Abnormal Estrogen Conjugation in Women at Risk for Familial Breast Cancer at the Periovulatory Stage of the Menstrual Cycle1

[CANCER RESEARCH 43,1884-1890, April 1983] 0008-5472/83/0043-0000$02.00 Abnormal Estrogen Conjugation in Women at Risk for Familial Breast Cancer at the Periovulatory Stage of the Menstrual Cycle1

Jack Fishman,2 H. Leon Bradlow, David K. Fukushima, John O'Connor, Robert S. Rosenfeld, G. Jay Graepel, Robert Elston,3 and Henry Lynch

The Rocketeller University. New York. New York 10021 Â¡J.F.. H. L. B.Â¡;Montefiore Hospital and Medical Center, Bronx. New York 10467 [D. K. F., J. 0., R. S. R.]; E. I. du Pont de Nemours, Wilmington, Delaware 19898 [G. J. G.]; Louisiana State University Medical Center, New Orleans, Louisiana 70112 [R. E.J; and Creighton University, Omaha, Nebraska 68178 [H. L]

ABSTRACT A number of such studies have been carried out on the population of the Isle of Guernsey, and the results to date have The present study was designed to establish whether women suggested the existence of endocrine differences residing prin with a family history of breast cancer exhibit endocrine abnor cipally in the androgenic hormones which appear to correlate malities which could be responsible for their increased risk for with increased risk for breast cancer (4, 5). Because these the disease. Plasma hormone levels were measured every sec studies were carried out on a very large heterogeneous popula ond day throughout the menstrual cycle in 30 women at risk for tion, an extensive library of hormones and their daily changing familial breast cancer and in an equal number of matched con levels during the menstrual cycle could not be assessed. Even trols. Thirteen of the 14 substances measured exhibited no now, detailed studies on a population large enough to yield a differences between the two populations, but plasma androster- meaningful number of positive cases would be prohibitive in one sulfate was significantly lower in the high-risk subjects. terms of time and effort. Therefore, a more realistic prospective Thirteen urinary hormones were measured every day throughout in-depth study would require a small select population of individ the cycle with only the mean estrone and estradiol glucuronide uals known to be at high risk for breast cancer which could be but not estriol glucuronide content being significantly lower in compared to a matched group of normal-risk women. Studies of the high-risk subjects. A compensatory increase in the urinary this type which have used racial or ethnic high- and low-risk estrogen sulfates was observed. Daily analysis of these differ populations can be faulted because the risks may incorporate ences showed that they were most pronounced in the periovu- environmental factors which may not be reflected in any endo latory period of the cycle. These results suggest that the genetic crine disturbances. Also, the environmental influences may alter risk for breast cancer is associated with an abnormality in the endocrine milieu in directions unrelated to breast cancer risk. estrogen conjugation at a specific time of the ovulatory cycle. Indeed, several such studies of endocrine differences between high- and low-risk racial groups have produced contradictory INTRODUCTION results (10, 32). One of the most secure epidemiological observations in breast The participation of endocrine factors in the etiology of human cancer is that a family history of the disease very significantly breast cancer is supported by several lines of evidence. These increases the risk for it (1, 23, 26). This makes a population at include a series of epidemiological studies and the well-charac genetic risk for breast cancer a particularly suitable group in terized impact of endocrine manipulation on the onset and prog which to attempt to identify hormonal factors which could be ress of the disease in animals and humans. Intensive efforts involved in the increased risk for the disease. Comparative have been devoted to identifying specific endocrine differences studies of such high-risk and low-risk women eliminate the between women with the disease and unaffected controls which question of environmental impact when these groups are could be involved in the disease process (2, 11, 15-17, 21, 25, matched also for geographic location, as well as dietary and 28, 29). Few, if any, clear-cut differences have been securely behavioral patterns. It is clear that any endocrine differences identified to date, and even these are subject to uncertainty as noted in such studies may represent factors which are associ to whether they were present prior to the disease or were a ated only with the risk for familial breast cancer and need not consequence of the illness. Furthermore, available epidemiolog necessarily apply to the population at large. Moreover, it is ical evidence suggests that it is the endocrine milieu in the early reproductive years of a woman's life which determines the risk certainly possible that endocrine abnormalities observed in our high-risk population may be overridden by other environmental of the development of breast cancer many years later (18), so factors in the population at large. It is, however, possible that that endocrine studies carried out after the diagnosis of the the individuals at genetic risk for breast cancer could constitute cancer could be irrelevant or even misleading insofar as the an enriched pool of endocrine abnormalities which could be endocrine role in the onset of the disease is concerned. These factors in nonfamilial disease as well. Therefore, disturbances in considerations have directed attention to prospective studies in endocrine profiles in a population at risk for familial breast cancer which endocrine profiles, obtained a considerable time prior to which are not present in a matched group free of genetic risk for the emergence of the disease, can be related to its subsequent the disease could provide leads to specific endocrine features incidence. linked to the incidence of breast cancer in all individuals. Such 1Supported by Grant CA 22795 from the National Cancer Institute. differences could provide a way of identifying women at risk for 2 To whom requests for reprints should be addressed, at The Rockefeller the disease and, more importantly, offer insight into the mecha University, 1230 York Avenue, New York. N. Y. 10021. 3 Recipient of Grant CA 28198 from the National Cancer Institute nisms of hormonal participation in the oncogenic process. Received August 19,1982; accepted December 10, 1982. Preliminary results of such an in-depth study in which hormonal

1884 CANCER RESEARCH VOL. 43

Table 1 Matching of high- and low-risk population statistics

Ofpairs3030302130303030Case case-controldifference0.23 mean1943.9712.331.7721.6727.3313.73137.5765.60Controlmean1943.7312.732.3721.6726.7313.20128.9764.70Mean Birth date(yr)Menarche Â±0.44"-0.36 (yr)ParityAge Â±0.36-0.60 Â±0.320.00 atfirstpregnancy(yr)Cycle Â±0.290.60

length(days)UH Â±0.850.53

peak(days)Wt Â±0.968.60 (Ib)HI Â±3.490.83 (inches)" Â±0.53t0.55-1.01-1.870.000.700.552.461.57PNS"NSNSNSNSNS<0.05NS S.E."Mean Â± NS. not significant.NO. levels throughout the menstrual cycle in 30 young women at an area as close as possible to the matching subject to avoid any high risk for familial breast cancer were compared with 30 possible microenvironmental effects. To avoid any possibility of seasonal matched controls have already been reported (13, 14). No sig changes, each subject and control pair was studied at the same time. nificant differences between these populations were observed in As shown in Table 1, the matching of the 2 populations for all of these the plasma levels of either prolactin, LH," FSH, estrone, estradiol, parameters was extremely close except for body weight where a small difference existed. Although the high-risk subjects were on average 3.97 or estriol at any stage of the menstrual cycle, although a con kg heavier than the controls, this is too small an amount to result in sistent trend toward lower values in all of these, except estriol, significant endocrine changes (12), particularly since it has been sug was noted in the high-risk populations (14). Analysis of urinary gested that height rather than weight is the more critical risk factor (9). metabolites, on the other hand, revealed highly significant differ Furthermore, our data show no correlation between body weight and ences in estrone and estradiol glucuronides, with the high-risk any of the significant hormonal differences observed, indicating that this individuals excreting lower amounts of these metabolites than matching factor had no influence on the results. Blood samples (15 ml) the controls (13). We now present further results from this study were obtained at the same time on alternate mornings from the start of which include differences in urinary estrone sulfate and in plasma one menstrual cycle to the start of the next cycle by a visiting nurse. Blood samples were promptly centrifuged, and the plasma samples were androsterone sulfate content. The temporal relationship of the kept at -20Â° until the analyses were carried out. Following careful various hormonal differences to the stages of the menstrual instructions, all subjects collected 12-hr urine samples (7 p.m. to 7 a.m.) cycle is also analyzed and discussed. These results provide every day starting from the cessation of one menstrual cycle to the compelling evidence that women at risk for familial breast cancer beginning of the next. On one day in the follicular phase and one day in display an abnormality in their metabolism of estradiol which is the luteal phase, a complete 24-hr urine collection was obtained to serve centered at the periovulatory period. as a check on the 12-hr analyses. All of the urine samples were frozen promptly and kept in this state until analysis. Each of the subjects kept METHODOLOGY a detailed diary during the study period, recording any unusual effects (colds, fever, emotional upset, or intake of any medications). Patient Selection. The high-risk subjects were selected from pedi Analysis. All analyses were carried out without knowledge of the patient's category. Plasma samples were analyzed for peptide and grees with a high incidence of hereditary cancer primarily on the basis of having one or more relatives (mother, sister, maternal aunt) with breast steroid hormones, estrone, estradiol, estriol, testosterone, dihydrotes- cancer from the Familial Breast Cancer Resource at Creighton University. tosterone 17(i-hydroxyandrostan-3-one, cortisol, androsterone, AS, de- Twenty-eight of the 30 high-risk patients had a mother or sister with hydroisoandrosterone,dehydroisoandrosterone-3-sulfate, progesterone, breast cancer, while the other 2 were twin daughters of a patient whose LH, FSH, and human prolactin. Peptide hormones were analyzed by mother had breast cancer. Seven had both mother and sister affected, direct RIAs on aliquots of the individual plasma samples. Steroids were while 19 had only a mother, and 2 had only a sister affected. Sixteen analyzed by RIA following extraction with ether and appropriate column had both a mother and maternal aunt affected; one had sister, mother, chromatography except for cortisol, which was measured using a corti- and grandmother with breast cancer; 4 had both mother and grand costeroid-binding globulin binding assay (18). Steroid sulfates (dehydro- mother affected; and one had mother and maternal uncle with breast isoandrosterone-3-sulfate, AS) were analyzed indirectly after solvolysis cancer. Finally, there was one case with sister, mother, and 2 paternal liberated the free steroids (20, 36). aunts with either breast or ovarian cancer or both (the mother). Second Urine samples were thawed, their volume was determined, and ali ary factors determining the eligibility of subjects for inclusion in the study quots were taken for creatinine measurements and urinary free cortisol included the absence of menstrual irregularities and the absence of (19). The urine specimens were adsorbed on XAD-2 (3), and the steroid thyroid or liver disorders or other health problems. In addition, subjects metabolites were eluted with methanol. The extracts were concentrated must not have taken thyroid drugs, tranquilizers, or birth control pills for in a vacuum, diluted with water, and hydrolyzed successively with 0- at least 6 months prior to the study. Each of the high-risk subjects was glucuronidase and sulfuric acid (pH 1) (13). After each hydrolytic step, matched as closely as possible to a control subject in terms of age, the urine was continuously extracted with ether, which was washed with parity, age at first pregnancy, age at menarche, weight, height, and 0.9% bicarbonate and saturated sodium chloride solution. The extracts ethnic origin. The control cases were recruited with the aid of an were analyzed for the compounds listed in Table 2. Aliquots of each extensive newspaper, radio, and television advertising program. In ad extract were analyzed separately by RIA with highly specific antibodies dition to the factors mentioned above, each control was selected from so that losses from separation procedures could be minimized. For the estrogen analyses, the extract was initially partitioned between water 4 The abbreviations used are: LH. luteinizing hormone; FSH, follicle-stimulating and benzene to separate out estrone and estradiol, and then the aqueous hormone, AS, androsterone 3-sulfate; RIA, radioimmunoassay. phase was extracted with ethyl acetate to obtain the estriol fraction. The

APRIL 1983 1885

2 crude fractions were then separately chromatographed on deactivated collection; and last urine collection to last plasma collection. The interval alumina to give eluates containing the purified estrogens. Recovery was for each individual was then adjusted to a standard median length. The monitored by addition of labeled ligands to the extracts using labeled median values for the 3 intervals were 8.25, 13.5, and 3.75 days, estrone or estradici in alternate samples. respectively, or 25 days for the period from the end of menstruation to To explore the possibility that there were reciprocal changes in the the start of the next cycle. The values for each of the parameters urinary estrogen sulfate fraction, pooled follicular- and luteal-phase urine measured were fitted between these fixed points using a procedure described in the "Appendix." There are 22 fitted points starting from the samples were first stripped of glucuronide conjugates by enzyme hy drolysis and solvent extraction and then hydrolyzed by solvolysis to first plasma sample (0.25 days to the last urine collection 21 adjusted liberate estrogens present as sulfoconjugates. The material extracted days later). Charts 1 to 7 are derived from these adjusted data. Com after solvolysis was analyzed by RIA for estrone and estradiol following parison of these values with the original raw data (13) showed good the same procedures as described above. agreement, indicating that the fitting procedure was successful. The coefficients of variation of the various assays were determined To examine individual daily values, urinary results in each subject by repeat assays of unknown samples and by analysis of known amounts normalized per g of creatinine were arrayed in a matrix, and the array of standards processed like the unknown samples (Tables 3 to 5). was aligned with the peak plasma LH day set as Day 0. Days prior to Analysis of Data. The data were initially analyzed by computing the this day were numbered T - 1, T - 2, etc., and the days following were average mean values of the hormonal concentrations in the various numbered T + 1, T + 2, etc. Similar examination of plasma hormone stages of the menstrual cycle. The complete menstrual cycle mean, the content was carried out with the values obtained every second day and follicular-phase mean, and the luteal-phase mean values for paired high- expressed as content per ml of plasma. The analysis of the daily values and low-risk subjects were then compared. The subjects and controls was carried out using a PDP-1170 computer and the BMDP P3D were paired on the basis of height, weight, age at menarche, parity, and statistical program to analyze the individual days of the matrix. length of cycle. The follicular phase consisted of all samples from the onset of menstruation up to the day of the plasma LH maximum. The RESULTS luteal phase included all of the samples from the LH peak to the onset of the next cycle. The mean values for each compound in each subpo- The plasma hormone data showed the presence of an LH and pulation were compared over the menstrual cycle to see if there were progesterone rise, indicating that ovulation had presumably oc- any cycle differences between the 2 populations. To facilitate comparisons between the results for the different sub Table 4 jects, the length of the cycle was standardized. For this purpose, the Recoveryof steroids from urine time of the first and last urine collections and the time of the LH peak All samples were added to 500 ml of urine, processed as described above, from an adrenalectomized subject. were considered to be fixed points in the menstrual cycle. It is assumed that it is valid to make comparisons among individuals at these times. added1500 found1600 (%)110 The time intervals between these points vary between individuals. The EstroneEstradiolEstriolng Â±195 standardization was performed by first calculating the median length of 3000 3000Â±275 100 60001500 5480Â±5581710 91.3114 the 3 intervals: first urine collection to LH peak; LH peak to last urine Â±194 Table 2 3000 2950Â±285 97.7 Compoundsanalyzed tor in plasma and urine 60001500 5500Â±3911050 91.7105 Plasma Urine Â±129 LH Estrone 2000 1870Â±148 93.5 FSH Estradiol 5000ng 5240Â±510Recovery 105 Human prolactm Estriol Estrone Cortisol Estradiol Tetrahydrocortisol Tables EstriÃ³! Tetrahydrocortisone Separation of 17tl-[3H]estradiol glucuronide and [3H]estrone sulfate all-Tetrahydrocortisol Progesterone The labeled compounds were added separately to control urine samples and Androsterone Testosterone processed by our standard procedure. AS Dihydrotestosterone Dehydroisoandrosterone Androsterone Dehydroisoandrosterone-3-sulfate Etiocholanolone 3n-hydroxy- 5|<-androsterone-17-one Cortisol Dehydroisoandrosterone l7,i-|3H|Estradiol 1052.00 x1051.0 1031.94X Testosterone glucuronide|3H|Estrone Dihydrotestosterone 170-hydroxyandrostan-3-one sulfateAdded1.00X x 105dpmGlucuronide9.2x 103SulfateVOX 10*

Tables Intraassayand interassay variability of plasma and urine PlasmaEstrone

creatinineAmountiig/g (%)8 recovered44.0 recovered57.6 (%)(%)8 recovered5.2 (%)9.6 Â±3.4" pg/ml (10)c + 4.8 pg/ml (14) 6.2 Â±0.5 (20) Estradiol 59.0(10)35.1 + 5.6 pg/ml 94 65.9(14)38.3 Â±2.98 pg/ml 5 4.1 3.8 + 0.42 (18) 11.1 Estriol 4.0 8.5 Â±0.89 (20)CV 10.5 LH Â±1.43mlU/ml (12) Â±2.11 mlU/ml (7) 6 ProgesteroneIntraassayAmount174 Â±1.78ng/dl (10)CV3 10InterassayAmount172.1 Â±6 ng/dl (7)Intraas-CV3UrineInterassay 3 CV, coefficient of variation. ' Mean Â±S.E. Numbers in parentheses, number of samples.

1886 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. Estrogen Metabolism in Women at Risk for Breast Cancer curred in each subject. Analysis of the plasma concentrations of 5.6 all the hormones measured showed no differences between the 5.4 2 populations except for AS, the mean values of which were lower in the high-risk group for the whole cycle as well as for the 5.2 follicular and luteal stages. Typical plots of plasma hormone levels for the whole cycle are given in Charts 1 and 2 for LH and 5.0 estradiol. These overlapping curves exhibited no difference be 4.8 tween the 2 populations. Comparable results (not illustrated) were obtained for cortisol, testosterone, dihydrotestosterone 4.6 17^-hydroxyandrostan-3-one, dehydroisoandrosterone, dehydroisoandrosterone-3-sulfate, progesterone, FSH, and prolactin. 44 In contrast, the curves for AS (Chart 3) showed a highly signifi cant difference with the levels in the high-risk group being lower. 42

The physiological significance of this difference is not clear, since 4O no biological role for this compound is known except to serve IO I5 20 25 30 as a source for free androsterone which is considered to be Chart 2. Mean plasma estradiol levels in control and high-risk subjects. Collec moderately androgenic. However, this difference in AS concurs tions were taken as described in Chart 1. Analysis was cameo out in extracts of the plasma samples. O. control; â€¢.highrisk. with the prospective studies of Bulbrook ef al. (4, 5) carried out in a population at large, where low androgens implied a greater risk for breast cancer. The initial analysis of the urinary values was done by combining the individual daily values and determining full-cycle and follicular- 3.8 and luteal-stage means. The only highly significant differences observed were in urinary estrone and estradiol glucuronides with 3.7 the levels for the high-risk subjects being lower for both com pounds (Table 6). Analysis by cycle stage showed that, in the 3.6 follicular stage, the difference was significant for estradiol glucuronide only. Both estrone and estradiol glucuronides were 3.5 significantly lower in the luteal phase, a result compatible with the results of the day-by-day analysis. It is particularly interesting 3.4 that no differences were seen in estriol glucuronide levels be tween the 2 populations (Table 6) in contrast to earlier reports (6-8, 10, 24, 27, 32, 33) that this metabolite was depressed in 3.3 IO I5 20 25 30 breast cancer patients. Analyses of the pooled sulfate fractions showed higher levels for estrone sulfate in the high-risk subjects, Charts. Mean plasma AS levels in control and high-risk subjects. Samples were collected as described in Chart 1. and the steroid sulfates were extracted, although the difference just escaped significance (p = 0.05) solvolyzed. and fractionated on partition columns prior to RIA using a highly specific because of the considerable scatter in the individual data. These antibody. The results were analyzed using a paired f test, and the results are significant (p = <0.01). O, control; â€¢,high risk. values were obtained on pooled and not individual samples, a circumstance which may further contribute to the absence of clear statistical significance in these differences. maximal differences in these 2 metabolites are clearly outlined Full-cycle plots of the daily urinary values of the estrone and with respect to the stage of the ovulatory cycle. In contrast, the estradiol glucuronides (Charts 4 and 5) are revealing, in that the corresponding plot for estriol glucuronide (Chart 6) shows no difference between the 2 populations, although a trend in the

7.0 direction opposite of the other estrogens is evident. The differ ences in estrone and estradiol glucuronides are clearly maxi mized in the periovulatory period from Day T - 2 to Day T + 3. 6.5 In the case of estradiol, the difference was observed from T â€”2 to T + 3, while for estrone, significant differences were observed 60 only from T - 0 to T + 3. The reasons for the distribution of the estrogen differences within the cycle are discussed below. 5.5 When the day-by-day results were examined statistically, the results observed in the mean values of the follicular and luteal

5.0 phases (Table 6) become clear. The statistical significance of the difference between the high-risk and the low-risk subjects for estradiol was located only in the Days T - 2 when the estrogen 45 surge starts and continuing to Day T + 3. No significant differ ence was observed in the early part of the cycle and the late- 40 luteal phase. Examination of the estrone results showed no IO 15 20 25 30 difference between the 2 populations until after the day of the Chart 1. Mean plasma LH levels in control and high-risk subjects. Plasma plasma LH peak between T - 0 and T + 3. These findings samples were taken every other day in the morning and analyzed directly for LH as described in "Methodology." O, control; â€¢.high risk. explain the absence of a difference in the mean estrone values

APRIL 1983 1887

24 1-Ã¶ Ã¶zVCM 22

CMÃ–1f 2.0

Ã¶ Ã¶^+1 1.8 UÃ¯ +1 +1 ffr511Ifa*~*.Â§sÂ»oâ€¢2!Hif o> coinÃ– CMÃ–1 16 1â€¢Â«t

f~inÃ¶ Ã¶^+1 1.4 +1-Hr- T-o(O Ã–COTj- 1 2L IO 15 20 25 incoÃ¶ Chart 4. Mean urinary estradici glucuronide levels in control and high-risk pa Ã¶ ^ +1 +1-Hh- tients. Twelve-hr urine samples were collected from 7 p.m. to 7 a.m. and hydrolyzed co m enzymatically. The phenolic extract was partitioned between benzene and water, in i^coPeo and estradici was determined by a specific RIA. O, control; â€¢,high risk.

30 co o ZZVh- i.s14)staoS10jegS.g{31Ã•13I"D1snDOÃ–1IIa-C'eâ€¢J3Â£s%Â¿Ã¶'v;28

T- CD â€¢

CO*-Ã– Ã–C\j+1 22 -H+1O) O>00000^ oâ€¢Â§io"IIQ) 2.0

CD1^Ã– 1.8 Ã–^â€¢H -H+1CO COT-r-^ 1.6 aiT^eo 1.4 t~-â€¢Â»Ã¶ 10 15 20 25 Ã¶ W +1 +1+1v Chart 5. Mean urinary estrone glucuronide levels in control and high-risk pa -0T a>a>CD tients. Collections and work-up were carried out as described in Chart 4. O, control; wo.***C'cIoIIIliMÃ¨g"t-CO^Â§:; â€¢high risk.

in the follicular phase, since there is a delay between estradici co . PzO 0"- secretion and estrone production, and therefore, the urinary difference is delayed until T - 0. Conversely, the significance of

CO CO *- COCMco the estrone differences exceeded that of estradici in the luteal c\jÃ¶1 1co state. The statistical observations are plotted in Chart 7. Several explanations can be offered for these differences in r^oÃÃ¶ urinary estrogen conjugates. There could be a decrease in Ã¶ ^ ovulating ovarian secretion in the high-risk subjects, resulting in +1 +1+1o en-Â»tâ€¢Â¿ WÃ¶1 decreased urinary metabolite excretion. Although no differences 1co have been reported in estrogen production rates between breast

cpin+1 cancer patients and control subjects (21), these studies have been limited to postmenopausal subjects and therefore did not +1+100 000CD include the ovulatory surge. When premenopausal subjects were OÃ•Csico examined, the studies were also not carried out specifically at the time of the ovulatory rise. In any event, decreased secretion incoÃ– "X Ã–r+1 would be expected to also result in a lesser plasma concentration +1 +1 co o>min of estradiol. This was not observed; thus, this explanation for 10CM r^eviSES(A the urinary changes lacks supporting evidence. An alternative possibility is that estradiol metabolism is altered in high-risk subjects, resulting in decreased conversion of estradiol and its metabolite estrone to glucuronide conjugates. This hypothesis (A(OLU UJ 111 is tentatively supported by our measurements of urinary estrone

1888 CANCER RESEARCH VOL. 43

3.0 Table 7 Urinary estrone sulfate in women at risk lor breast cancer 2.8 The differences between the subjects and controls are statistically not signifi cant. 2.6 i/g/g creatinine

2.4 Subjects Controls Folicular Luteal Follicular Luteal 22 Estrone sulfate 5.32 + 4.16 5.73 Â±4.73 3.83 Â±2.99 4.72 + 4.0

20 Mean Â±S.E.

1.8 31 ). This hypothesis postulates that, unlike estrone and estradiol, 16 estriol is protective against breast cancer. A considerable body of biological and epidemiological evidence has accumulated 1.4 which casts doubt on this hypothesis (see Ref. 35 for a review), 10 15 20 25 and our study also fails to support it. Both plasma and urinary Chart 6. Mean urinary estriol glucuronide levels in control and high-risk patients. estriol concentrations of the high-risk subjects were equal to or Collections and work-up were carried out as described in Chart 4. O, control; â€¢, high risk. greater than those of controls. Moreover, the estriol quotient was elevated in the high-risk subjects rather estrone + estradiol than the lower values called for by the estriol hypothesis. A number of factors provide a measure of confidence in the results and conclusions obtained in this study. The analyses were carried out blindly in one laboratory on body fluids obtained from patients selected by another institution. Assays were per formed daily throughout the menstrual cycle, and of the numer ous steroid and peptide hormones measured, only the urinary estrogens showed clear-cut significant differences, with these differences being maximal at the time of a secretory surge of the hormone. The decrease in glucuronide excretion in the high-risk individuals is balanced by a corresponding elevation in estrogen sulfate excretion, further supporting a shift in conjugative path -2 -I 0 +1 +2 +3 +4 +5 +6 ways. A critical question is whether such a shift is linked to the Day from plasmo LH peak risk for breast cancer. Intuitive reasoning would suggest that an Chart?. Statistical significance of daily differences in urinary estradici and alteration in estrogen physiology is the most probable endocrine estrone glucuronides between women at risk for breast cancer and controls (Student's 2-tailed ( test). E2, estradici; E,, estrone. factor to be involved in breast cancer incidence, since the breast is target tissue for the hormones. A change in conjugative pathways clearly can result in alterations in estrogenic physiolog and estradici sulfates in the 2 population groups (Table 7). While ical processes. The nature of the biological changes induced by the difference in these conjugates between the 2 groups just the conjugative shifts and the relationship of these to other than escaped statistical significance, possibly because of methodo familial breast cancer are questions of high priority in our inves logical difficulties, their direction does indicate that there is a shift tigation. from glucuronide excretion to sulfate excretion. In fact, the increase in estrogen sulfates in the high-risk group almost pre In a recent study, we have found that women with breast cancer exhibit an increase in 16o-hydroxylation of estradiol rel cisely matches their glucuronide deficit. Since estrogen sulfates ative to normal controls (30). Oxidative transformations of estra have a greater quantitative and qualitative biological potency diol, as measured by the radiometrie method used in this study, than do the glucuronides (34), such a metabolic shift could be are not affected by age, and therefore, the measurements in construed as producing a greater biological impact for the same postmenopausal women reflect such relative enzymatic activity amount of hormone secreted. Implicit in such a concept Â¡sa in their earlier reproductive period. If the increased 16Â«-hydrox- relationship between genetic risk for breast cancer and the ylation in the cancer patients is shown to precede overt presence enzymatic mechanisms regulating relative conversion to glucu of the disease, it is possible that this metabolic alteration could ronide and sulfate conjugates. These mechanisms include the be linked to the conjugative defect noted in the high-risk subjects. corresponding transferases and hydrolases, and the differences Such a connection would provide valuable insight into the ques may reside in either or both. These enzymatic changes may tions of the biological effects of the conjugative disturbance and become elevated at substrate levels which ensure enzyme ve the extension of its relevance to breast cancer in the population locities at which the differences become manifest. Such sub at large. strate levels are clearly maximal at the periovulatory stage of the cycle, accounting for the experimental detection of the differ ences at that time. ACKNOWLEDGMENTS For some 20 years, attention has been focused on estriol as We are greatly indebted to our cases and their controls without whose extraor a key estrogen involved in breast cancer risk analysis (10, 22, dinary cooperation this study could not have been carried out

APRIL 1983 1889

APPENDIX 38.'745-748, 1978. 8. Cole, P., and MacMahon, B. Oestrogen fractions during early reproductive life Method of Standardization in the aetiology of breast cancer. Lancet, 1: 604-606. 1969. 9. DeWaard, F. Breast cancer incidence and nutritional status with particular The first step in the standardization process was to calculate the median intervals reference to body weight and height. Cancer Res., 35: 3351-3355,1975. among the 4 fixed points. In calculating the medians, the time of the LH peak was 10 Dickenson, L. E., MacMahon, B., Cole, P., and Brown, J. B. Estrogen profiles obtained by interpolation from a quadratic fit around the observed maximum. The of Oriental and Caucasian women in Hawaii. N. Engl. J. Med., 297: 1211- median time from the first urine collection to the LH peak was 8.25 days, from the 1213, 1974. LH peak to the last urine collection was 13.47 days, and from last urine collection 11. Everson, R. B., Li. F. P.. Fraumeni, J. F., Jr., Fishman, J., Wilson. R. E., Strout, to last plasma collection was 3.75 days. The calculated minimum, median, and D., and Morris, H J. Familial male breast cancer. Lancet. 7: 9-12, 1976. maximum time from the first urine collection to the LH peak for the cases are 1.45, 12. Fishman, J , and Bradlow. H. L. Effect of malnutrition on the metabolism of 8.25, and 21.99, respectively; the corresponding values for the controls are 4.97, sex hormones in man. Clin. Pharmacol. Ther., 22: 721-728. 1977. 13. Fishman, J.. Fukushima, D. K., O'Connor. J. L., and Lynch, H. T. Low urinary 8.33, and 18.21. A f test of a difference between the means of these 2 groups is not significant (t = 0.30). Therefore, it is reasonable to assume that it is valid to estrogen glucuronides in young women. Science (Wash. D. C.), 204: 1089- use the same fitting procedure for both groups. These median times were then 1093, 1979. 14. Fishman, J., Fukushima, D., O'Connor, J., Rosenfeld, R. S.. Lynch, H. T., adjusted so that the time from FP (first plasma) to LP (last plasma) equaled 25 days. The adjusted medians are taken as the length of time to which the original Lynch. J. F., Gurgis, H.. and Maloney, K. Plasma hormone profiles of young observations are scaled. The corresponding adjusted times are 8.18, 13.35, and women at risk for familial breast cancer. Cancer Res., 38: 4006-4011,1978. 3.72 days. Although points were fitted after the last urine collection, they were not 15. Hoover. R . Gray. L. A., Cole, P.. and MacMahon. B. Menopausal estrogens used in any of the analyses because only plasma parameters were measured then. and breast cancer. N. Engl. J Med., 295. 401-405. 1976. 16. Jick, H., Walker. A. M.. Watkins, R. N., D'Ewart, D., Hunter, J. R., Danford. The first point is the time of the first plasma collection, time zero, and is 0.25 days after the first urine collection and 7.93 days before the LH peak. The fitted points A.. Madsen, S., DiÃ±an,B., and Rothman, K. J. Replacement estrogens and before the LH peak were obtained as though the observed days are 7.93/1 days breast cancer. Am. J. Epidemici., 772: 586-594, 1980. long, where / is the actual number of days between these 2 points for an individual. 17 Kelsey, J. L. A review of the epidemiology of human breast cancer. Epidemiol. For points beyond the LH peak, the days are 13.35// days long, where /' is the Rev., 7: 74-109, 1979. actual number of days between the LH peak and the last urine collection for each 18. Korenman, S. G. Oestrogen window hypothesis of the aetiology of breast individual. Demonstrating the fitting procedure is most easily done by a numerical cancer. Lancet. 7 700-703. 1980. example. Suppose for a particular woman that her LH peak occurs after 10 days; 19. Kream. J., Fukushima, D. K., and Zumoff, B. A rapid competitive protein she had 25 unne collections and 14 plasma collections; and we want to calculate binding radioimmunoassay of urinary cortisol. Clin. Chem., 24: 994, 1978. a parameter value for her at adjusted Day 5 (i.e., 5 adjusted days after the first 20. Kream, J., Hellman. L., and Rosenfeld, R. S. Radioimmunoassay of androster- plasma collection). Thus, we want the value that occurs 5 x 7.93/10 = 3.965 days one and androsterone-3-sulfate in plasma. Steroids, 27. 727-739. 1976. after the first plasma collection or 3.965 + 0.25 = 4.215 days after the first urine 21. Kwa, H. G., Cletin, E., deJong-Bukker, M., Bulbrook, R. D., Hayward, J. L.. collection. For a urine parameter, we then obtain 2 parabolas, one fitted through and Wang, D. Y. Plasma prolactin and its relationship to risk factors in human the values she has for 3, 4, and 5 days after the first urine collection and one fitted breast cancer. Int. J. Cancer. 77: 441-447, 1976. through her values for 4, 5, and 6 days after her first urine collection. The value of 22. Longcope, C., and Pratt, J. H. Relationship between urine and plasma estrogen these parabolas evaluated at 4.215 days which is closest to the value obtained by ratios. Cancer Res.. 38: 4025-4028, 1978. linear interpolation between Days 4 and 5 is taken as the desired value. For a 23. Lynch, H. T , Krush. A , Lemon, H , Kaplan. A. R., Condii, P. T., and Bottomley, plasma parameter, we obtain 2 parabolas, one fitted through the values she has R. Tumor variation in families with breast cancer. J. Am. Med. Assoc., 222: for 2, 4, and 6 days after the first plasma collection and one fitted through her 1631-1635.1972. values for 4, 6, and 8 days after the first plama collection. Similarly, the value of 24. MacMahon. B.. Andersen, A. P., Brown. J., Cole, P., DeWaard, F., Kauraniemi, the parabolas evaluated at 3.965 which is nearest the value obtained by linear T., Ravnihar, B., Stormby, N., Tnchopoulos, D., and Westlund, K. Urine interpolation between Days 4 and 6 is the fitted value. A similar procedure is used estrogen profiles in European countries with high or low breast cancer rates. for fitting points beyond the LH peak. Suppose we wish to calculate her value after Eur. J. Cancer, 76: 1627-1632, 1980. 15 adjusted days. For the urine parameter, we then want the observation 10 + (15 25. MacMahon, B.. and Cole, P. The ovarian etiology of human breast cancer. - 8.18) x (24 - 10)/(13.35) = 17.05 days after the first urine collection. For her Recent Results Cancer Res.. 39. 185-192. 1972. plasma parameters, we want the observation 10 + (15 - 7.93) x (24 - 10)/13.35 26. MacMahon, B., Cole, P., and Brown, J. Etiology of human breast cancer. A = 17.41 days after the first plasma collection. For the urine parameter, parabolas review. J. Nati. Cancer Inst., 50: 21-42, 1973. are fitted with the observations after 15, 16, and 17 days and after 14, 15, and 16 27. MacMahon, B.. Cole, P., Brown, J. B., Aoki. K., Lin, T. M., Morgan, R. W.. days and evaluated as before. The parabolas for the plasma parameters are fitted and Woo, N. C. Urine oestrogen profiles of Asian and North American women. on the observations after 14, 16, and 18 days and after 16. 18, and 20 days and Int. J. Cancer, 74: 161-167, 1974. again evaluated and compared with the value from linear interpolation. Near the 28. Pike, M. C., Cassagrande, J. T., Brown, J. B., Gerkins, V., and Henderson, B. end points, where parabolas could not be fitted, linear interpolation was used. E. Comparison of urinary and plasma hormone levels in daughters of breast Quadratic interpolation was used because it uses more information than just linear cancer patients and controls. J. Nati. Cancer Inst., 59: B51-B55. 1977. interpolation; 2 quadratics were calculated, and the one giving a result closest to 29. Ross. R. K.. Paganini-Hill. A.. Gerkins, V. R., Mack, T. M.. Pfeffer, R., Arthur. that from linear interpolation was used to prevent any undue influence of outliers. M.. and Henderson, B. E Replacement estrogens and breast cancer. Am. J. Epidemiol., 772: 586-594, 1980. 30. Schneider, J., Kinne, D., Fracchia, A., Pierce. V., Anderson, K. E., Bradlow, H. L., and Fishman, J. Abnormal oxidative metabolism of estradiol in women with REFERENCES breast cancer. Proc. Nati. Acad. Sei. U. S. A., 79: 3047-3051,1982. 31. Trichopoulos, D , Brown, J B., Garas, J., Papaioannou, H.. and MacMahon, 1. Anderson, D E. Genetic study of breast cancer identification of a high risk B. Elevated urine estrogen and pregnane diol levels in daughters of breast group. Cancer (Phila.), 34: 1090-1097, 1974. cancer patients. J Nati. Cancer Inst., 67: 603-606, 1981. 2. Armstrong, B. K. Diet and hormones in the epidemiology of breast and 32. Trichopoulos, D.. Cole, P., Brown, J. B., Goldman, M., and MacMahon, B. endometrial cancers. Nutr. Cancer, 7: 90-95.1979. Estrogen profiles of primiparous and nulliparous women in Athens, Greece. J. 3. Bradlow, H. L. 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1890 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. Abnormal Estrogen Conjugation in Women at Risk for Familial Breast Cancer at the Periovulatory Stage of the Menstrual Cycle

Jack Fishman, H. Leon Bradlow, David K. Fukushima, et al.

Cancer Res 1983;43:1884-1890.

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