant papillomas offamilial intestinal polyposis give origin to carcinoma byprogression. Many other examples could be cited but, ingeneral, neither clinical observers nor laboratory investigators have recorded the developmental histories and fatesof individual lesions in sufficient detail.

The 2nd important rule is the rule of independent progression of characters ; different characters of a particulartumor undergo progression independently of one another.This rule leads to the more general proposition that thestructure and behavior of tumors are determined bynumerous unit characters that, within wide limits, areindependently variable, capable of highly varied combinations, and apt to progress independently. This rule hasproved valid and useful in diverse studies of neoplasia inanimals and in man. The associations of characters suchas growth rate, invasiveness, powers of metastasis, responsiveness to hormones, and morphologic characters in tumors of one general kind are highly varied and includemany anomalous associations or “dissociations―of characters, as exemplified by the “locallymalignant― and“metastasizing benign― tumors of man. The rule stillholds good when the analysis is pushed to the level ofenzymes and histocompatibility genes, and investigationsat this level confirm earlier suspicions about the individuality of tumors ; probably no two tumors are exactly alikein every respect even when they are evoked by similarmeans from the same tissue and have the same generalproperties (18, 39, 46, 57). Many wide generalizationsabout “cancer―have broken down, as most of themeventually do, because they have not taken into accountthe independent variability of characters and the mdi

viduality of tumors. Moreover many characters oftenprominent in tumors are not essential to the neoplasticdisease but are only incidental consequences or accompaniments of it; they may be wholly lacking from certainotherwise typical growths described by Potter (46) as“minimal deviation― tumors. The wider “deviations,―although incidental, might be exploited nevertheless inprophylaxis or, as Potter notes, in the therapy of particularkinds of neoplasia.

Progression is an important mechanism in the clinicalcourse of many neoplastic diseases, but it is not applicable,without redefinition, to the critical changes that take placeduring the preclinical or induction period. Visiblelesions, even the microscopically visible ones, do not adequately reveal the whole nature and extent of the neoplastic disease. The experimental analysis of skin carcinogenesis, briefly reviewed in the following paragraphs,has provided most of the available information about theearliest steps.

SKIN CARCINOGENESIS

The skin, because it is accessible to direct observationduring life, is the ideal site for studying neoplastic development throughout its course. Moreover observations onoccupational neoplasias of human skin correspond with,and in some respects extend in an important way, theextensive investigations of experimentally induced neoplasia in the skin of animals. It is possible here to summarize only briefly and somewhat dogmatically the vastamount of information that has been accumulated. Theprimary aim is to infer broad general principles that arewidely applicable to other sites where direct observationis more difficult.

Experiments on rabbits and mice have shown thatchemical carcinogens applied to the skin quickly effect apersistent, probably irreversible, change in the skin thatis not manifested by enduring histologic or cytologic signsand that, in particular, does not entail proliferation ofcells. This first and decisive step of initiation changesthe functional reactivity of the skin and establishes a newcapacity for neoplastic development that is revealed at alater time when varied lesions emerge focally from theexposed region.

It is convenient to divide the emergent lesions into 3main groups—A, B, and C. The Group A lesions aretrivial ones exemplified by freckles and other familiareffects of overexposure to sunlight. Many, perhaps all,of them are side effects due to nonspecific irritation by thecarcinogen. They are not neoplastic by the usual clinicalor microscopic criteria and have no capacity for neoplasticdevelopment, but they deserve notice because they provideevidence of substantial exposure to a potentially carcinogenic stimulus.

The Group B lesions are the earliest lesions recognizableas neoplastic by the usual criteria. They are mostlybenign or imperfect neoplasms, the term imperfect beingapplied to tumors that do not allow an unequivocal diagnosis of benign or malignant neoplasm because of anomabus associations, or “dissociations,―of unit characters.These lesions in animals and in man include the following:(a) conditional papillomas that grow only when stimulated

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by certain extrinsic factors, regress when those factors arewithdrawn, amid recur when they are restored ; (b) spontaneously regressing papillomas ; (c) indolent papillomasthat emidure for a long time with minimal growth ; (d)papillomas that grow progressively without changing theircharacteristics; (e) papillomas in which carcinoma originates by progression ; (f) carcinomatoids or keratoacanthomas that resemble invasive carcinomas histologicallybut regress spontaneously ; (g) noninvasive carcinomasin situ, in which invasive carcinoma may, or may not,originate by focal progression.

The Group B lesions include the varied “precanceroses,―‘‘precancerous lesions, ‘‘and ‘‘morphologic precursors of

cancer― observed on human and animal skin. The basicobjection to these terms is that many of the lesions towhich they are applied are not “pre-―anything; they aretermuimial lesions with no future. Group B lesions are subject to at least 4 different fates—namely, (a) regression;(b) indolent persistence ; (c) progressive growth withoutqualitative change ; and (d) progression to true, invasive,carcinoma. Progression to carcinoma is often the leastlikely outconie. The fate of a particular lesion is rarelypredictable. Progressiomi seems to be random in time andsite. Even when it is almost certain that true carcinomawill emerge somewhere in a region exposed to optimal carcinogenic stiniulation, the tinie and site of progression arenot predictable.

Group C lesions comprise the malignant, mainly epidermoid, carciflolnaS which can emerge in at least 2 differentways—namely, as a result of progression in a Group Blesion, or directly from a place in the exposed region whereno Group lesion is, or ever has been, present. Most carcinomas in mouse skin probably originate by progressionin a Group B lesion, but it seems to be a general rule towhich no certain exception has been found that, in everytissue in which carcinoma usually originates by progressiomiin a Group B lesion, the same sort of carcinoma sometimes enierges from the exposed region as a carcinoma ohinitio with mm spatial relationship to past or presentGroup B lesions. Briefly summarized, not all carcinomasoriginate by progression in a Group B lesion; not allGroup B lesions have the capacity for progression tocarcinoma, and of those with the requisite capacity only afraction, usually a small fraction, manifest it by actualprogression.

In certain circumnst.ances the neoplastic lesions are verynumerous, and they emerge both concurrently and consecutively. The lesions tend to emerge in an ascendingorder of gravity, but there is no fixed rule and often theemergence of the various types seems to be random ; lesionsof varied types may be present contemporaneously andan ulcerating carcinoma may be the first, although moreprobably it will be the last, type to emerge (53). It isimportant to note in man, as well as in animals, that thefirst carcinoma may emerge long after the last knownexposure to carcinogen and after all Group B lesions havedisappeared and that carcinomas are apt to emerge consecutively throughout remaining life.

The experimental analysis of epidermal neoplasia hasled to a 2-stage hypothesis of carcinogenesis, the 2 stagesbeing described as initiation and promotion. Rous and

his colleagues studied early conditional tumors in rabbitsand inferred that initiation establishes a subthreshoklneoplastic stale characterized by the presence of latenttumor cells that proliferated to form a visible tumor onlyas a result of promotion by various extrinsic stimuli not,

by themselves, carcinogenic (48). Berenblum and Shubik(3), using suboptimal or subcarcinogenic applications tomice, inferred similarly that the carcinogen initiated latentor dormant tumor cells and that these cells proliferated inresponse to promotion by certain noncarcinogenic, or veryweakly carcinogenic, agents of which croton oil was themost effective. Both groups of investigators maintainedthat initiation and promotion were different and distinctconsecutive processes.

The accuracy of the observations leading to the 2-stagehypothesis is not questioned, but the interpretation isstill doubtful. The rabbit tumors studied by Rous wereconditional papillomas or carcinonnatoids, whereas thetumors evoked by croton oil or other promoting agentswere mostly spontaneously regressing papillomas orpapillomas with a low capacity for progression to carcinomas. The initiation-promotion experiments seemto apply predominanatly to special cases of imperfectneoplasia, and their wider relevance to carcinogenesis ingeneral is questionable. In particular, it is doubtful ifpromotion substantially increases the eventual yield ofcarcinomas (13, 45, 50, 54).

The evidence for a crucially important first step ofinitiation is extremely strong, but the further assumptionsthat it affects only a few isolated cells scattered throughthe exposed area and that promotion merely stimulatesthese cells to proliferate are open to criticism. The originof tumors from single cells has not been demonstratedand the histology of early lesions, especially carcinomain situ, does miot support it (45). There is good evidence,moreover, that the early visible lesions, whether evokedby promotion or not, do not reveal the full neoplasticpotentialities of the exposed region. The latent or dormant tumor cell hypothesis does not account satisfactorily for the variety of the early lesions or for the latetumors that emerge consecutively long after the withdrawal of known initiating or promoting stimuli. Toaccount for the consecutive emergence of tumors for up to2 years after withholding carcinogens from rabbit skin,Friedewald and Rous (27) proposed that, as well as initiating latent tumor cells, the carcinogen initiates latentneoplaslic potentialities in cells, which then advance atvaried, often extremely slow, rates towards the neoplasticstate.

Berenblum (2) has rightly emphasized that tumorsemerge focally and not diffusely, but it is not obligatoryto presume that initiating carcinogenic action is limitedto the places where tumors emerge or that the time andsite of emergence and the qualitative variety of the tumorsare determined, once and for all, at the time of initiation.An alternative and more elastic hypothesis is that initiation establishes a region of incipient neoplasia (meaning,literally, “thebeginnings of neoplasia―) that is coextensive with the area of exposure to carcinogen and has apermanent new capacity for neoplastic development. Theterm capacity is used here in the special sense in which

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Grobstein (31) applies it to embryonic development torefer to “therange and character of the demonstrable andimmediate developmental alternatives.― The concept issimilar to that of embryonic competence, but it is moregeneralized. An essential feature of competence andcapacity is the element of choice between 2 or more developmental paths available in a region; individual cellsmust choose one of them. The initiation-promotionexperiments disclose 1 or 2 of the available paths of neoplastic development without revealing the total capacityof the region of incipient neoplasia. The concept ofincipient neoplasia can accommodate the latent tumorcells as a special case, with the reservation that they aremore likely to be cell aggregates from the first., rather thanisolated cells (23—25).

The concept of a regional type of neoplasia based on asubstantial expanse of incipient neoplasia from whichmultiple tumors of varied types can emerge both concurrently and also consecutively over long periods of time isapplicable to neoplasia in various other tissues.

REGIONAL NEOPLASIA

NEOPLASIA IN THE URINARY BLADDER

Neoplasia of the vesical epithelium illustrates severalimportant aspects of the operation of multiple etiologicfactors including some not yet mentioned, and there is anindustrial regional form that is reproducible in laboratoryanimals and clinically similar to the sporadic disease in thegeneral population.

Epidemiologic studies of the industrial disease in manand experiments on animals alike indicate that the disease is induced by aromatic amines, notably 2-naphthylamine. These substances are not carcinogenic at theirpoint of entry, but after absorption they act selectively onthe bladder in man and in some, but riot all, species oflaboratory animals. The probable chain of events is asfollows : The amines are absorbed through the skin, digestive tract, or lungs and carried to the liver, where theyare detoxified by oxidation to orthoaminophenols orarylhydroxylamines. The latter substances are believedto be the effective carcinogens, but they are immediatelyconjugated in the liver with glucuronic acid and excretedin this noncarcinogenic form by the kidneys. The effective carcinogen is liberated from the glucuronide in thebladder by hydrolysis effected by f3-glucuronidase presentin the urine. If the effective carcinogen released in the

liver is not quickly conjugated, it is likely to induce hepatoma. If it is conjugated but excreted mainly in the bile,it may induce neoplasia in the intestines. Even if conjugated and excreted mainly by the kidneys, it will notinduce neoplasia in the bladder unless the appropriatehydrolytic enzyme for liberating the effective carcinogenis present in the urine or, perhaps, in the vesical mucosa

(6, 8, 9).The pattern of induced neoplasia varies from one species

of laboratory animal to another as a consequence of genetically determined differences in the biochemical processesinvolved in the detoxification and excretion of the absorbed a.mines. Genetically determined differences, ifless in degree, may be expected also in man. Further

more, within the limits imposed by genetic factors, thebiochemical processes will be subject to enhancement orrepression by a variety of nonspecific factors, such as thepH of the urine and the time it remains in the bladder.A variety of genetic and physiologic factors must operatein sequence before the effective carcinogen begins to acton the mucosa of the bladder. After it has begun to act along induction period antecedes visible neoplasia, whichmay not be apparent, in man, until many years after thelast exposure to a known carcinogen, and then tumorsmay emerge consecutively throughout. remaining life.

Neoplasia of the human bladder is of 2 main types. Onetype is characterized by solid tumors, usually solitary,that are carcinomas ab initio. The other regional type ischaracterized by multiple papillary tumors. Those thatemerge early seem to correspond with the Group B lesionsof the skin because they are benign or imperfect tumorswith a low capacity for progression to carcinoma. Thedisease can often be controlled for many years by fulgurating individual tumors, but new primary tumoi-s areapt to emerge consecutively and as time passes to beincreasingly dangerous until, eventually, some of themgrow invasively, disseminate, and kill the patient.. Thepapillary type of disease in man and in animals is a regional one affecting a wide expanse of vesicle mucosa, andperhaps all of it (7, 47, 49). The origin of carcinoma byprogression in a papilloma has been described in theindustrial disease in man and in the induced disease inanimals, but the more conspicuous phenomenon seems tobe the consecutive emergence of primary tumors that., astime passes, are of increasing gravity or malignancy whenthey first emerge (7, 29, 30, 56).

NEOPLASIA OF THE UTERINE CERVIX IN Wo@1EN

The etiology of carcinoma of the uterine cervix in womenis obscure, but the pattern of neoplastic development isremarkably similar to the regional type observed in theskin. There is substantial evidence for widespread incipient neoplasia affecting much or all of the cervicalepithelium and extending into the vagina. The outstanding feature is the origin of carcinoma by progressionin Group B lesions comprising the varied dysplasias andcarcinomas in situ. The Group B lesions in the cervix aresubject to the same fates as those in the skin. It is widelyagreed that most invasive carcinomas originate in GroupB lesions, especially carcinoma in situ, but there is lessinformation about the proportion of Group B lesions thatgive origin to invasive carcinomas. Clemmeson (11)estimates that 25—33% of such lesions will give origin tocarcinoma within 15 years; the others persist harmlesslyor regress.

It must be emphasized that carcinoma in situ does riotturn into invasive carcinoma by mere extension in spaceand time; it gives origin to invasive carcinoma only as aresult of a decisive change by progression, whereby neoplastic cells acquire powers of invasion which they did notpossess before (22, 34, 35).

REGIONAL NEOPLASIA IN OTHER SITES

The examples of regional neoplasia quoted so far relateto surface epithelia that are accessible to direct observa

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tion during life. It is much more difficult to study thiskind of neoplastic development in less accessible sites, butevidence is accumulating to show that it is more commonthan formerly supposed. The regional pattern is evidentin familial intestinal polyposis in man (16). It seems tobe the usual type of induced neoplastic development inendocrine tissues of animals (5), and good examples havebeen described in thyroid neoplasia in man (26). Mammary neoplasia in “high-incidence― strains of inbred mice,already discussed, conforms with the same pattern.1\IanTimary neoplasia in women, also, is probably a regional disease, but the regional pattern is clearly evidentonly III the development of carcinomas that originate inchronic cystic disease. Cutler (12) believes that about20 % of all breast cancers develop in this way. At thepresent time, attemition is being given to a similar patternof neoplastic development imithe lung (1).

CONDITIONS FAVORING REGIONAL NEOPLASIA

The regional pattern of neoplasia, of which there aremany variants, is conspicuous in animal tissues that havebeen subjected to optimal carcinogenic action and inhuman tissues that, by reason of occupation or environmemit., have been exceptionally exposed to chemical orphysical carcinogens. The duration or repetition of thecarcinogenic stimulus is probably more important thanits intensity, and a strong element of promoting actionmay help significantly to evoke a multiplicity of variedGroup B lesions. The same regional pattern is evident,even when the carcinogenic stimulus is minimal or unrecognized, where an exceptional, genetically determinedliability to a particular kind of neoplasia has been demonstrated, as in xeroderma pigmentosum and familial intestinal polyposis. In many experiments on animals aknown carcmogen is applied optimally to tissues whosegemietic sensitivity is exceptionally high as a consequenceof inbreeding, so that the extrinsic carcinogenic factorand the intrinsic genetic factor act cooperatively to produce a maximal effect. This combination of circumstancesfavorable t.o the manifestation of regional neoplasia maycome about, by chance, in man but i)rObably riot often to acomparable degree.

When carcinogenic action is suboptimal or geneticsensitivity is low, neoplasia is more likely to be localized.Applicatiomis of the potent carcinogen 9 , 10-dimethyl-1 , 2-benzanthracene to the skins of rats and guinea pigs, 2species notoriously refractory to chemical induction ofepidermal neoplasia, evoke mainly solitary carcinomasthat emerge as such after long delay. Solitary tumorsdo not necessarily indicate narrowly localized carcinogenicaction ; they are interpretable with equal or greater probability as localized manifestations of the neoplastic capacity of a region of incipient neoplasia established by widespread carcinogenic action. Both localized and regionaltypes of neoplasia of the urinary bladder are encounteredin workmen exposed to dangerous aromatic amines andapparently with the same relative frequency as in thesporadic disease in the general population. These empirical facts justify 2 reasonable but as yet unsubstantiatedinferences: 1st, that. the localized and regional types ofmieoplasia iii the urinary bladder, although clinicallydistinct, may be initiated by the same or similar carcino

gens acting diffusely on the mucosa of the bladder; and2nd, that the sporadic disease, of unknown etiology, maybe attributed to the action of a chemical carcinogen,possibly of endogenous origin, excreted in the urine (6, 9).

IMPLICATIONS OF REGIONAL NEOPLASIA

The finding of an overtly regional type of neoplasiaimplies that a region of incipient neoplasia has been established coexistensive with the action of a known or, asin the uterine cervix, unknown carcinogenic stimulus ofsubstantial duration and intensity. The early emerginglesions of Group B are significant in 2 ways : some of themare potential sites of progression to malignant neoplasia;and, together, they reveal the existence of a region ofincipient neoplasia within which malignant tumors mayemerge with no spatial relationship to a precursor lesion.Furthermore the Group B lesions provide informationabout the extent of the incipient neoplasia and, to somedegree, about its capacity, which determines the range andcharacter of the emerging lesions. It is probable, but notyet proved, that solitary tumors as well emerge from aregion of incipient neoplasia whose neoplastic capacity ismanifested at only one place.

INITIATIoN

The crucial event in carcinogenesis is initiation ; factorsthat operate before or after this event are different innature although they may be decisive. As exemplifiedin neoplasia induced in the bladder by remotely administered carcinogens, numerous physiologic and geneticfactors influence the access of administered materials tothe target tissues in an effectively carcinogenic form.Numerous factors also affect the consequences of directapplication of effective carcinogens to sensitive tissues(53), amid genetic factors control the sensitivity of thetissues.

Initiation is effected quickly. It establishes a region ofincipient neoplasia with a permanent new capacity forneoplastic development, but it is not manifested by durablecharacteristic morphologic changes. Empiric observations show that initiation is riot an all-or-none phenomenon. The quality or capacity of the incipient neoplasia,and consequently the quality of the emergent lesions, isdirectly related to the intensity and, probably to a greaterdegree, to the duration or repetition of the initiatingstimulus. Repeated stimuli summate to augment thecapacity for malignant neoplasia (41). The course ofxeroderma pigmentosuin can be retarded to an importantextent by using barrier creams to reduce continued exposure of the skin to ultraviolet irradiation (55). Thereis ami important possibility that subeffective carcinogenicstimuli, not necessarily identical with the initiating stimulus, might summate with that stimulus dangerously toincrease the capacity of the incipient neoplasia. If thisis true, it would be logical and prudent to eliminate, sofar as possible, exposure of the tissue to all kinds of potentially carcinogenic stimuli that might summate with theinitiating stimulus.

The concept of incipient neoplasia, implying thatinitiation effects a diffuse regional change, justifies theinvestigation of exposed tissues in bulk by chemical orother methods; according to the dormant tumor cell

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hypothesis, such investigations are unlikely to be rewarding (2). The information obtained from chemicalstudies is not yet decisive, but it is not inconsistent withthe widely held opinion that carcinogens bring about in adirect way a permanent heritable change in the structureor utilization of genetic materials. It is questionable,nevertheless, if all forms of neoplasia are attributable to adirect sj)ecific action of carcinogens, chemical, physical,or viral, on the genetic material of sensitive cells. Theaction of some carcinogenic agents may be indirect, neoplasia being a secondary consequence of, for example,“frustrated repair― of injuries caused by the agents (28,38, 44). It has been questioned, moreover, if carcinogenstruly initiate neoplasia or merely accelerate or enhance aIrocess that starts and adavances slowly without them(40). The most reasonable view, as expressed by Shubik(53), is that some carcinogens truly initiate neoplasia butthat often the basic action of a carcinogen is to enhance apre-existing latent. predisposition. The basic questionis whether or not genetic constitution per se can lead toneoplasia without an extrinsic carcinogenic stimulus; itcami certainly determine to an important or decisive extent the response to such a stimulus. The interplay ofmultiple genetic and extrinsic factors is complex and needsre-evaluation in the light of the newer knowledge aboutthe structure of the genetic material and the effects ofextrinsic or extragenetic factors on its utilization. Onlybrief mention can be made here of matters that will surelybecome increasingly important in future discussions ofmultiple etiologic factors.

GENETIC AcrloN IN NORMAL ANDNEOPLASTIC DEVELOPMENT

Evidence has been accumulating to show that only aportion of the genetic information coded in the nuclearDNA' is in effective use at a particular time, other portions being available for use in different circumstances.The “expression―of the selected portion to yield specificphenotypic characters requires 2 main steps : in the 1ststep, called transcription, the genetic information is recoded in RNA, and in the 2nd step, called translation,the coded RNA directs the synthesis of a specific protein.Genetic action and the expression of genetic potentialitiesthus depend on 3 consecutive processes namely selection,transcription, and translation of a portion of the total DNAcode, and these processes are differently and elaboratelyregulated.

To facilitate discussion, distinctions have been drawnbetween (a) the total genome carrying the whole of thegenetic information ; (b) the effective genome, the portionof the total genome that is in effective use in a particularcell at a particular time; and (c) the facultative genome,comprising those portions of the total genome that areavailable for use as effective genomes in various circumstances (24, 25). The facultative genome provides forthe choices of alternative utilizations of the total genomethat are implied by competence and capacity in embryonicdevelopment and by metaplasia in both embryonic andadult tissues. It is an important empiric fact that theavailable choices are discrete, mutually exclusive, andlimited in number, being few in comparison with the number that random sampling of the total genome could pro

vide. The term “facultative genome― acknowledges theselimitations without specifying the mechanisms underlyingthem, which are obscure. It remains to be seen whetheror not they are attributable to a sort of nuclear “different.iation― or chromosomal organization such as Brink(10), for example, has proposed, but the facultative genomeseems to be an intrinsically stable and replicable entity.Another important characteristic is that the choice of aneffective genome is often biased or heavily weighted infavor of one of those available in the facultative genome.Many embryologists @OWrecognize that the apparentspecificity of embryonic induction depends as much on thecompetence of the reacting tissue as on the quality of theinducing stimulus. In some forms of induction the inducing stimulus is qualitatively specific in that it selectsone of the effective genomes available in the facultativegenome of the reacting cells. Nevertheless autonomouschanges in competence may lead to a state of developmentalImminence in which a new differentiation takes placewithout the customary inducing stimulus, either spontaneously, as it seems, or in response to a wide variety ofnonspecific stimuli (31, 32).

The fundamental differences between neoplastic andnormal development are probably established at the outsetby the process of initiation. In some forms of carcinogenesis, at least, potent carcinogens may be supposed toalter the total genome drastically, perhaps by direct action on DNA or indirectly by interfering with its accuratereplication. The alteration, it is reasonable to suppose,is not a mere selection of genetic patterns already presentin the total genome of cells of the reacting tissue. Thisinterpretation does not necessarily apply to enhancementby carcinogens, which is especially conspicuous in inbredstrains of animals. The total genome may be altered bysummation of ordinarily ineffective carcinogenic stimuli,but there is a strong suggestion that developmental imminence is involved; initiation either happens “spontaneously― or, as in xeroderma pigmentosum, is effectedby extrinsic stimulation no greater than every member ofthe population receives.

Initiation having been accomplished, neoplast.ic development reveals several phenomena that have beenrecognised in normal development. The cells of a regionof incipient neoplasia are characterized by a newfacult alivegenome, which provides the basis for the diversity ofGroup B lesions. The factors that determine the localization and particular characters of individual lesions arenot well defined. Promoting agents seem to act selectively to favor the proliferation of cells that form certainkinds of benign and imperfect neoplasms. Promotingstimuli may also summate, as Nakahara (41) believes, toincrease the capacity of the incipient neoplasia. A cornparable selective action may account for the directiveaction of certain agents, notably hormones, on neoplasticdevelopment. These agents act specifically in the samesense as certain inductors act specifically in embryonicdevelopment by selecting one of a number of possible pathsof development. Nevertheless there is a possibility withsome evidence to support it that the capacity increasesautonomously with time as embryonic competence isbelieved to do and that the incipient neoplasia reaches astate of developmental imminence, when lesions emerge

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1346 Cancer Research Vol.25,September1965

without extriiisic stimulation or in response to minor andnonspecific stimuli which may account for the localization.The emergence of tumors at sites of recent trauma recognized in animals as the “Deelman phenomenon― andreported from time to time in man may be a special case ofnonspecific stimulation of tissue in a state of developmental imminence. In general the localization andparticular characters of emergent tumors seem to berandom in time amid site except in the special cases al

ready mentioned, but this may reflect only ignorance ofthe significant, although perhaps nonspecific, stimuli thatcommonly evoke iieoplastic lesions from tissue in a stateof developmental imminence.

Progression requires certain permissive factors, as doall stages of neoplastic development, but attempts to inciteprogression have given erratic results. The few positiveresults may be attributed to nonspecific stimulation of

neol)laStiC tissue iii a state of developmental imminemice.

More generally, progression seems to be random in timeamid site vlt.hough its consequences are circumscribed bythe capacity of the tissue in which it takes place (24, 25).

CONCLUSIONS

Overt neoplasia depends on multiple factors which actsimultaneously or consecutively and which vary at differemit stages of the neoplastic disease. At each stage thereis a complex interj)lay of genetic and nongenetic factors.The action of t he recogmiized carcinogenic, inductive orinitiating agents is conditioned by varied permissive factors, and the quality of the resulting neoplasia may bedetermined in part by directive factors. Varied geneticand physiologic fsctors are involved in delivering aninitiating carcinogen to the target tissue in effective formamid amoumit., and others control the responsiveness of thetarget tissue.

Initiation is effected quickly but is not manifested bypersistemit characteristic signs or by proliferation of cells.

Overt mieoplasia is first evident after a substantial latentor induction period. Initiation establishes a new reactivity and capacity for neoplastic development hereinreferred to as incipient neoplasia. When the initiatingstimulus is optimal iii quality, intensity, and duration,or when genetic factors are especially favorable, multiplediscrete neoplastic lesions emerge concurrently or consecutively from a region of incipient neOj)lasia coextensivewith the exposure to the initiating agent. Many of theearly lesions are benign or imperfect neoplasms; they aresignificant because progression to malignant neoplasiamay take place in one or more of them and because theydisclose a region of incipiemit. neoplasia within whichmalignant neoplasms may develop without. spatial relationship to precursor lesions. Solitary tumors do notnecessarily result from strictly localized carcinogenic action ; with equal or greater probability they are localized

manifestions of an extensive region of incipient iieoplasia.Initiation almost certainly brings about a l)errnanent

replicable change in the genetic material, or at least in thepattern of its utilization; it. establishes a new capacityfor neoplastic developmemit based on a new facult.ativegenome which circumscribes the range and character of

emergent mleOl)lastic lesiomis. The capacity, and coii.sequemitly the type, of overt iieoplasia depends on the nature

and intensity and, probably most of all, on the durationor repetition of the initiating stimulus. Initiation is apparently irreversible, but the capacity of the incipientneoplasia is not static ; it can be enhanced by summation ofinitiating stimuli, and it probably increases autonomouslywith time so that eventually a state of developmentalimminence may be reached. A similar state may be established by genetic factors as in xeroderma pigmentosum,in certain tissues including mammary tissue in selectivelyinbred mice, and perhaps in the subcutaneous tissues ofrats.

Overt neoplasia, which is nearly always focal or multifocal depends basically on 3 consecutive processesnamely selection, transcription, and translation of one ofthe effective genomes available in the facultative genomeestablished by initiation. The 3 steps are differently andelaborately regulated by genetic and extragenetic mechanisms, most of which are as yet undefined. The directivefactors presumably operate at the level of selection, asmay some promoting factors. Other promoting factorsmay favor transcription arid translation of certain effective genomes that have been selected already. Somepromoting factors, which are also weak carcinogens, mayact by summating with the initiating carcinogen to increase the capacity of the incipient neoplasia to a state ofdevelopmental imminence. The material basis of thisstate is unknown but the phenomenon seems to be a realone and important or, often, decisive in neoplastic development. It may account for the erratic emergence ofprimary tumors at sites of recent trauma and for theequally erratic progression induced by trauma in established tumors. As a rule progression seems to be randomin time and site although its consequences are circumscribed qualitatively by the capacity of the tissue inwhich it occurs.

The aim of prophylaxis must clearly be to protect frominitiating carcinogens and from other initiating agentspossibly of a different kind that might summate to enhance the capacity of the incipient neoplasia. At presentthere seems to be little chance of rectifying the geneticchange effected by initiation but, as witnessed mostencouragingly by the limited success of endocrine therapyin cancer of the human breast and prostate, the effects ofthe genetic change might be mitigated by controllingpermissive and directive factors and factors regulating theselection, transcription, and translation of genetic patterns. The most effective way of interfering with neoplastic development is likely to differ from one type ofneoplasia to another and to depend on a more exactknowledge of multiple factors than is yet available.

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1965;25:1339-1347. Cancer Res   Leslie Foulds  Multiple Etiologic Factors in Neoplastic Development

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