progress in understanding breast cancer: epidemiological and biological interactions

Breast Cancer Research and Treatment 11:91-112 (1988) © Kluwer Academic Publishers - Printed in the Netherlands

Review

Progress in understanding breast cancer: epidemiological and biological interactions

Peter Boyle 1'3 and Robin Leake 2 1 Unit of Analytical Epidemiology, Div&ion of Epidemiology & Biostatistics, IARC, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France; 2 Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Great Britain

Key words: breast neoplasms, epidemiology, biology, estrogen, prolactin, dietary factors, alcohol consumption, intervention

Summary

Little progress has been made recently in our understanding of the epidemiology of breast cancer. While results from epidemiologic studies regarding reproductive factors remain fairly reproducible from one study to another, other associations such as that between breast cancer risk and dietary fat intake, although biologically plausible, are not consistently found in direct study of humans, while yet other associations, which appear less plausible biologically, become stronger (such as the increased risk associated with modest levels of alcohol consumption). In this paper we attempt to review the epidemiology and biology of breast cancer jointly and describe possible mechanisms of breast cancer induction, the cellular composition of the breast, the epidemiology of breast cancer, and salient biological features, and attempt to reconcile the biology and epidemiology. It becomes obvious that future progress depends on better biological thinking by epidemiologists, and vice-versa. Areas of further research are suggested and discussed, concluding that the ability to measure diet with greater precision could have an important role to play in clarifying our understanding of breast cancer.

Introduction

Descriptive epidemiology has provided compelling evidence that the majority of human cancer, perhaps as much as ninety per cent, may be avoidable [1-3]. Analytical epidemiology has provided var- ying degrees of insight into the causes of cancer at different sites. For example, while the etiology of lung cancer is understood to the extent that preven- tive strategies can be determined, since an estimate

of up to 85 per cent of lung cancer could be avoided by elimination of cigarette smoking [4], it is not clear whether alteration or avoidance of proposed risk factors for breast cancer [5] would reduce incidence. It has been postulated that a population which 'achieved a five-year reduction in age at first delivery might achieve a 30% reduction in breast cancer incidence' [6]. Even if this statement were not tentative, such an alteration in lifestyle would be much more difficult to implement than a reduc-

Address for offprints: Peter Boyle, Unit of Analytical Epidemiology, The International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France 3 (Previous address: Departments of Epidemiology and Biostatistics, Harvard School of Public Health; Division of Biostatistics and Epidemiology, Dana-Farber Cancer Institute; Boston, MA 02115, USA)

92 P Boyle and R Leake

tion in cigarette smoking. Biological analysis of breast cancer indicates

that, in fact, we are dealing with several different diseases. For example, breast cancer can be hormone sensitive or hormone independent; individual tumors can be highly aggressive or slow growing; primary tumours can rapidly metastasize or remain local over a long time.

Thus, while it can be argued that some limitation of our understanding of human breast cancer may be attributed to deficiencies in epidemiological methodology, another part may be attributable to problems of biological classification. Clarification of current problems may well be dependent upon communication between epidemiologists and basic scientists.

It seems appropriate, therefore, to attempt to provide an outline of both the salient epidemiologic and elementary biologic features of breast cancer in the hope that a scientific basis for the observed epidemiologic associations can be provided. A joint approach might help to identify areas for future etiologic studies which should have a better a priori chance of providing useful information - 'useful' in the sense of clarifying our understanding of the etiology of breast cancer and increasing prospects of prevention. We propose to [1] outline possible mechanisms of breast cancer induction, [2] discuss the cellular composition of the breast, [3] summarize the descriptive and analytic epidemiology of breast cancer, [4] outline the biological features of breast cancer, [5] attempt to reconcile the epidemiological factors with known biological factors, and [6] present some conclusions.

Possible mechanisms of breast cancer induction

The vast majority of breast cancers are cancers of epithelial cells. Transformation of breast cells and promotion of tumor growth are thought to involve promotion of DNA synthesis and cell division. Thus, it is reasonable to postulate a role for both carcinogens and co-carcinogens (growth promoters) in the establishment of at least some classes of breast cancer.

Carcinogens in breast cancer are thought to

range from chemical carcinogens in the diet to radiation damage. Growth promoters include both hormones and locally produced growth factors. For this reason, we must examine the structure of normal breast epithelium in relation to the supply of hormones (by the bloodstream and/or local production) and growth factors.

Cellular composition of normal breast

The human mammary gland is composed of 15-20 interconnected ducts which arise from the nipple and areola, and radiate along the anterior and lat- eral thoracic wall (Fig. 1). Overlying the glandular tissue is a layer of adipose tissue. Additionally, connective tissue, vessels, nerves, and lymphatics are found in different parts of the breast. The nipple is a concentration of epithelial cells into which the ducts drain and is surrounded by the pigmented skin which forms the areola. The breast is highly vascularized and well provided with lymphatic drainage, leading mainly towards the axilla.

Normal development of unique mammary gland structures is seen within six weeks post-fertilization of the ovum. The main lactiferous ducts become canalized close to term, but the majority of ductal development and virtually all the development of glandular secretory components depends on hormonal changes at puberty. It is important to note that, in the fetus, the ectodermal cells, destined to became myoepithelial cells, develop in direct contact with both the developing ducts and the me- senchyme. Fibrous connective tissue and adipocytes also appear at this time. The major development of the intralobular alveolar elements awaits the hormonal changes of puberty, and full development is not initiated until pregnancy.

At puberty (specifically, the ovarian production of estrogen and progesterone), the 15-20 embryon- ic ducts undergo extensive arborization into separate lobes. Only 10-15 of these may be functional. Each functional lobe is, in effect, a separate gland operating within the environment of the adjacent fat and stromal cells.

After puberty, the mammary gland is a complex arrangement of inter-connected secretory units

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surrounded by fat and connective tissue, and ser- viced by an extensive blood, lymphatic, and nerv- ous system. The fundamental secretory units of each lobe are the many alveoli (typical structure shown in Fig. 2) which show maximum growth and regression during various hormonal changes of puberty, pregnancy, and menopause. These alveoli are surrounded by a mesh of myoepithelial cells which show some of the properties of epithelial and muscle cells and provide the power needed for milk ejection. The whole structure is surrounded by the basal lamina which acts as a boundary between epithelium and stroma. The boundary is permeable to growth factors and metabolites but is normally breached only by migratory blood cells.

Although breast size varies considerably from individual to individual, and within an individual is capable of changing in the course of a lifetime, such alterations are due to changes in the content of connective tissue and fat rather than to changes in the amount of epithelial tissue.

Breast tumor growth

The majority of breast tumors result from trans-

Breast cancer epidemiology and biology 93

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formation of ductal or lobular epithelial cells. Many of the clinical tumors are multicentric [7] and it is difficult to say whether or not all transformation events take place at the same time. Certainly, many individual tumors comprise both biologically differentiated and undifferentiated epithelial cells. Once the tumor is established, it continues to grow as carcinoma in situ, i.e. a confined group of cells still linked by cell-cell contact and contained within a basement membrane. At some stage, usually pri- or to the tumor being clinically detectable, cell-cell interaction begins to break down. At the same time the tumor cells secrete digestive enzymes and the metastatic process begins.

Breast tumors are usually slow growing. It takes about 10 years (by extrapolation) for a tumor to grow from the initial transformation event until the time that it becomes palpable. This means that the metabolic and hormonal conditions which may be related to the incidence of breast cancer are those in existence at the time of transformation and these may not be the same as those at the time of diagnosis.

Epidemiology of breast cancer

Descriptive epidemiology

As for every form of cancer, the descriptive epidemiology of breast cancer is hampered by the lack of incidence and mortality data from Africa and Asia. Analysis of available international data indicates that the incidence of breast cancer is generally increasing with the average increase of the order of 2 per cent per annum. Only in Bombay (India) is the rate of increase in incidence" low (0.5% per an-


hum). The greatest increases in risk are occurring in areas or population-groups where the risk was initially low, i.e. the risk of breast cancer is appar- ently becoming more uniform on an international basis.

The mechanisms underlying these temporal changes appear to be cohort effects rather than the effects of calendar time. Increases in the overall rate are being produced by the emergence of birth cohorts with an increasing risk of breast cancer over the previous cohort at the same age [8, 9]. This generation (cohort) effect is mainly evident in women over the age of 45, but in the Mersey Re- gion (England) and the German Democratic Re- public, there are also generation effects among premenopausal women.

Black women in Alameda County appear to demonstrate premenopausal generation effects while white women in Alameda demonstrate postmenopausal effects. It has previously been shown that while blacks have breast cancer incidence rates consistently lower than whites, black women under the age of 40 have a higher incidence rate than white women [10].

It is difficult to make a general statement about trends in breast cancer mortality rates (Figs 3, 4 and 5) with, for example, high-risk Anglo-Saxon and Scandinavian populations showing very little change in mortality while several countries outside Europe show large increases in mortality rates e.g. Singapore and Puerto Rico (not depicted).

Increases in overall incidence rates have been observed in the United States, Canada, New Zea- land, Scotland, and Scandinavia, while there has been little, if any, concomitant increase in mortality in these countries. Assuming little or no im- provement in survival is brought about by treatment, only 1 in 10 of these 'excess' cancers is lethal, which, if true, would suggest major changes in the presentation, diagnosis, or definition of breast cancer. The first mentioned is a reasonable supposi- tion in view of the increasingly widespread use of mammography and the other forms of breast examination.

The most probable explanation for this apparent discrepancy may be the improved and/or earlier diagnosis over the last 10 years which might lead to increased surgical 'cure'.

Reproductive history and menstrual factors

Changes in the female body which are associated with sexual intercourse (pregnancy, childbirth, lactation, the use of contraceptives) are regarded as extrinsic factors by the World Health Organization Expert Committee on the prevention of cancer [11]. As already indicated, normal breast epithelial cells respond markedly at puberty and during pregnancy. This indicates sensitivity to the hormones and growth factors whose concentrations change at these times. Other treatments influencing hormone and growth factors levels (e.g. oral contraceptives, additive hormone therapy etc.) might also influence breast epithelial cell division and, therefore, increase or decrease the chances of cell transformation.

An early age at first birth is associated with a substantial reduction in risk of breast cancer. Women who have a child before the age of 18 have about one-third the risk of women whose first children are born after age 30 [12, 13]. i t is probable that full-term pregnancies are necessary to confer this protection [14] which applies to breast cancer occurring at any age. Recently a case-control study of breast cancer cases under 33 found a relative risk of 2.4 among women who had a first trimester abortion (either spontaneous or induced) before their first full-term pregnancy [15], these results being similar to those obtained from animal experi- ments [16].

Childlessness is in itself a risk factor for breast cancer. However, if age at first birth is delayed beyond age 30 there is no protective effect and some evidence of enhanced risk even over childless women [17]. Furthermore, it has been shown that the decreasing risk of breast cancer with increasing parity is an effect independent of age at first birth [18].

Breast feeding was once considered to reduce the risk of breast cancer [19] but it has since been suggested that this is not so once parity has been accounted for [20-22]. Its role remains controver- sial in that there is no real consistency in results obtained by various studies.

Artificial menopause reduces the risk of breast cancer [23, 24] and there is evidence that an early


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time of their own birth, having adjusted for the age at which the cases themselves had their first live birth. These findings have been confirmed [12, 36] but their interpretation in biological terms remains unclear. The possibility that this might be related to the number of pituitary cells secreting prolactin is worth consideration.


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Benign breast disease

Women who have had fibrocystic breast disease are found to have 2-3 fold increased risk of breast cancer [37-39] which persists for up to 30 years after the fibrocystic disease has been diagnosed [38]. Risk varies according to the type of fibrocystic disease diagnosed, being greatest in lesions charac- terized by some form of epithelial disturbance [39, 4O].

However, the etiology of fibrocystic disease appears to differ from that of breast cancer. For example, the risk of fibrocystic disease is increased by an artificial menopause [41] and is not found to be associated with age at menarche [41, 42]. The risk is considered to be real in view of the agreement of observations between case-control and cohort studies and the magnitude of the risk. The latter precludes the possibility of women in higher social


class goupings beings more likely to have their fibrocystic disease investigated and diagnosed, and it would appear that they have independently an increased risk of developing breast cancer.

The relationship between fibroadenoma and breast cancer remains unclear. Hutchinson et al. [39] found no independent risk elevation due to fibroadenoma alone but, when diagnosed concom- itantly with fibrocystic disease, it produced a greater risk than that found to be associated with fibrocystic disease on its own. Other studies, however, have reported no association between fibroadenoma plus fibrocystic disease and subsequent breast cancer risk [37, 43]. In a large study of consecutive breast biopsies it was demonstrated that the majority of women (70 per cent) who underwent a biopsy for benign breast disease were not at an increased risk of cancer [44]. Risk was increased by the presence of atypical hyperplasia [44] and women having this feature had an approximate 4-fold increase in breast cancer risk over a general population [45].

Diet

Dietary practices are becoming increasingly the focus of attention in studies of human cancer epidemiology [46, 47]. The concept is not new; Tannen- baum [48] clearly demonstrated the effects of dietary deficiency and excess on carcinogenesis in 1940. Since then, there has been a search for pre- formed carcinogens in food items and, more recently, it has been postulated that metabolic effects of diet may influence the occurrence of cancer by indirect means and that carcinogens may be produced from dietary substrates [49].

Much of the evidence linking breast cancer risk to dietary parameters has been derived from correlation studies between breast cancer incidence and mortality with per capita intake of total fat and other nutrients in a variety of countries [50-53]. This was related to the concept that fat intake correlated with cholesterol intake and that cholesterol, as the parent molecule of steroids, might be the source of increased estrogens. A direct correlation has been reported between breast cancer mortality and intake of milk, table fats, beef, calories,

and protein, as well as fat [54], although the same study reported an inverse correlation with intake of eggs. Even after controlling for age at first mar- riage, the association between breast cancer risk and milk consumption (direct) and consumption of eggs (indirect) remained.

The incidence of breast cancer among premenopausal Japanese-American women in California is now almost as high as that for Causasian women [55]. The low Japanese rates are thought to be associated with dietary practices and this has been investigated [55] by comparison of the diet of Japa- nese men whose wives had developed breast cancer with that of other Japanese men. It was reported that husbands of the cases consumed more beef and other meat, butter/margarine/cheese, corn, and wieners than the controls, and that they ate less Japanese foods. If the diets of husbands and wives are similar, this would suggest that the 'Western Diet' poses a risk of breast cancer to women ex- posed to it.

Data are available from one large prospective study and several case-control studies to shed some light on the dietary hypotheses. These studies have concentrated, directly or indirectly, on dietary fat intake, and include the implication that increased fat intake leads to increased fat content and size of breasts. Fat cells can provide the enzyme aromatase which converts adrenal androgens to estrogens, the principal source of estrogens in postmenopausal women.

Relative risks of between 1.6 and 2.6 were reported from a case-control study involving 77 cases for five categories of food: fried foods, fried pota- toes, hard fat used for frying, dairy products (ex- cept milk), and white bread [57]. This study was limited by the small sample size and interpretation was hampered by the failure to calculate indices of nutrient intake.

A larger study (400 cases) found total fat consumption to have the strongest association with breast cancer risk in premenopausal women, although there were weaker associations found for saturated fat and cholesterol [58]. Controlling for the effect of each nutrient merely increased the risk associated with total fat consumption, whereas the effects of saturated fat and cholesterol diminished.

The association of total fat consumption with breast cancer risk was the only consistent finding in the postmenopausal group.

Another large study (577 cases) found that the relative risk increased significantly with increasing frequency of consumption of beef and other red meat, pork, and sweet desserts [59], and supported a link between breast cancer and the consumption of animal fat and protein. This study did not, however, calculate nutrient densities.

A case-control study based on 2,024 cases found no difference in risk between cases and controls produced by differences in ingestion of fat. Simi- larly, there was no difference in risk of breast cancer associated with diets containing Vitamin C or cruciferous vegetables. Risk for breast cancer in women over 55 years of age increased with a lower reported intake of vitamin A in the diet [60]. A smaller case-control study from Greece found no association with fat but a significant protective effect associated with increasing consumption of sal- ad-type vegetables [61].

Recent case-control studies have been providing only marginal evidence in favour of the association between dietary fat intake and breast cancer risk. Lubin and her co-workers reported that women in the highest quartiles of fat and animal protein intake and in the lowest quartiles of fiber intakes, had about twice the risk of women in the lowest quartiles of fat and protein and in the highest quartile of fiber intake [62]. A case-control study from Hawaii reported only a suggestion that cases consumed more saturated fat and oleic acid than neighbor- hood controls, although the authors stated that the differences were not impressive [63]. A similar study from France found risk increased with increasing consumption of cheese and decreased with increasing consumption of yoghurt [64].

In summary, diet, but especially a diet high in fat, is thought from the results of animal studies and on biologic principles to influence the risk of breast cancer. Results of direct studies on humans provide conflicting and weak evidence of an association although results from some studies are so clear and internally consistent that they cannot be ignored. The association has been described as being 'weak in individuals as opposed to populations'

Breast cancer epidemioIogy and biology 99

[47]. While biologically plausible, there seems to be no consistent evidence available from direct studies of breast cancer cases to support the association of current fat intake with breast cancer risk. The most powerful study to date, the prospective Nurses Health Study, reported no evidence of any increased risk for total fat, saturated fat, linoleic acid, or cholesterol in either pre-menopausal or post-menopausal women [65]. It may well be that the dietary influences in early life, especially at puberty (compare radiation), are important deter- minants of breast cancer risk, influencing risk by affecting endocrine function [6].

Alcohol consumption

Several studies have shown that alcohol consumption increases the risk of female breast cancer and only one or two studies contradict these findings. The evidence of an association is becoming firmer although the risks reported are generally small (odds ratios of between 1.0 and 2.0) [see for example 71-74]. However, the alcohol habit is so widespread that the possibility remains that it is an important determinant of breast cancer risk. The literature has been summarized as suggesting a need for women to be recommended to moderate their alcohol consumption until the situation is clar- ified [75]. If such an association is real it may not be direct. It could arise from the direct action of alcohol on endocrine function (although low levels of alcohol intake are not at present thought to alter this) or through an association between alcohol consumption and unmeasured causative agents. For low levels of alcohol intake the latter option could be a possible, but speculative, explanation of these results.

Obesity

De Waard and his colleagues in the Netherlands first described the association between overweight and obesity and increased breast cancer risk in 1964 [76], and he has subsequently refined and cham- pioned his original hypothesis [7%79]. Weight has


since been shown to be an independent risk factor for breast cancer in a wide variety of population settings: in women over 50 in Taiwan [80]; in Ath- ens [81]; in women over 49 in Brazil [82]; in Japan [83]; in Canada [84]; in post-menopausal women in Israel [85]; and in others, all of which have recently been reviewed by Rose [86]. However, the author of this latter review [86] admits that not all studies have been confirmatory [for example 32, 8%89]. However, the body of the evidence points to an increased risk of breast cancer at least in postmenopausal obese women.

Genetics

It is almost uniformely reported that women with a first degree relative with breast cancer are at an increased risk of developing breast cancer themselves. Anderson [90] has noted that the risk is most strongly influenced by the mother since the risk is much higher in women with a mother and a sister affected than a sister alone. There appears to be a 2-3 fold risk associated with having a first degree relative with breast cancer [5].

Radiation

Radiation to the breast in high doses has been shown to increase the risk of breast cancer whether the irradiation was from nuclear explosions [91], from treatment for acute postpartum mastitis [92], or from fluoroscopy [93]. Confirmatory results have been found in animal studies [94]. Exposure of the breasts to radiation at age 10-19 is associated with a particularly high risk [93] with the greatest risk associated with perimenarcheal exposure [95]. This suggests that breast tissue is particularly vul- nerable at this time either because the breasts are developing rapidly or because most women have not given birth. The best explanation could be the former, since exposure to radiation prior to the age of 10 has consistently been shown to carry no increased risk while the greatest risk has been associated with exposure during the first pregnancy [95].

Other factors

A variety of other putative risk factors, such as thyroid hormone production, reserpine use, and use of hair dyes have been associated with breast cancer risk and have been reviewed elsewhere [5, 96].

There is no good evidence to suggest that human breast cancer may have a viral origin although viral gene sequences have been detected in some breast cancers [97]. Furthermore, even in susceptible mice, estrogen stimulation may be necessary for mouse mammary tumor virus to produce tumors [98].

Effect of dietary fat intake on the prognosis of breast cancer

It has been shown that survival rates from breast cancer are higher among women in areas where breast cancer rates are low [99, 100] and twenty years ago Haenszel [101] suggested that factors which determine tumor development may also effect the course of the disease. This, in turn, suggests that different risk factors may put different cell populations (undifferentiated or highly differentiated) at risk of transformation.

A study of the possible influence of dietary fat intake on survival was stimulated following a comparison of survival rates among breast cancer cases in Boston and Tokyo. The higher survival rates in Tokyo could not be explained by differences in age, stage, histology, parity, or age at first full-term pregnancy [102]. The major distinguishing variable between Japanese and American women which is also thought to be a breast cancer risk factor, is dietary fat intake (see above). As suggested earlier, the role of fat has been related to possible stimulation of tumor growth by estrogens.

Abe et al. [103] reported a 5 year survival rate of 56 per cent in obese women as compared to 80 per cent in non-obese women, and Boyd et al. [104] examined the relationship between breast cancer risk factors and subsequent prognosis and found that body weight was the only risk factor associated with survival.

A recent review of the relationship between obesity and breast cancer recurrence after mastec- tomy found that nine studies were supportive and two others non-supportive [105]. The evidence of association appears strong.

Biological features of breast cancer

The four cell types which are most closely involved in breast cancer are the glandular epithelial and myoepithelial cells, the stroma, and the fat cells (see Fig. 2). The growth of glandular epithelium depends not only on the free hormonal content of the blood supply, but also on the output of the other cell types, either through direct functional contact (myoepithelium), secretion of growth factors (stroma), or activation of precursors (stromal and fat cells). These will be considered in turn.

Hormones

The principal hormones involved in mammary growth are the lactogenic hormones, which are polypeptides, and the steroids (estrogens, progesterone, and glucocorticoids). The lactogenic hormones are growth hormone, placental lactogen, and prolactin. Prolactin is released by specific cells in the anterior pituitary. Its release is mainly con- trolled by factors from the hypothalamus, the principal one of which is prolactin inhibitory factor (PIF). Evidence is strong that PIF is simply dopamine. However, the inhibitory effects of dopamine can be reversed by estrogen acting either directly on the pituitary or via the hypothalamus. Both stress and opiate agonists are promoters of prolactin release.

Prolactin is involved in the control of growth, development, and differentiated function of the mammary gland. There is considerable evidence [106] that mammary growth in humans can be induced by prolactin in the absence of steroids. For this reason, prolactin may be very important in sustaining the growth of malignant epithelium. Blood levels of prolactin and placental lactogen rise throughout pregnancy. A direct mitogenic ef-


fect of prolactin has been shown on the human breast cancer cell line MCF-7 [107] and on rat mammary cells [108]. An indirect mitogenic effect of prolactin was also reported by Shafie and Brooks [107] who demonstrated a prolactin-induced increase in available estrogen receptors. Prolactin also has the ability to modulate gene transcription and mRNA stability, as has been reported for casein mRNA [109, 110]. There is also evidence for translational and post-translational effects of prolactin [111-114].

The exposure of the breast to high levels of prolactin during pregnancy does not, in itself, seem to be a risk factor. This is probably because, at the same time, there are high levels of progesterone and progestins which are known to reduce DNA synthesis and cell division in breast cancer cells in vitro.

The specific effects of prolactin are very much dependent on the stage of differentiation of the mammary epithelium [115-118]. Prolactin action is, of course, modified by changes in the levels of other hormones. For example, prolactin effects on lipid metabolism are dependent on the concomitant presence of insulin and glucocorticoid [113, 119,120]. Further, there is evidence of an indirect effect of estrogen and a direct effect of progesterone [121] on the number of available prolactin receptors.

Estrogens are the steroids normally associated with mitogenic events in female reproductive epithelium. In early studies, it was thought that estriol (E3) might be protective since it bound the estrogen receptor much less tightly than estradiol (E2). It was also shown that a single dose of E 3 was not fully estrogenic. However, several laboratories [122, 123] have now shown that sustained estriol, at physiological levels, is fully estrogenic. Estrogens have a well-est~blished [124] effect on mammary gland growth and differentiation. They also promote prolactin secretion from the anterior pituitary [125-127]. A tight correlation between the onset of ovarian function and mammary growth was established as long ago as 1905 (128). Injection of estrogen was soon shown to promote ductal proliferation and the formation of mammary cysts [129, for review]. However, attempts to show direct effects


of estrogen on mammary gland epithelial proliferation have been relatively unsuccessful [130-134] although proliferative responses can be induced in vitro using estrogen and glucocorticoid [135]. In the intact animal, estrogen-induced responses require an intact pituitary gland, indicating either an indirect role for estrogens or a synergistic role for one of the products of the anterior pituitary (e.g. prolactin). Indeed, in ovariectomized mice prolactin stimulation of mammary epithelial proliferation re- quired a prior injection of estrogen and progesterone [131]. The role of estrogen-induced growth factors must also be considered [136 and v.i.].

Whereas injection of estrogen alone into virgin mammals induces ductal proliferation, addition of progesterone at physiological doses produces lobuloalveolar development similar to that in pregnancy. It has been suggested [137] that, while estrogen plus prolactin stimulated proliferation of ductal epithelium, progesterone plus prolactin also stimulated alveolar epithelial proliferation. The effects of progesterone are not seen on mammary epithelial cells in culture [138] and may well involve induction of growth factor secretion by the surrounding stroma. An important physiological role of progesterone may be to prevent terminal differentiation of mammary epithelium during pregnancy [124].

Although specific effects of glucocorticoids on the mammary gland are ill-defined, they do appear to have significant influence on the initiation and maintenance of differentiation [139-140]. For example, prolactin-induced differentiation is enhanced by glucocorticoids [141-145].

It is well established that insulin is an essential pre-requisite for demonstrating proliferative responses of mammary epithelium in vitro. However, the mechanism(s) is obscure and the role of insulin or insulin-like factors in maintaining alveolar cell proliferation in vitro is not proven.

There is some evidence from studies in mice [146] that lobuloalveolar development is proportional to the plasma level of tri-idothyronine (the most active thyroid hormone). However, the overall role of thyroid hormones on mammary development is probably only a permissive done.

Several different prostaglandins have been shown to affect mammary gland development but

precise data on their roles is limited. More information is needed on the specific roles of each type of prostaglandin in mammary gland function before any statement can be made with regard to either tumor growth or therapy. A recent observa- tion [147] that the breast tumor content of prostaglandin E 2 was inversely related to the presence of estrogen receptors is of interest. High levels of estrogen receptor are usually associated with low proliferation [148]. Hence, there is a possible correlation between elevated levels of prostaglandin E2 and increased proliferation. It may be worth noting that tamoxifen (which may be effective in autonomous as well as hormone dependent breast cancer [149]) inhibits prostaglandin synthesis [150].

Cell-cell interactions

The integrity of epithelium depends to a great extent on specialized cell junctions. There are gap junctions which, as the name suggests, involve the coalescence of neighboring cell membranes and the formation of a small gap such that ions and small molecules may be exchanged between the two cells. Tight junctions, in which the outer layers of the two cell membranes are fused, occupy a larger area of the normal mammary epithelial cell surface than do other junctions and so act as the major force in anchoring cells together. Desmosomal junctions are also part of the cell-cell adhesion mechanism but, in this case, there is no contact between cell surfaces and material in the space between the membrances is responsible for adhesion [151]. Changes in the distribution of these junctions have been associated with increased metastatic potential, and long term prognosis might be provided by measuring the ease of dis- aggregation of epithelial cells in an in vitro test.

Epithelial cell-stromal interaction may involve transmission of growth factors but is also dependent on the structure of the basal lamina. Changes in the composition of basal lamina are associated with the metastatic potential of some mammary tumors [1521.

Growth factors

Although evidence relating growth factors to breast cancer proliferation is limited, there are several indications that they may have a role to play. Epidermal growth factor (EGF) is detectable in medium in which breast stromal cells have been cultured. Breast cancer cells have been reported to contain EGF receptors possibly in inverse proportion to estrogen receptors [153], although not all studies support this [154]. EGF receptors are capable of modifying progesterone receptor activity [155] and contain sequences closely related to those in the v-erb-B oncogene protein product [156] such that, once established, tumors might be able to sustain their own growth factor stimulus. The mechanism by which growth factors produced by the stoma might stimulate epithelial cell growth has been reviewed by Cuhna et al. [157].

Other factors

Both normal and malignant breast epithelium are dependent on an adequate blood supply and the appropriate hormones and nutrients supplied therefrom. Inadequate blood supply may result in insufficient supplies of factors essential for normal cell growth control. One possible difference between normal and malignant cells has been suggested [158] to be the level of interchange of metabolites, including metabolites of vitamins, through the gap junctions. A relationship between vitamin intake and protection against some cancers is well recognized [159].

Role of fat cells

Several different hormones have been cited as having mitogenic potential for components of breast epithelium. Although estrogens are only mitogenic in conjuction with pituitary factors (particularly prolactin) and, possibly, growth factors, it has been common to cite estrogens as critical promoters of breast cancer. However, breast cancer is most frequently a disease of postmenopausal women, in


whom the normal source of estrogens, the ovary, is effectively non-functional. The estrogens thought to promote breast cancer in these women are those derived from the adrenal androgens which are made estrogenic by an aromatase usually associated with peripheral fat cells and readily demonstrated in breast fat cells [160]. Aromatase action is not, however, limited to breast adipocytes but can also be demonstrated in stroma. However, it is interesting to note that the steroid content of postmenopausal breast tissue reflects the plasma steroid content characteristic of premenopausal women. This implies that critical steroid metabolism or selective steroid concentration occurs in normal breast tissue [162].

Histology and prognosis

An understanding of the biology of breast cancer involves examining not only the initial causes but also the growth patterns of different types of breast cancer. It is generally accepted [162] that tumors containing mainly undifferentiated (Grade III) cells will be more aggressive than those containing mainly highly differentiated (Grade I) cells. The differentiated cells are, of course, associated with normal features of breast epithelial cells such as presence of functional estrogen receptors [163]. This would suggest that the hormone dependent disease should be more slowly growing than the autonomous disease, e.g. ER+ disease carrying a better prognosis than E R - disease [164]. One possible interpretation of such a conclusion is that breast cancer can arise from transformation of either a fully differentiated epithelial cell or a stem cell (a relatively undifferentiated precursor cell). Whatever the validity of this suggestion, differentiated breast cancer does tend to become less well differentiated as the disease progresses. As has already been suggested, separate factors may promote stem cell transformation compared to those needed for differentiated cell transformation.


Mammographic observations

One result of the current screening programs is that information is accumulating about the incidence of high risk patterns in relation to subsequent survival. Women who develop aggressive disease frequently do so without having shown a high risk pattern on previous mammography. Conversely, women with high risk patterns who do develop breast cancer are more likely to have the relatively differentiated, hormone dependent disease with good prognosis. Since stromal changes contribute to the high risk pattern, there is again the suggestion that stromal-epithelial interaction is important at least in the development of the differentiated form of breast cancer. Analysis of risk factors should, therefore, be related to modulation of bio- chemical behavior of stroma as well as epithelium.

Reconciliation of biology and epidemiology

The etiology of breast cancer may be considered under three headings: reproductive factors, dietary factors, and another category composed of diverse elements, discussed above, superficially unrelated to the two foregoing categories. Furthermore, it has been demonstrated that breast cancer is not a biologically homogenous entity, with the possibility of, at the very least, two axes of classification of breast tumors, i.e. by the hormone dependence of the tumor and by the cell type of origin of the tumor. How can what is known about the biology of breast cancer explain the etiology?

Reproductive factors

Breast cancer risk is increased in nulliparous women over parous women unless the first birth (or full term pregnancy) occurred after the age of 30 to 35. The risk decreases with an increasing number of children and, despite earlier reports, does not appear to be influenced by breast feeding. An early age at menarche, and a late age at menopause, probably serve to increase risk, as do pregnancies of less than 5 months duration and, perhaps, irreg-

ular menstrual cycles. Artificial menopause, however, decreases breast cancer risk.

Such observations led initially to formulation of the 'estrogen-window' hypothesis, i.e. extended periods of exposure to unopposed estrogen caused excessive epithelial proliferation. Such extended periods are seen during menarche, irregular periods, and menopause, particularly late menopause. During regular periods, progesterone is secreted after about 14 days of estrogen secretion such that it can act as an anti-estrogen and prevent the excessive proliferation.

Recent work has indicated that estrogen alone has little mitogenic potential for breast epithelium and may well act via the hypothalamus and/or by modulating stromal secretion of growth factors. For this reason, it is interesting to note that serum prolactin levels are suppressed in young women in proportion to the number of children to which they have given birth [165]. There is also increasing evidence that estrogen receptor levels are inversely proportional to epidermal growth factor receptor levels [153].

Attempts to analyse the influence of steroids contained in oral contraceptive pills have been con- tradictory [166, 167] but by and large negative [168, 169]. Problems remain in the difficulty for investi- gators to establish accurately the pattern of use and the steroid content of the pills taken by women [170]. Furthermore, it is possible that it is still too soon for most oral contraceptive studies to detect a difference in risk between users and non-users (if one does exist), in view of the extended period between the initial transformation event and the clinical manifestation of breast cancer.

There is increasing evidence that tamoxifen has a beneficial effect, with minimal side effects, when used as adjuvant therapy of breat cancer [171-173]. This value is greatest for patients with ER-rich (greater than 100 femtomols/mg cytosol protein) tumors. However, at least a proportion of patients with ER-poor tumors (less than 20 fmols/mg cytosol protein) also benefit in terms of increased disease-free interval (although it is not yet clear whether this leads to an overall survival benefit). For this reason, intervention studies with tamoxifen have been proposed for selected high-risk pa-

tients. Unfortunately, we currently lack sufficient information on the effects of long term tamoxifen, in otherwise well women, in terms of osteoporosis (data available so far is of course on cancer patients, many of whom already have abnormal calci- um metabolism), myocardial function, and other accumulated side effects, such as benign or malignant disease. The partial agonist activity of tamoxifen may, in fact, prove beneficial in terms of maintaining both bone structure and blood supply (the cells of the major blood vessel walls are known to contain functional estrogen receptor). However, it must be established whether tamoxifen should be given to the well woman as a single agent or in conjunction with progesterone. The high-risk group must be carefully selected (only post-menopausal?) and the length of therapy must be established (forever?).

Dietary factors

Breast cancer risk in populations appears related to the per capita consumption of both fat and cholesterol. Although this association is biologically plausible it is an association not generally found in direct study of breast cancer cases (see above). This appearent inconsistency demands extensive epidemiological investigation in diverse populations. Recently, it has been suggested that breast cancer risk may be increased by a low dietary intake of Vitamin A and elevated by modest consumption of alcohol.

Dietary intake of both fat and cholesterol has been associated with elevated plasma estrogen levels since increased fatjntake leads to increased fat cell production which, in turn, leads to increased peripheral aromatization of adernal androgens to

/

estrogens. Estrogen is, of course, structurally derived from the cholesterol nucleus.

Two factors, however, suggest that this interpretation may be oversimple. Firstly, elevated plasma estrogen levels are not generally reported from series of breast cancer patients although it has been reported [174] that it is the free estradiol concentration, rather than the total, which is critical. Second- ly, it has been suggested that a high intake of eggs


may be protective against breast cancer although egg yolk is high in cholesterol content.

A dilemma exists over obese patients. In simple terms, excess fat cells contribute to increased amounts of 'available' estrogen. This should increase the chances of growth of hormone-dependent tumors. However, such tumors are associated with good prognosis, whereas obese patients with breast cancer have a shorter life expectancy than thin people. One explanation is that elevated available estrogen is necessary for initiation of all breast tumors even though some of these then become ER-negative by the time the tumor is an overt clinical problem. The high level of estrogen at the 'nascent' stage of the tumor may even prompt growth of 'aggressive' cells. Of course, there are several confounding factors such as the increased weight corresponding to reduced blood circulation which actually increase the chances of premature death in the obese breast cancer patients compared with the thin patients. Nevertheless, it remains to be seen precisely by what mechanism the excess estrogen stimulates the early growth of the tumor.

The protective role of vitamin A has been associated with its undoubted ability to act as a free radical sink. However, more recent evidence has demonstrated that vitamin A has a profound effect on cyto-skeletal structure [175], and independent observations have indicated that changes in cyto- skeletal organization are associated with malignant transformation [176].

Although it is not yet fully appreciated what role alcohol plays in the determination of breast cancer risk, it is well-recognized that the metabolism of alcohol can have a significant effect on the NAD/ NADH ratio. Such a change will influence many oxido-reduction reactions, which would include the conversion of estradiol to estrone by the 1713- steroid dehydrogenase. Estrone is known to be less estrogenic than oestradiol.

Conclusions

We have summarized widely-accepted evidence regarding the biology and epidemiology of breast cancer. It is clear that 'breast cancer' is not a homo-


geneous entity and that several distinctive axes of classification are possible. Furthermore, we have tried to give an indication that knowledge of the etiology of breast cancer is relatively poorly understood, relative to the number of studies undertaken to seek the cause.

The very fact that some breast cancers may be aggressive while others are less so, some may depend strongly on estrogen for growth while others may not, and that breast cancer may well arise from cells at different points down the differentiation pathway clearly indicates the heterogeneity of the disease. Given the propensity of carcinogens to produce specific tumors (vinyl chloride monomer/ angiosarcoma of the liver, asbestos/mesothelioma of the pleura, aflatoxin/hepatocellular carcinoma, etc.), it is a logical conclusion that each type of breast cancer may have a different 'cause' or, at least, one etiologic factor may have more influence on one form of breast cancer than another.

To continue treating breast cancer as a homogeneous entity for epidemiological purposes would clearly be a mistake in view of what is known about the biological heterogeneity of this disease. Breast cancer should be considered, at least, on the basis of the hormone dependence of the tumor [177] and the cell-type of origin of the tumor [178] although due to the predominance of intraductal carcinoma the effect of this second classification may not be great; clinicians have recognized the importance of these two features for several years [179]. In addition to the potential range of transformation factors (initiators), the roles of potential promoters deserve consideration.

It is clear that great progress will not be made without due interaction between biological and epidemiological thought. The prize is great: the ability to prevent one per cent of breast cancer would mean a reduction of over 5, 000 cases per year worldwide. However, it is not entirely clear that any feasible intervention strategy which could be implemented in the general population has a guar- antee of reducing the occurrence of this disease. The proposed randomized trial of reduction in dietary fat intake in United States women appears a long-shot from the available epidemiologic evidence derived from case-control and cohort stud-

ies. Paradoxically, there appear good reasons from international comparisons , such as [50], and our appreciation of the biology of the disease, for thinking that such an intervention could be success- ful. It is clear, however, that more work needs to be done in the area of nutrition and breast cancer to investigate the roles of the intake of total fat, saturated fat, total calories, fiber, and vitamins on influencing the risk of disease. Parallel studies, such as that being organised by the SEARCH pro- gram of IARC [180], using identical protocols to conduct population-based case-control studies in geographically separated populations at disparate risk of disease, could potentially throw some light on these aspects.

While the use of tamoxifen has been promoted on a trial basis as a prophylactic in high-risk women [181] it may yet be premature to undertake such an intervention. The potential side-effects of tamoxifen in well women are not yet clearly understood and it would be precipitous to give a compound, which could possibly cause serious side-effects, to a large number of otherwise well women without having much more information regarding toxicity in animals and man.

There is a measure of consistency about findings of increased breast cancer risk in obese women which, perhaps, offers some prospect for prevention. It would seem timely, in theory, to undertake a randomized trial of weight-reduction in obese women to reduce their breast cancer risk. While theoretically attractive in view of the accumulated evidence, the logistics are much more complicated. However, in view of the evidence, it could be more likely to succeed than either trials of fat reduction (on an isocaloric diet) or tamoxifen.

While it remains our view that, despite such a large number of studies, progress in the understanding of the epidemiology of breast cancer has been disappointingly slow, a view which also comes across from the second UICC Multi Disciplinary Breast Cancer report [182], there remains opti- mism that by bringing better biological insight to epidemiological studies, our understanding can be improved. This will be greatly assisted by the im- proving ability of the epidemiologist to obtain dietary information with greater precision on better

defined, biologically homogeneous groups of breast cancer cases.

Acknowledgements

This investigation was supported by Grant CA-06516, awarded by the National Cancer In- stitute, DHHS.

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