Hormone Assays in Human Breast Cancer

Hormone Assays in Human Breast Cancer R . D. BULBROOK Imperial Cancer Research Fund. Lhwln’a Inn Fields. London. England Page

I. Introduction . . . . . . . . . . . . . . I1 Methods . . . . . . . . . . . . . . A. Chemical . . . . . . . . . . . . . . B. Clinical . . . . . . . . . . . . . . C . Nomenclature . . . . . . . . . . . . . 111. Hormone Imbalance and Hormone Dependency in Human Breast Cancer . I V. Urinary Estrogen and Progesterone Metabolites . . . . . . A Prognosis . . . . . . . . . . . . . . B . Effects of Treatment . . . . . . . . . . . C Comparison with the Normal Population . . . . . . . V. Urinary Androgen and Corticosteroid Metabolites . . . . . A . Prognosis in the Advanced Disease . . . . . . . . B. The ‘‘Free Period” and Other Host Factors . . . . . . C. Prognosis in the Early Disease . . . . . . . . . D . Prophylaxis . . . . . . . . . . . . . E . Comparison with the Normal Population . . . . . . . F The Effects of Mastectomy on the Endocrine System . . . . VI. Hormones in Plasma . . . . . . . . . . . . A. The 17-Hydroxycorticosteroids . . . . . . . . . B . The 17-Oxosteroids . . . . . . . . . . . VII . Secretion Rate Studies . . . . . . . . . . . VIII. Pituitary Hormones . . . . . . . . . . . . A . Gonadotropins . . . . . . . . . . . . B. Growth Hormone and Prolactin . . . . . . . . . C. Thyroid Function . . . . . . . . . . . . I X . Endocrine Status after Endocrine Ablation . . . . . . . X . Epidemiological Studies . . . . . . . . . . . X I . Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

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I . INTRODUCTION This article is mainly concerned with work published after 1955 on the role of the hormones in human breast cancer. Many of the methods used for the determination of the hormones or their metabolites before that date are not acceptable by present standards . It was also thought at that time that large differences might exist between the endocrine environment in 329

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patients with breast cancer and in normal women. Consequently, there was a tendency t o study small numbers of patients and to ignore the great clinical variability that exists within the disease. Present evidence points to the probability that hormonal abnormalities in this disease are small and exert their effects over long periods of time. If this is so, then highly specific and reliable routine assays are required; these must be applied to large groups of patients, and the results require refined statistical techniques for their evaluation. A main theme of this paper is an attempt t o adduce evidence that two groups of patients can be distinguished, from the time that the first diagnosis of breast cancer is made. It is postulated that one of these groups possesses hormone-responsive tumors, associated with a normal hormone environment; the second group possesses unresponsive tumors, and it is in this group that hormonal abnormalities are t o be found. Thus, an essential feature in any study of the endocrine aspects of breast cancer is a measure of the hormone-responsiveness of the tumor. Without this information, the normal hormonal environment found in so many patients may obscure the fact that an abnormal environment may be found in the rest. The clinical effects of endocrine therapy are not described. In spite of the fact that breast cancer has been known and written about for over two thousand years, the confusion in the clinical literature is such that a Kafka would be required t o do justice to the subject. Studies on the metabolism of various steroids by patients with breast cancer, or the effects of additive therapy (estrogens, androgens, and corticosteroids) on the endogenous hormonal environment are not discussed, except for one or two papers that appear to be relevant t o the main theme of the review. References to work in these fields include Bayer et al. (1957, 1960), Breuer (1961), Breuer and Nocke (1959), Schubert (1957, 1958), Schubert and Bacigalupo (1960, 1962), Schubert and Schroder (1960), Segaloff (1961, 1964). Several general reviews and proceedings of symposia are available, and these cover the ground not dealt with in this review (Bielschowsky and Horning, 1958; Bulbrook and Strong, 1959; Shimkin, 1957; Pincus and Vollmer, 1960; Strong, 1958). Two symposia sponsored by the American Cancer Society (1957, 1965) provide much information.

11. METHODS A. CHEMICAL The quality of many of the analytical methods used in the early work in breast cancer left a good deal to be desired. Frequently, compounds derived from different secretory products were measured in groups, little attention being paid t o preliminary hydrolysis of conjugates or purification.

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Data on the accuracy, precision, sensitivity, and specificity of the methods used were omitted. International standards were not used in biological assays. A marked improvement occurred starting in the early 1950’s, and much of the credit for this is due t o the influence of the Edinburgh School (see Brown, 1955; Brown et al., 1957; Marrian, 1957; Loraine and Brown, 1959; Loraine, 1958) and t o Borth (1950, 1952) and Diczfalusy (1957). It soon became apparent that if hormonal abnormalities existed in women with breast cancer they would be small, and, to add to the difficulties, the enormous variation in the normal population began t o be appreciated. For example, Brown (1955) showed that in normal women there was at least a fivefold variation in the excretion of estrone, estradiol-17& and estriol in the menstrual cycle. Other workers found an equally large variation in the excretion of 1l-deoxy-17-oxosteroids (Kellie, 1954; Brooksbank and Salokangas, 1959; Bulbrook, 1965) and in the corticosteroids (Borth et al., 1957; Level1 et al., 1957). In the last five years, however, it has become clear that even the best of the methods for the analysis of urinary steroids may give rise to misleading conclusions about the environment from which the steroids were derived. Two examples may emphasize this point. There is considerable evidence bearing on the reliability of Brown’s method (1955) for measuring urinary estrone, estradiol-l7p, and estriol: but it is now known that there are at least fifteen urinary metabolites of the estrogenic hormones (see Diczfalusy et al., 1961) and there is evidence for the presence of several more. It is almost certain that measurement of only three of these compounds is not sufficient if deductions are to be made about the secretion and metabolism of their precursors. At the time of writing, there is no generally accepted method for the measurement of the majority of the urinary estrogen metabolites that would be suitable for the massive studies required in breast cancer (see Paulsen, 1965). The second example concerns the C19steroids. It has only recently been appreciated that dehydroepiandrosterone is secreted into the blood partly as the sulfate (see Baulieu, 1962; Vande Wiele et al., 1963). Assays of urinary dehydroepiandrosterone, as currently performed, provide no information as to the proportions of free and conjugated steroid actually secreted. Again, it has recently been shown that A4-androstenedione may be converted to testosterone by the liver. However, some of this testosterone does not then mingle with the body pool, but is promptly conjugated and excreted into the urine as the glucuronide (Korenman and Lipsett, 1964; Tait and Horton, 1964). Thus any correlations found between the amounts of urinary hormones or metabolites and any particular aspect of breast cancer must be regarded

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at the moment with (tolerant) suspicion as far as physiological deductions are concerned. It is a little early to judge the potential usefulness of assays of plasma steroids, By analogy with the history of the development of meaningful urinary assays, the present spate of plasma methods may require substantial modification as experience is gained. It is of interest that reported levels of plasma testosterone in normal women fall sharply with every improvement in methodology (see Hudson et d., 1963;Forchielli et d ,1963;Riondel et al., 1963;Lobotsky et al., 1964). Immunological assays for growth hormone and insulin in plasma now appear to be satisfactory, and those being developed for ACTH and the gonadotropins show considerable promise (see Berson and Yalow, 19G4). Future advances in our knowledge of human breast cancer are entirely dependent on the availability of analytical methods designed t o fit the particular problem. There are certainly no grounds for optimism that rapid advances will occur in this field. B. CLINICAL

It has taken a long time to realize that the results of the most refined biochemical procedures are of little use unless they are matched with an equal degree of accuracy and sltill on the part of the clinical investigator. Many studies are highly suspect in this regard. There are, however, signs of change, and much of the current American work is based upon a common clinical protocol (see Segaloff, 1961,1964). Another clinical problem that is by no means solved is the selection of appropriate controls. These have ranged from hospitalized patients with any disease other than breast cancer, to patients with benign breast disease, or to normal healthy women. These choices are all open to criticism since there is little doubt that even entry into hospital will affect some hormone levels (see Apostolaltis and Loraine, 1960;Mason, 1959)and that diseases other than those of the endocrine or target organs may also affect the hormonal environment (see, for example, Chou and Wang, 1939; Shuster, 1960;Green and Pulvertaft, 1962). C. NOMENCLATURE The most common trivial names for the steroids are used throughout; these are: 1. dehydroepiandrosterone : 2. androsterone: 3. etiocholanolone: 4. androstenedione :

3@-hydroxy-androst-5-en-17-one 3a-h ydroxy-5a-androstan-17-one 3or-hydroxy-5@-androstan-l7-one androst-4-ene-3,17-dione

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The ll-deoxy-17-oxosteroids are defined as Cl0 compounds of the androstane series, lacking a ketone or hydroxyl group at C-11. The urinary fraction is mainly made up of compounds 1, 2, and 3 above. They are derived from secreted testosterone, androstenedione, dehydroepiandrosterone and its sulfate. The 1l-oxygenated-17-oxosteroids are as above except for the presence of a ketone or hydroxyl group at C-11. These compounds are derived mainly from cortisol. The 17-hydroxycorticosteroids (17-OHCS) refer to compounds in urine measured as 17-oxosteroids after reduction of the residual 17-oxosteroids with sodium borohydride followed by oxidation of Cpl steroids with sodium bismuthate (Appleby et al., 1955) or with sodium periodate (Few, 1961). The plasma 17-OHCS are compounds measured by the reaction of Porter and Silber (1950). “Early breast cancer” is defined t o include any cases in stages I or 11; that is, patients with operable lesions confined to the breast and axilla. “Advanced breast cancer” is defined to include patients with inoperable local lesions and/or those with distant metastases. Breast tumors are referred to as “responsive” or “unresponsive” in this text. These terms usually have highly specific meanings, but their precise definition varies from worker to worker (see Bulbrook et al., 1960a; Hayward and Bulbrook, 1965; Segaloff, 1961). I n the absence of any general agreement in classification of response to treatment, the term “responsive tumor’’ is broadly defined as one in which all lesions diminish in size upon treatment: “successful response” is synonymous. Where authors have not provided strict evidence, their clinical evaluation has been taken on trust. Unresponsive tumors are those in which there is evidence of continued growth after treatment.

111. HORMONE IMBALANCE AND HORMONE DEPENDENCY IN HUMAN BREASTCANCER It has been demonstrated repeatedly in laboratory experiments that tumors can be induced in almost all organs that respond to hormones, by alteration of the hormonal environment; the alterations may be brought about by hormone excess or by deprivation. Examples of such results are the induction of pituitary tumors with large doses of estrogen, or the induction of thyroid and pituitary t,umors by administration of antithyroid drugs such as thiouracil. In these cases, a common mechanism of excessive stimulation of secretory cells exists. This type of experiment led t o the concept of hormone imbalance as an important factor in endocrine carcinogenesis (Gardner el al., 1954; Furth, 1955, 1957; Hertz, 1957). A second concept, based on clinical observations and on laboratory experiments, concerned the behavior of tumors arising in organs that are

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responsive to hormonal stimuli. Briefly, if normal cells are dependent on a continuous supply of hormones for their growth, rate of metabolic activity, and continued existence, then tumor cells arising from these normal cells may be dependent on the same hormones, a t least for a period in their life span. While in this dependent state, the tumor cells behave as do normal cells when hormonal stimuli are withdrawn-that is, they atrophy. The fact that endocrinological treatment is rarely, if ever, curative and that renewed tumor growth eventually occurs has led some workers to prefer the term “hormone-responsive” in place of “hormone-dependent .” However, it may very well be that dependency is absolute at some stage in the progression from the normal to the neoplastic cell and that “responsiveness” describes a later stage in the process (see Huggins, 1960; Furth, 1957). The two concepts, that of hormonal imbalance as a factor in the etiology of breast cancer and that of the responsiveness of the established tumor, underlie almost all the investigations described in the following sections.

IV. URINARYESTROGEN AND PROGESTERONE METABOLITES It has been known for nearly seventy years that oophorectomy may bring about regression of advanced breast cancer. Subsequently, it was shown that administration of estrogens to several animal species results in carcinoma of the breast (see Lacassagne, 1955). Furthermore, normal breast growth can be induced in hypophysectomized rodents with estrogens together with progesterone, growth hormone, prolactin, and cortisol (see Hadfield, 1958). Thus, the estrogens appeared to be of primary importance in human breast cancer, and there are frequent references in the literature to the “estrogen-dependency” of human tumors. However, there is no direct evidence that the estrogens are carcinogenic in man, and they have not been shown to be carcinogenic in the monkey (Engle et al., 1945; Pfeiffer and Allen, 1945). The indirect evidence cited above that the estrogens might play a key role in breast cancer and the availability of useful methods for urinary estrogen analyses (Brown, 1955; Bauld, 1956) gave a great stimulusIto investigators. A. PROGNOSIS Most of the effort spent on investigations of the part played by the estrogens was devoted to establishing a possible relation between the amounts of estrogen in the urine and the subsequent response of the patient to treatment by stilbestrol, adrenalectomy, or hypophysectomy. The most common finding was that the urinary titers of estrone, estradiol, or estriol were not related to response (Strong et al., 1956; Birke et al., 1958; Bul-

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brook et al., 1958a,b,c; Brown et al., 1959; McAllister et al., 1960; Irvine et al., 1961; Palmer and Hellstrom, 1962), or were only poorly correlated

(Jull el al., 1963). In only two instances (Huggins and Dao, 1954; Bergenstal et al., 1955; Block et al., 1959) was a good correlation found between estrogen excretion and response t o adrenalec tomy. Shucksmith et al. (1960) claimed that the estrogens were valuable in prediction of response providing that the results were supported by biopsy findings. Thus, if a “high” estrogen output was found in conjunction with stimulated breast tissue in the biopsy section, the patient responded t o either adrenalectomy or hypophysectomy : a high estrogen excretion with unstimulated breast tissue was associated with response to adrenalectomy. A “low” estrogen output and a stimulated breast indicated a response to hypophysectomy, and a low estrogen excretion with an unstimulated breast, response neither t o adrenalectomy nor to hypophysectomy. While the bulk of the evidence does not show a relation between estrogen excretion and response to treatment, the subject is by no means closed, for a variety of reasons. The criticism leveled at Casanova can be applied (“the statistics are impressive but you should have seen some of the women!”) : in other words, some of the authors studied extremely heterogeneous populations and usually only small numbers of patients. Estrogen excretion throughout complete menstrual cycles was rarely measured. Finally, in many cases only estrone and estriol were measured (the estradiol levels were below the limits of sensitivity of the method), and these metabolites may not give an accurate enough index of estrogen status (see Section 11, A). B. EFFECTS OF TREATMENT A second line of research on the role of the estrogens was an attempt to correlate changes in estrogen excretion, brought about by treatment, with the resultant clinical effects. The treatments were oophorectomy, adrenalectomy, hypophysectomy, cortisone administration, and androgen administration. No convincing relationship between changes in estrogen levels and the response of the patient to treatment has emerged from this work. Objective regression of the disease has occurred in the presence of continued excretion of estrogens in the urine; and patients have failed to respond to treatment even when estrogen excretion has fallen to amounts indistinguishable from zero (Bayer et al., 1957, 1958; Bulbrook and Greenwood, 1957; Greenwood and Bulbrook, 1957; Hellstrom et al., 1957; Bulbrook et al., 1958a,b,c; Scowen, 1958; Block et al., 1959; Brown et al., 1959; McAllister et al., 1960; Hayward et al., 1961; Nissen-Meyer and Sanner, 1963; Persson and

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Risholm, 1964). Estrogen excretion following adrenalectomy or hypophysectomy is discussed in Section IX. The results of Nissen-Meyer and Sanner (1963) are interesting in that they show a very slow decline in estrone and pregnanediol excretion after the menopause with a secondary peak of estrone excretion 10-15 years later. These authors also point out the effectiveness of administered cortisone in depressing the excretion of these compounds in postmenopausal patients, a finding confirmed by Persson and Risholm (1964), who also noted that estrogen excretion rose again when cortisone therapy was withdrawn. Jull et al. (1963) also found a secondary rise in estrogen excretion after oophorectomy. C. COMPARISON WITH THE NORMAL POPULATION The third line of approach t o the problem was to compare estrogen excretion in patients with breast cancer with that of normal women. Brown (1958) found that postmenopausal patients with breast cancer excreted slightly larger amounts of urinary estrogens than did controls. This increase was due mainly to an increase in the estriol fraction. This finding has been confirmed by Nissen-Meyer and Sanner (1963), by Stern et al. (1964), and, in a very extensive study, by Persson and Risholm (1964). Jull et al. (1963) noted, however, normal estrogen excretion in 9 of 10 untreated premenopausal women. None of the authors cited has cared to speculate on the possible physiological significance of the raised proportion of estriol, nor is there evidence that the degree of abnormality in estriol excretion is correlated with the course of the disease. Stern et al. (1964) used discriminant functions for differentiating between normal women and pre- and postmenopausal patients with early breast cancer. Before the menopause, a combination of assay results on “gonadotropin residue carbohydrate,” estrone, and etiocholanolone gave the most powerful discrimination: after the menopause, gonadotropin, estriol, and androsterone. Using the latter three variables, these authors achieved a very impressive separation between cancer cases and controls. In some of the studies referred to above, urinary pregnanediol excretion was measured. No results of interest emerged, To summarize, work on the urinary estrogens in breast cancer has produced inconclusive and unsatisfactory results. At the time of writing new methods are required for the measurement of the majority of the estrogen metabolites in urine, for the determination of plasma estrogens, and for the calculation of secretion rates. When generally accepted methods are available, investigations should be carried out on homogeneous groups of patients and controls and for periods of time sufficient to encompass any cyclic variation.

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V. URINARYANDROGEN AND CORTICOSTEROID METABOLITES A. PROGNOSIS IN THE ADVANCED DISEASE The first indication that urinary androgen and corticosteroid metabolites might be of use in the prediction of the results of adrenalectomy or hypophysectomy came from Allen et al. (1957). They reported a close correlation between a ratio of what they called the ll-deoxy fraction t o the ll-oxy fraction of the urinary 17-oxosteroids (for definitions, see Section 11, C). The method they used can only be described as unusual. Plantin and his colleagues (1958), using a more reliable method, failed to confirm these findings. I n both instances the number of patients studied was very small. Bulbrook et al. (1960a) reinvestigated the problem using yet another method for the analysis of the individual compounds in the urinary 17oxosteroids (Kellie and Wade, 1957). This study included assays of urinary estrogens, pregnanediol, 17-OIICS1 and gonadotropins in addition t o the 17-oxosteroids. Their thesis was that previous failures t o achieve accurate prognosis might have been due to insufficient measurements of enough aspects of the hormonal environment. They found that the amounts of urinary 17-OHCS were usually low and those of the 1l-deoxy-17-oxosteroids (dehydroepiandrosterone, androsterone, and etiocholanolone) were usually high in patients who subsequently responded to adrenalectomy or hypophysectomy, compared with the amounts of these steroids found in the urine of patients who subsequently failed t o respond. Neither group of steroids, used alone, gave a reasonable prediction of response, but when expressed as a ratio there was a clear difference between responsive and unresponsive patients (Hayward et al., 1961). A more efficient division between the patients was achieved by the use of a discriminant function as follows: 80 - 80 X 17-OHCS(mg/24 hours)

+ etiocholanolone (pg/24 hours)

When the results of the relevant assays were substituted in the formula and the answer was a positive number (a positive discriminant), the clinical response to adrenalectomy or hypophysectomy was usually good. When the answer was a negative number (a negative discriminant) the response was usually poor. The discriminant function was calculated from the results of a retrospective study, and it did not seem reasonable to deny treatment to patients on the basis of the discriminant alone. It was decided, therefore, to test the practical use of the discriminant in a forward study in which patients with positive discriminants were t o be recommended for hypophysectomy and those with negative discriminants, for adrenalectomy. If the previous findings of Bulbrook et al. (1960a, 1962a) were valid, the re-

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mission rate should rise in the hypophysectomy series and fall in the adrenalectomy series. At the outset of the trial, it was decided that if a regression rate significantly below 20% were encountered, then neither adrenalectomy nor hypophysectomy would be justified, bearing in mind the severity of these operations, the availability of other forms of treatment (such as cortisone), and the short duration of remission even in successful cases. Clearly, the 20% limit was a subjective decision. Other workers might accept a higher limit or insist on a lower one. One half of this trial has now been completed (Atkins and his colleagues, 1964). It was shown that when patients were treated by adrenalectomy on the basis of a negative discriminant, their regression rate was 9%, a rate that was significantly lower than the 20% limit described above. These authors concluded that adrenalectomy could not be recommended to patients with negative discriminants. The termination of the trial at this point left the position with regard to hypophysectomy in some doubt. The regression rate for patients with positive discriminants was five times that of the negative discriminant group and almost double that achieved by selecting patients for hypophysectomy on a random basis. However, this last finding did not achieve formal significance. A new trial has now been initiated in which patients with negative discriminants are to be treated by hypophysectomy. If the regression rate is low, then endocrine ablation will offer little hope to these patients. It would be miraculous, in this field, if there were complete agreement among workers. Sim et al. (1960) found that assays of 17-OHCS and 11deoxy-17-oxosteroids were of no value in predicting the clinical outcome of pituitary destruction by yttrium-90 implantation. On the other hand, Juret el al. (1964) have found a remarkable correlation between the amounts of androsterone and etiocholanolone in the urine and the patients’ response to this treatment. When the sum of these two compounds was below 0.3 mg/24 hours the regression rate was 7y0. When the sum of the titers was above 2 mg/24 hours, the rate was 70%. Hayward and Bulbrook (1965) have recently summarized their total experience with the discriminant in predicting the results of hypophysectomy or adrenalectomy in advanced breast cancer. The regression rate in 77 patients with positive discriminants was 47%, compared with that of 11% in 82 patients with negative discriminants. The mean survival of the former group was almost 18 months compared with 10 months in the latter. Their personal evidence is thus very strong that the discriminant is useful, but what is now required is evidence that it will work in other hands, on patients drawn from other areas. If it does, then a considerable stride

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will have been taken toward efficient palliation for patients with advanced metastatic breast cancer. B. THE “FREEPERIOD” AND OTHERHOST FACTORS

It is unfortunate that so many workers have chosen to concentrate on one single aspect of the endocrine environment in their search for prognostic tests. It is inconceivable that changes in the secretion of one hormone could occur without affecting the secretion or metabolism of others. One of the most striking examples of this interrelation is the influence of the thyroid hormone on the metabolism of androgens, corticosteroids, and estrogens (see Hellman el al., 1959, 1961; Fishman et al., 1962). Information from a wide variety of assays might thus be additive. In the same way, it seems unreasonable t o concentrate on the endocrine system t o the exclusion of the host and her tumor. At the most simple level, there are obvious differences between patients in the site or sites of their secondary growths. At one end of the spectrum, patients may be found with extensive skin lesions (and few in other sites) while other patients may present with what appears to be a single cerebral metastasis. There is no information whether the hormonal environment influences the site at which the dominant metastases will appear. At a more complex level, there are obvious differences in the degree of differentiation of the tumors. At one time it was claimed that response to endocrine therapy could be correlated with the histology of the tumor (Huggins and Dao, 1952)) but subsequent work has not confirmed this (Allen et al., 1958). This may imply that differentiation of the tumor is independent of the endocrine milieu, but a re-examination of this problem, combining histological and endocrinological investigations, would not be amiss. There is good evidence that the period between the first definitive treatment of the tumor (usually mastectomy) and the time of first recurrence is related t o the hormone-responsiveness of the tumor and t o the length of survival of the patient (Bergenstal et al. 1955; Hellstrom and Franksson, 1958; Luft et al., 1958; Juret et al., 1964). This so-called “free period” was found to be about 40 months in patients responsive to adrenalectomy or hypophysectomy and 30 months in unresponsive cases; the difference is highly significant (Macdonald, 1962). Juret (personal communication, 1964) suggested that the predictive power of the discriminant might be improved if the length of the free period were also taken into account. When this was done, it appeared that the length of the free period was irrelevant t o the remission rate in patients with positive discriminants. Whatever its length, the rate remained fairly constant at about 40-50%. However, in patients with negative discriminants, no regressions a t all were observed in those with a free period shorter than two years. The few regressions recorded

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in this group all occurred in patients with long free periods (Bulbrook and Hayward, 1965). Another method of taking into account the biological properties of the tumor has been described by Folca et al. (1961). Radioactive hexestrol was administered t o patients with advanced breast cancer, and skin secondaries were removed later. The amount of radioactivity found within the metastases, when expressed as a ratio of that found in muscle gave a clear differentiation between patients subsequently responsive or unresponsive to adrenalectomy. A similar approach has been described by Deshpande et al. (1963) and by Ellis et al. (1965) in the early disease using radioactive testosterone, and by Demetriou et al. (1964) using labeled estrogens.

C. PROGNOSIS IN THE EARLY DISEASE There is a widespread belief that breast cancer can be cured if radical surgery can be carried out early enough. The data of Schwartz (1961) and Gershon-Cohen et al. (1963) indicate that this concept may be overoptimistic. The belief in the efficacy of surgery is probably why so little attention has been paid to early breast cancer by endocrinologists and why a disproportionate effort has been made in the advanced disease. I n the light of what little information is now available, the early phase of the disease may be a much more rewarding field for hormone studies. Hayward and Bulbrook (1962) and Bulbrook et al. (1964a) found that approximately half of their patients with the early disease had negative discriminants 10 days after mastectomy. In a three-year study, the rate of recurrence in this group of 26 patients was almost three times as great as that of 21 patients with positive discriminants (54% compared with 20y0). The death rate was six times as great in the patients with negative discriminants as in those with positive discriminants. These results were obtained from a study of patients aged from 31 to 78 years. When the results for women aged 65 and over were omitted (on the basis that tumor growth tends to be slower in elderly patients) and the stage of the disease was also taken into account, a group of ten patients with negative discriminants and stage I1 carcinomas was isolated of whom nine died within three years. These findings might have been obtained if patients with negative discriminants had a greater degree of advancement of the disease when first seen but analysis showed that this was not so: the stage of the disease and the grade of the tumor (Bloom, 1950) were almost identical in patients with positive and with negative discriminants. Two points may be made about this work: in the first place, the number of patients studied was small; in the second place, the urine was obtained

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10 days after mastectomy. The first point can only be answered by investigating more patients; the second point is dealt with at length in Section V, F. When the results described above are considered in conjunction with those obtained in patients with the advanced disease, it seems possible that patients with breast cancer may be divisible into two broad groups from the time of first diagnosis. The first of these groups is characterized by an abnormal excretion of 11-deoxy-17-oxosteroids and 17-OHCS after mastectomy (the negative discriminant group). In this group, the recurrence rate after mastectomy is high and, if the patient survives long enough, response to adrenalectomy or hypophysectomy is poor (see Section V, A). The second group of patients consists of a population with a normal hormonal environment (positive discriminants), a slow recurrence rate, and a good chance of response to endocrine ablation. To obtain direct proof for these assumptions, Bulbrook and Hayward (1965) followed up a group of 47 women with the early disease. They found that of 26 patients with negative discriminants at mastectomy, four died without any endocrine treatment, four died while on additive treatment, and only four were submitted to adrenalectomy or hypophysectomy. None responded t o these forms of treatment. Three of 21 patients with positive discriminants at mastectomy were subsequently treated by endocrine ablation, and two had a successful response. Thus, what little evidence is available supports the thesis that two groups of patients may be defined a t the time of mastectomy and that response to subsequent ablative therapy might be predictable at this time. Bulbrook and Hayward (1965) noted that in the patients with negative discriminants who died before adrenalectomy or hypophysectomy could be performed, the interval between recurrence and death was very short. This point is of particular importance because it indicates that the type of patient submitted to these operations will be selected to a certain extent by the intensity of the follow-up after mastectomy and the therapy instituted a t recurrence. A center where patients were followed up at very short intervals of time, where additive therapy was not commonly used, and where there were adequate facilities for adrenalectomy or hypophysectomy might treat a different population compared with a center without these characteristics. If the results of Bulbrook and Hayward (1965) are a fair sample of the general population, any delay in treating recurrence by endocrine ablation may allow the less favorable cases to succumb. A particularly surprising finding was that patients with positive discriminants, in stage I1 and with tumors of a high grade of malignancy (both of the latter factors indicating a grave prognosis) nevertheless had a low recurrence rate. This implies that the hormonal environment may be of

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more importance in prognosis than the degree of spread of the disease or the degree of dedifferentiation of the tumor cells.

D. PROPHYLAXIS When 'the beneficial effects of oophorectomy in advanced breast cancer became generally appreciated, it was thought that the use of this treatment at a very early stage in the disease might prevent or delay recurrence. A great deal of wholly unnecessary confusionarose because many of those who used prophylactic oophorectomy failed to randomize their patients between this treatment and no treatment in addition to mastectomy. The few workers who carried out properly designed trials failed to show a significant advantage in prophylactic oophorectomy although there was a consistent trend toward a better prognosis in patients so treated (see Lewison, 1962) and impressive results have recently been reported by Nissen-Meyer (1964). In the last decade, a very large, randomized trial has been undertaken by the Christie Hospital in Manchester (see Cole, 1964), where patients have been treated by mastectomy or by mastectomy plus ovarian irradiation. Treatment was decided by random selection. The only significant effect so far noted was an improvement in the rate at which distant metastases occurred at five and at seven years. Thus, the beneficial results of prophylactic oophorectomy or X-ray castration are small and it still has to be shown that the benefits of late oophorectomy are inferior in the aggregate. The results of Bulbrook et al. (1964a) may have a considerable bearing on this problem. They have suggested that two groups of patients may be distinguished at first diagnosis; one with negative discriminants, a rapid recurrence rate, and tumors unresponsive to hormones; the other with positive discriminants, a slower rate of recurrence, and hormone-responsive tumors (see Section V, C). If this is so, prophylactic oophorectomy may be of little use in the former group, and for the first three or four years of any trial of such therapy, these will be the patients who make up the majority of the cases with recurrence. The lack of beneficial effect in this group would tend to conceal a valuable effect in the few cases of recurrence in patients with positive discriminants. These latter patients would tend to have a late recurrence and the benefits, if any, of prophylaxis might only become apparent after a trial had been running for many years. Bulbrook and Hayward (1965) have described the establishment of a trial on prophylaxis at the time of mastectomy in which the discriminant function is measured before therapy is instituted. This trial should eventually provide direct evidence concerning hormone responsiveness in early breast cancer and whether or not a group of patients can be identified for whom prophylactic treatment is useless.

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Similar hormone studies might be desirable where prophylactic adrenalectomy or hypophysectomy are considered.

E. COMPARISON WITH

NORMAL POPULATION Kellie (1954) noted that patients with breast cancer excreted amounts of urinary dehydroepiandros terone, androsterone, and etiocholanolone that were within, but at the lower end of, the normal range. However, the patients were not divided according t o the responsiveness of their tumors to endocrine therapy. Bulbrook et al. (1962a) compared the excretion of the 1l-deoxy-17-oxosteroids in the urine of normal healthy women with the amounts found in the urine of patients with advanced breast disease who had either responsive tumors or who had tumors that did not respond to subsequent adrenalectomy or hypophysectomy. After due allowance for the fact that in normal women and in responsive patients the excretion of the 1l-deoxy-17-oxosteroids is age related, they showed that the excretion of androsterone and etiocholanolone was usually normal in responsive patients but was significantly low in unresponsive cases. They pointed out that without the knowledge of the responsiveness of the tumor, it would be difficult to make a significant comparison with the normal population. In patients with the early disease, the mean excretion of androsterone and etiocholanolone was significantly subnormal. The scatter of the results indicated that it was quite possible that two populations existed, one with a normal excretion of these compounds and another with a subnormal excretion (Bulbrook e2 al., 1962b). In a study with carefully selected controls Stern et al. (1964) reported similar findings. In premenopausal patients with early breast cancer the abnormality was greater than in postmenopausal women. The differences between the various groups were shown much more clearly when the discriminant function described in Section V, A was used (Bulbrook et al., 1962a)b) or when a three-variate discriminant based on androsterone, estriol, and “gonadotropin residue” titers was employed (Stern et al., 1964). The findings of Bulbrook et al. (1962b) that patients exhibited abnormalities in the excretion of some steroids at an early stage of the disease raised the possibility that the abnormalities might have preceded the clinical diagnosis of breast cancer. The precursors of the urinary steroids could conceivably be linked with the causative processes leading to neoplasia, or could influence the rate of growth of an established tumor, or could affect the biological properties of the tumor with respect to hormone responsiveness by influencing selection processes during the preclinical lifespan of the tumor. It now seems clear that perhaps two-thirds of this lifespan occurs before diagnosis of the disease is made (Gershon-Cohen et al., THE

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1963; Schwartz, 1961). Thus, if the precursors of the urinary androgen metabolites affected the rate at which tumor cells divided, a deficiency in the secretion of these compounds might have a profound effect on the time at which the tumor became large enough for discovery. A prospective study has been initiated to test the hypothesis that a group of women exist among the normal population who have a high risk of breast cancer, and that this group may be identified by the amounts of 11-deoxy-17-oxosteroids and 17-OHCS in their urine. So far, a single 24hour urine sample has been collected from each of 3500 normal women, aged 35-55 years. I n this group, an annual incidence of breast cancer of one per thousand is to be expected. So far, ten cases of breast cancer have been diagnosed at varying times after the collection of their urine but no statement can be made on such a small group (see Bulbrook et al., 1962b; Hayward, 1964; Bulbrook, 1965). Similar prospective studies may be needed to obtain information on other aspects of the hormonal environment in women who will eventually develop breast cancer. As far as the estrogens and progesterone are concerned, day-to-day variation would make such an investigation a formidable undertaking. In other parts of this paper, workers whose deductions have been based on the results of estimations in single samples of urine have been castigated. It must, therefore, seem unreasonable to base a large experiment of this nature on the results of single 24hour urine samples. However, such evidence at present available shows that the day-to-day variation in 17-OHCS and 11-deoxy-17-oxosteroids within normal women is small compared with the variation between women (see Kappas and Gallagher, 1955; Brooksbank, 1961; Bulbrook, 1965). More information on this point would not be amiss. Another explanation for the results of Bulbrook et al. (1962b) and Stern et al. (1964) may lie in the fact that tumors induced experimentally in rodents may affect steroid metabolism (Goodlad and Clark, 1961). It seems unlikely that a tumor weighing from 1 to 5 gm in a woman weighing 70 kg would bring about the profound changes in steroid excretion so far described, Finally, there remains the problem of selecting the best controls for such studies: this is discussed in Section 11, B.

F. THEEFFECTSOF MASTECTOMY ON

THE

ENDOCRINE SYSTEM

The results of Bulbrook el al. (1962b, 1964a) were obtained from determinations carried out on urine collected 10 days after mastectomy. It may be argued that such estimations do not give an accurate estimation of steady state levels; that is, an estimate of the hormonal environment that existed in the patient over a considerable period of time before the diagnosis

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was first made and which may have been of crucial importance in the preclinical disease process. The type of forward study described in the preceding section is the only way to resolve this question, but there are several points of interest in this field that bear examination. Estimations carried out on urine obtained from patients before mastectomy may be further from steady state levels than postoperative specimens. This is particularly true of 17-OHCS levels (see Moore, 1957). What is now required is a prolonged study of endocrine status after mastectomy to see whether the amounts of hormones found remain constant throughout the subsequent course of the disease. Even if the postoperative levels are not closely related to preclinical levels, providing they remain fairly constant they may be of considerable importance in determining some of the characteristics of the disease during the postmastectomy period. In one follow-up that has been reported briefly, it was not appreciated that so many patients would die within a year of mastectomy, and these were lost from the study. This gave a considerable bias to conclusions based on results obtained on the survivors. Nevertheless, there were indications that as the disease progressed, hormonal changes did occur and that some patients with positive discriminants after mastectomy later had negative ones (Bulbrook and Hayward, 1965). I t may be a mistake t o regard mastectomy simply as a surgical maneuver which causes only transient alterations in hormone production and metabolism. Grossman et al. (1950) showed that lactation could occur in men following injury to the chest wall. This may occur via a neural pathway analagous to the alterations in pituitary secretion brought about by the suckling stimulus. Although lactation has not been reported to occur in the remaining breast after mastectomy, there is no evidence that changes in pituitary function do not occur to a degree that is insufficient to cause lactation but which may nevertheless alter the effective stimulus to the other breast and to occult metastases. Altered thyroid function may be yet another mechanism whereby mast,ectomy brings about changes in the hormonal environment. In short, it cannot be assumed that mastectomy is necessarily beneficial in all cases, and hormone studies coupled with an intensive follow-up might be instructive. IN PLASMA VI. HORMONES

A. THE17-HYDROXYCORTICOSTEROIDS The availability of methods for the assay of plasma cortisol (or corticosteroids) has led to several studies of the levels of these compounds in

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the plasma of patients with breast cancer. BBnard et al. (1962a) noted great variability between the plasma corticosteroid levels, but nevertheless found that the mean level was significantly raised above that in their controls. Schubert et al. (1961) found high resting levels of plasma corticosteroids in advanced cases, and this was associated with a marked response to ACTH. Deshpande et al. (1965) have reported high corticosteroid levels in about half of their advanced breast cancer patients. This remarkable and highly unusual unanimity is not shared by Beck et al. (1965), who found normal plasma corticoid levels in their patients. Both Beck et al. (1965) and Deshpande et al. (1965) have commented on the lack of correlation between plasma levels and the amounts of 17-OHCS in the urine. The urinary amounts were lower than would have been expected from the plasma levels. Three explanations have been put forward to account for these findings: thyroid abnormalities, hepatic malfunction with regard to cortisol metabolism, and a raised level of transcortin. Raised plasma 17-OHCS and high cortisol binding have been reported to occur in patients with breast cancer (Sandberg et al., 1960). It may be significant that similar findings have been reported for pregnancy and for women treated with estrogens (see Peterson, 1959; Layne et al., 1962). However, as already noted in Section IV, high estrogen levels have not been demonstrated in patients with breast cancer. In only 2 of 28 patients studied by Deshpande et al. (1965) were raised plasma 17-OHCS associated with raised amounts of urinary compounds and with high plasma and urinary 17-oxosteroids.I n these cases, it was supposed that these results were due to stress-induced ACTH secretion. Schubert et al. (1961) suggested that the effects of ACTH on plasma corticosteroids might be a useful diagnostic test for the recognition of various stages of the disease, but the variability of response to ACTH in the advanced disease shown by Beck et al. (1965) makes this suggestion of doubtful value. B. THE17-OXOSTEROIDS The main compounds in the plasma 17-oxosteroid fraction are the sulfates of dehydroepiandrosterone and androsterone (see Deshpande and Bulbrook, 1964). BBnard et al. (1962b) found no differences between the plasma 17-oxosteroid levels in normal premenopausal women and patients with breast cancer, although there was a tendency for the latter to have slightly higher levels. However, in postmenopausal women with breast cancer, the plasma 17-oxosteroids were significantly raised above those of the controls. The findings of Deshpande et al. (1965) are not in agreement with these results: no significant divergence from normality was found, but

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there was a tendency for the plasma 17-oxosteroids to lie a t the lower end of the normal range. The main difference between the results of BQnard et al. (1962b) and those of Deshpande et al. (1965) is that the former workers found that several of their controls had little or no 17-oxosteroid in their plasma whereas the latter workers did not. Another interesting claim by BQnard et al. (1964) is that a substantial proportion of the plasma 17-oxosteroids was present in the free form in patients with breast cancer whereas these compounds were conjugated in the controls. Confirmation of this finding would be desirable. There is no information on plasma levels of testosterone, A4-androstenedione, and estradiol-17/3 in patients with breast cancer.

RATESTUDIES VII. SECRETION Pearlman (1957) described a method for the measurement of the amount of progesterone secreted into the blood stream. The method depended on the administration of a tracer dose of radioactive progesterone, which was assumed to mingle with the body pool of progesterone, and the subsequent isolation from the urine of a unique metabolite derived from this pool. The specific activity of the metabolite was determined, and this gave the degree of dilution of the administered hormone by the endogenous production of progesterone and, hence, the secretion rate. Using this approach, Vande Wiele and Lieberman (1960) reported results of measurements of the secretion rates of dehydroepiandrosterone following the administration of a tracer dose of this compound. Their method was used t o measure secretion rates in patients with advanced breast cancer (Bulbrook el al., 1963). While the specific activities of urinary dehydroepiandrosterone, androsterone, and etiocholanolone were similar in the cancer patients, those found in the control series did not agree well. Lieberman and his colleagues (Vande Wiele et al., 1963) had meanwhile noted similar discrepancies and found that the cause was due to the fact that dehydroepiandrosterone sulfate was also a secretory product and that a radioactive tracer dose of this compound had to be administered simultaneously with the tracer dose of dehydroepiandrosterone itself. This finding invalidated previous work with a single radioactive steroid. However, the degree of dilution of radioactive dehydroepiandrosterone by endogenous precursors was found to be highly correlated with the actual amounts of dehydroepiandrosterone, androsterone, and etiocholanolone found in the urine (Bulbrook et al., 1963). This finding provides some evidence that the abnormalities in the urinary 1l-deoxy-17-oxosteroid fraction which correlate so highly with the clinical course of breast cancer,

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are due to a failure of secretion of either dehydroepiandrosterone and its sulfate or of both of them. A discussion of the possible physiological implications of the results described in Sections V-VII has been given by Bulbrook (1965). Briefly, it is thought that the results indicate a low androgenic stimulus in patients who have tumors that are unresponsive t o therapy. If androgens act as antiestrogens as far as human breast growth is concerned, then there may be an excessive estrogenic stimulus to the breast in these cases. Direct evidence bearing on the hypothesis would be desirable. The role of the corticosteroids might be as antiandrogens or through an effect on the immunological mechanisms. These compounds can certainly increase metastatic spread and alter the distribution of the sites of secondary growth (Sherlock and Hartmann, 1962). The combination of androgen and corticoid metabolites in a discriminant function is empirical, but there is a possibility that this function may give a better indication of androgen status than measurement of the androgen metabolites alone. It has to be admitted, however, that inferences about the net hormonal stimulus to the target organ derived from results of assays of groups of hormones with synergistic or antagonistic actions have as much reliability at the moment as weather forecasts.

VIII. PITUITARY HORMONES A. GONADOTROPINS There is a particularly interesting observation on a relationship between the urinary excretion of gonadotropin and the clinical course of breast cancer. Loraine et al. (1957) measured gonadotropin excretion in 47 postmenopausal patients with advanced breast cancer before the patients were treated with stilbestrol. After therapy, the patients’ response to treatment was classified a8 “worse,” “no apparent change,” or “improved.” The amounts of gonadotropin excreted by these groups were compared with those excreted by 37 women with diseases other than cancer. The mean amount of gonadotropin excreted by the group whose disease progressed while on stilbestrol was significantly higher than that of the other groups and of the controls. These results are not in agreement with those of Segaloff el al. (1954), but Loraine el al. (1958) are critical of the assay methods used by the former workers. Boyland el al. (1958) also noted that no remissions occurred following irradiation of the pituitary gland in patients who excreted very high amounts of gonadotropins before treatment. The results of Loraine at al. (1957) showing high gonadotropin excretion

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in patients unresponsive to stilbestrol are the third instance where hormonal abnormalities have been found only in the unresponsive patients. It will be recalled that the excretion of subnormal amounts of androgen metabolites and negative discriminants were found in patients who did not respond to adrenalectomy and hypophysectomy (see Section V, A). Kushinsky et al. (1960) noted abnormalities in the metabolism of estradiol in patients who did not respond to the administration of stilbestrol. In each of these examples, the responsive patients were similar t o the controls as far as these parameters of endocrine function are concerned. This is a remarkable finding when it is considered how advanced was the disease in some of these patients (see Bulbrook, 1961).

B. GROWTHHORMONE AND PROLACTIN Growth hormone and prolactin are both necessary for breast growth in hypophysectomized rodents (Lyons et al., 1955). In man, however, there is controversy about the separate identity of growth hormone and prolactin (see Berson and Yalow, 1964). Beck et al. (1965) found that human growth hormone and ovine prolactin bring about virtually identical metabolic effects in man except that the latter hormone does not mobilize free fatty acids. Considerable advances have been made in methods for the measurement of growth hormone in human plasma (Hunter and Greenwood, 1964), and it is now clear that plasma growth hormone levels are dependent on the metabolic state of the patient. The plasma concentrations of growth hormone, insulin, and glucose are interdependent: both growth hormone and cortisol secretion can be stimulated by insulin-induced hypoglycemia (see Roth et al., 1963; Landon et al., 1963). Administration of glucocorticoids or ACTH for long periods of time diminishes the response (Hartog et al., 1964). Beck et al. (1965) have demonstrated that the metabolic response of the subject t o both growth hormone and prolactin is modified by estrogens and androgens and comment on the intriguing questions raised by this finding. The results described above illustrate the very great difficulties in obtaining meaningful results from measurements of growth hormone in patients with breast cancer. At the moment, the only available data are a preliminary study by Hunter and Greenwood (1964), who found no significant abnormalities in plasma growth hormone levels in patients with breast cancer from whom samples of blood were obtained 2 or 3 hours after a meal. Future studies should prove interesting, especially in view of the lesions in carbohydrate metabolism known to occur in breast cancer patients (see Glicksman and Rawson, 1956).

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C. THYROID FUNCTION There is controversy over the relation between previous thyroid disease and its effect on the incidence of breast cancer; on thyroid function in patients with the established disease; and on the usefulness of thyroid extract or triiodothyronine in the treatment of the disease. Repert (1952) found the incidence of thyroid disease to be ten times greater than expected, in a group of 306 patients with breast cancer. Loeser (1954) claimed that breast cancer was more common in hypothyroid patients (or hyperthyroid patients made hypothyroid by treatment) than in euthyroid women. Wynder et al. (1960) found a previous history of hyperthyroidism was more common among his controls than in his cancer patients. The conclusion to be drawn from these results is that thyroid function is unlikely to be of primary importance in affecting the incidence of breast cancer; if it were, a much clearer trend should have emerged from the work described above. Nevertheless, a secondary role is not excluded by these results. It is known that the level of the thyroid hormone affects androgen, estrogen, and cortisol metabolism (see references in Section V, B). It is conceivable, therefore, that the steroidal abnormalities which occur in hypothyroidism might predispose a patient to breast cancer, and those occurring in hyperthyroidism might have a protective effect. These effects on the incidence of breast cancer would depend on the severity of the thyroid disease, the length of time the disease existed before treatment and the efficiency with which the patient was treated. A prospective study of thyroid function in the normal population in relation to subsequent breast cancer might be useful, especially if women with subclinical thyroid abnormalities could be detected. Carter et al. (1960) found no difference between serum protein-bound iodine (PBI) levels in healthy postmenopausal women and postmenopausal women with “cured” breast cancer but noted a highly significant increase in PBI levels in patients with progressive metastatic breast cancer. Edelstyn et al. (1958) found normal thyroid function in localized breast cancer, no matter how far advanced, but found a lower thyroid function in the disseminated disease. The latter finding might be expected from the results of Sommers (1955), who reported thyroid atrophy in patients with advanced breast cancer. The usefulness of thyroid hormone in the treatment of either early or advanced breast cancer is not clear. Loeser (1954) and Lemon (1955, 1957) recommend its use, with or without prednisone; Edelstyn et al. (1958) and Emery and Trotter (1963) using thyroid extract or triiodothyronine alone found them useless. Thyroid effects on breast cancer may be mediated via alterations in

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steroid production and metabolism. If this is so, measurements of thyroid function alone may be misleading, especially in the light of the results of Schubert and Bacigalupo (1963). These authors found that triiodothyronine failed to alter steroid metabolism in a case of advanced breast cancer although it had profound effects in women with the early disease or benign breast disease. IX. ENDOCRINE STATUSAITER ENDOCRINE ABLATION One red herring, vigorously pursued, that emerged from the work described in the preceding sections was the finding that removal of the ovaries and adrenal glands or the hypophysis did not abolish hormone production in every case. Estrogenic vaginal smears were noted; estrogens, androgen metabolites, pregnanediol, and gonadotropins were found in the urine; and 17-OHCS were found in the blood after withdrawal of cortisone support (Struthers, 1956; Bulbrook and Greenwood, 1957, 1958; Greenwood and Bulbrook, 1957; Bulbrook et al. 1958a,b,c; Baron et al., 1958; Lipsett and Pearson, 1956). These findings were so contrary to the canons of classical endocrinology that a considerable and acrimonious controversy arose. It was claimed that the small quantities of hormones usually found might be artifacts, and several workers thereupon went to considerable lengths to substantiate their original claims (Diczfalusy et al., 1958; Bulbrook et al., 1960b). In the uproar, it passed almost unnoticed that the relation between the small amounts of hormones found after endocrine ablation and the clinical course of the disease was poor or nonexistent. Regression of breast cancer could clearly occur even in cases where later postmortem disclosed relatively large amounts of apparently viable pituitary tissue (Luft, 1958; Van Buren and Bergenstal, 1960) and in cases excreting detectable amounts of various hormones or their metabolites. In retrospect, this is not surprising since oophorectomy alone will bring about at least 30y0 of remissions in the advanced disease and there is good evidence that this operation does not abolish estrogen secretion (see Bulbrook et al., 1958a; Nissen-Meyer, 1964). Although this field has not been very fruitful, it should perhaps be borne in mind that hypophysectomy may occasionally bring about temporary remission in patients who have already been oophorectomieed and adrenalectomieed (Hellstrom and Franksson, 1958), and the mechanism for this effect could operate via the removal of the last traces of circulating hormones.

X. EPIDEMIOLOGICAL STUDIES The incidence of breast cancer in women varies considerably in different countries (see Dunham and Dorn, 1955; Shimkin, 1963). It would be logical

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to carry out comparative hormone studies in various ethnic groups with different incidence rates, but this field has so far received little attention. The low incidence of breast cancer in Japan (compared with Great Britain or America) is very striking. Even when corrections are made for differences in such factors as pregnancy, nursing habits, etc., the low incidence rate remains, and Wynder el al. (1960) have suggested that there may be basic endocrinological differences between Japanese women and women from races with a high incidence of the disease. Bulbrook et al. (1964b) have reported briefly on a comparison between the amounts of 1l-deoxy-17-oxosteroids excreted in the urine by Japanese and by British women. When corrections were applied for differences in body size, there was no appreciable difference between the amounts of these compounds excreted by the two races. However, the Japanese women excreted more androsterone than etiocholanolone (5a:5p ratio of 1.3) compared with the British women (5a:5p ratio of 1.0); the difference was highly significant. These authors speculated on the possibility that the Japanese women might have a greater thyroid function than the British women, at least as evidenced by the 5a:5@ratio, and that this increased thyroid function might play some part in reducing the incidence of breast cancer (see Section VIII, C). XI. CONCLUSIONS

It would be optimistic to expect advances in the field of human breast cancer to occur at a faster rate than they do in endocrinological studies of the normal population. New findings are rapidly exposing more and more gaps in our knowledge, and any expectations that the tools a t present available are suitable for making accurate and meaningful measurements of the steroid hormone environment have been dashed by recent discoveries of unexpected secretory products, new urinary metabolites, and the fact that compounds such as testosterone glucuronide may appear in the urine, derived not from testosterone circulating in the peripheral blood, but from conversion of androstenedione in the liver. Immunological methods for protein hormones are of recent introduction and have not been generally applied. A further disadvantage in this field is the fact that many worthwhile studies have not been attempted with the older methods that were available. Thus, there is no really substantial body of work on many of the endocrine aspects of human breast cancer, and the reports on this vast subject are scattered and often contradictory. In spite of this, it is possible to discern one or two leads which might be worth exploiting. These concern the findings that point to the existence of two groups of patients at the time the disease is first diagnosed: one with

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an apparently normal hormonal environment, slow growing and hormoneresponsive tumors; the other with abnormalities in the environment which are correlated with rapid recurrence and hormone-unresponsive tumors. There is also the hopeful possibility that in the latter group of patients, these abnormalities precede the disease and may be useful for the identification of a group of women with a high risk of subsequent breast cancer. ACKNOWLEDGMENTS The author would like to thank his friends in the Imperial Cancer Research Fund for their help and Miss Daphne Clarke for the trouble she took in preparing the typescript. REFERENCES Allen, B. J., Hayward, J. L., and Merivale, W. H. H. 1957. Lancet i, 496-499. Allen, B. J., Hayward, J. L., and Merivale, W. H. H. 1958. I n “Endocrine Asperts of Breast Cancer” (A. R. Currie, ed.), pp. 253-263. Livingstone, Edinburgh and London. American Cancer SOC.Symp. 1957. Cancer Res. 17, 422-546. American Cancer SOC.Symp. 1965. In press. Apostolakis, M., and Loraine, J. A. 1960. J . C1i.L. Endocrinol. Metab. 20, 1437-1444. Appleby, J. I., Gibson, G., Norymberski, J. K., and Stubbs, R. D. 1955. Biochern. J . 60, 453-460. Atkins, H., Bulbrook, R. D., Falconer, M. A., Hayward, J. L., MacLean, K. S., and Schurr. 1’. H. 1964. Lancet ii, 1133-1136. Baron, D. N., Gurling, K. J., and Radley Smith, E. J. 1958. Brit. J . Surg. 46, 593-606. Bauld, W. S. 1956. Biochem. J . 63, 488-495. Baulieu, E. 1962. J . Clin. Endocrinol. Mehb. 22, 501-510. Bayer, J. M., Nocke, W., and Breuer, H. 1957. Klin. Wochschr. S6, 682-687. Bayer, J. M., Breuer, H., and Nocke, W. 1958. Bull. SOC.Intern. Chir. 17, 146-155. Bayer, J. M., Breuer, H., and Nocke, W. 1960. Klin. Wochschr. 38, 1143-1146. Beck, J. C., Blair, A. J., Griffiths, M. M., Rosenfeld, M. W., and McGarry, E. E. 1965. Proc. Can. Cancer Res. Conf. 6, In press. Bbnard, H., Bourdin, J. S., Saracino, R. T., and Seeman, A. 1962a. Ann. Endocrinol. (Paris) 23, 15-22. BBnard, H., Bourdin, J. S., Saracino, R. T., and Seeman, A. 1962b. Ann. Endocrinol. (Paris) 23, 525-532. Bbnard, H., Bourdin, J. S., Saracino, R. T., and Seeman, A. 1964. Compt. Rend. h a d . Sd.268, 6511-6515. Bergenstal, D. M., Huggins, C., and Dao, T. L.-Y. 1955. Ciba Found. Colloq. Endocrinol. 8, 415-433. Berson, S. A., and Yalow, R. S. 1964. In “The Hormones” (G. Pincus, I<. V. Thimann, and E. B. Astwood, eds.), Vol. 4, pp. 575-630. Academic l’resp, New York. Bielschowsky, F., and Horning, E. S. 1958. Brit. Med. Bull. 14, 106-115. Birke, G., Dicafalusy, E., Franksson, C., Hellstrom, J., Hultberg, S., Plantin, L.-O., and Westman, A. 1958. I n “Endocrine Aspects of Breast Cancer” (A. R. Currie, ed.), pp. 213-220. Livingstone, Edinburgh and London. Block, G. E., McCarthy, J. D., and Vial, A. B. 1959. Surg. Forum 9, 619-624. Bloom, H. J. G. 1950. Brit. J . Cancer 4, 259-288. Borth, R. 1950. Intern. Z. Vihminforsch. 22, 226-235. Borth, R. 1952. Ciba Found. Colloq. Endocriaol. 2 , 45-53.

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