[CANCER RESEARCH 41, 105-107. January 1981] 0008-5472/81 /0041-OOOOS02.00 Steroid Hormone Accumulation in Human Breast Cyst Fluid1
H. Leon Bradlow,2 Robert S. Rosenfeld, Jacob Kream, Martin Fleisher, John O'Connor, and Morton K. Schwartz
The Rockefeller University [H L B ] and Memorial Sloan Kettering Cancer Center. New York. New York 10021 [M. F.. M. K. S.], and Institute for Steroid Research. Montefiore Hospital and Medical Center. Bronx. New York 10467 [R. S P., J. K.. J. O.}
ABSTRACT MATERIALS AND METHODS
Elevated concentrations of peptide hormones have been described previously in human breast fluid. In the current study, Cyst fluid was obtained from patients as they presented to the levels of cortisol, progesterone, testosterone, dihydrotes- their physician for treatment, at which time simultaneous blood tosterone, androsterone, androsterone sulfate, dehydroisoan- samples were collected. Plasma and cyst fluid were frozen at drosterone, dehydroisoandrosterone sulfate, estradiol, es —20°until they were assayed. The samples were analyzed by trone, estradici sulfate, and estrone sulfate were measured. RIA3 procedures originally devised for the analysis of plasma. The levels of the four 17-ketosteroids and the two estrogen Because the assays of these compounds involve solvent ex sulfates were markedly elevated over the plasma level, while traction and column chromatography prior to RIA, no problems that of the other compounds was the same or only slightly in applying this method to the analysis of cyst fluid samples higher than the plasma levels of the same compounds. were anticipated. Nevertheless, applicability of these proce dures to the analysis of steroids in cyst fluids was validated by INTRODUCTION serial dilution and by recovery studies in which known amounts of steroids were added to aliquots of cyst fluids. Serial dilution Repeated reports have demonstrated an increased risk of studies for all of the steroids studied showed good linearity, breast cancer in women with cystic breast disease (1, 3, 5, 8, with r values greater than 0.9 in all cases. The increment values 10, 11, 20, 21). As part of a program to explore whether a in the recovery experiments were within 15% or less of the relationship existed between the compounds present in breast amount added to the individual samples. These results show cyst fluid and the risk for breast cancer, the concentrations of that our RIA procedures were applicable to the analysis of cyst a wide variety of hormones, enzymes, tumor-associated anti fluid samples. Intraassay variability was less than 10%, and gens, and ions in breast cyst fluid were determined. The relative interassay variability was less than 15%. Control samples were concentration of luteinizing hormone, follicle-stimulating hor run with each batch of samples. Further support was provided mone, and human prolactin has been reported previously (3, by analysis of the same samples by RIA and by the double- 4, 18). The present report deals with the concentrations of isotope derivative method which gave results agreeing within steroid hormones in cyst fluid samples from the same popula ±10% (S.D.). Cortisol was determined by a modification of the tion used in the peptide hormone studies (4). The presence of procedure of Murphy (12). Testosterone and dihydrotestoster elevated concentrations of 17-ketosteroids has been noted one were analyzed on the same aliquot (2) using Celite partition from this laboratory in a small group of patients (7). In an chromatography to separate the 2 compounds which were then attempt to definitively determine the generality of this finding, quantitated using the same antibody. Dehydroisoandrosterone the levels of a wide variety of steroids and their conjugates and androsterone were determined on a benzene extract of (cortisol, testosterone, progesterone, dihydrotestosterone, an cyst fluid followed by chromatography on a short aluminum drosterone, dehydroisoandrosterone, androsterone sulfate, column (9, 13). Dehydroisoandrosterone sulfate and andros dehydroisoandrosterone sulfate, estrone, estradiol, estradiol terone sulfate were measured by the same procedure following sulfate, and estrone sulfate) in cyst fluid were determined and solvolysis of a 0.05-ml aliquot of each cyst fluid specimen (15). compared with the levels of the compounds in plasma. The For the assay of estrogens, the cyst fluid extracts were chro- concentrations of cortisol, progesterone, testosterone, and matographed on Sephadex LH-20 to remove interfering andro- dihydrotestosterone in cyst fluid were approximately equal to gens. The estrogen fractions were then analyzed separately for their concentrations in plasma obtained at the same time. estrone and estradiol using specific antibodies (22). For the In marked contrast, the concentrations of dehydroisoandros estrogen sulfates, an aliquot containing [3H]estrone sulfate as terone, androsterone, and their sulfates in cyst fluid were a marker was first solvolyzed and then extracted with benzene. markedly elevated over the plasma level of these compounds An aliquot was counted as a measure of the extent of solvolysis, in each patient studied. While the concentrations of free estro the remainder was chromatographed on LH-20, and the appro gens in cyst fluid and plasma were essentially identical, their priate column fractions were analyzed in the same manner. sulfates were found in much higher concentrations in breast Progesterone was measured by the method of deVilla era/. (6). cyst fluid. Steroid-binding proteins were determined by standard meth ods: corticosteroid-binding globulin by a minor modification of 1This work was supported by Contract NCI-CB 53853-37, "Biochemical the method of Trapp and West (19), and steroid hormone- Analysis of Human Breast Cyst Fluid and Its Correlation with Development of Breast Cancer," awarded by the National Cancer Institute. Department of Health, binding globulin by the method of Rosner (16). Education, and Welfare, and Grant CA 22795 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at: The Rockefeller University, 1230 York Avenue, New York. N. Y. 10021. 3 The abbreviations used are: RIA. radioimmunoassay; c/p. cyst fluid/plasma. Received June 13. 1980; accepted September 26. 1980.
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RESULTS AND DISCUSSION were found in cyst fluids. In the 25 samples analyzed, the values were in no case greater than 10% of the corresponding Mean concentrations and c/p ratios for cortisol, testoster plasma levels. one, progesterone, and dihydrotestosterone are summarized To establish the uniqueness of these elevated breast cyst in Table 1. The mean cortisol and testosterone levels were fluid levels, these same compounds were measured in fluids essentially identical to plasma levels. The mean c/p ratio for obtained from a variety of other cysts. As shown in Table 6, progesterone was only slightly elevated (2.66). However, both the concentrations of these compounds in pleural effusions, the cyst fluid and the simultaneous plasma concentrations synovial fluid, renal, ovarian, and hepatic cysts were compa showed large standard deviations reflecting the fact that sam rable to plasma levels showing that the elevated breast cyst ples of both were collected in all phases of the menstrual cycle. fluid levels do not represent a general phenomenon of enclosed Cyst fluid dihydrotestosterone levels were significantly elevated cystic spaces. over plasma levels (p < 0.05), with the mean level 3 times While the breast cyst fluid concentrations of testosterone, greater than the plasma level; and the c/p ratio was 5.41. dihydrotestosterone, cortisol, and progesterone are compatible The C-19 ketosteroids and their sulfates were consistently with diffusion as a mechanism for exchange of these com elevated in cyst fluid over blood, in some cases by as much as pounds into and out of cyst fluid, the results for the 17-keto- several thousand-fold. In the 76 paired samples in which both steroids cannot be accounted for by any such simple diffusion- cyst fluid and plasma were obtained, blood levels of the free related process; and only active transport or synthesis in situ compounds were lower in all but 4 cases. In all cases, the could account for the accumulation of such high levels. From sulfates were present in higher concentration in cyst fluid. The those cases in which cyst fluid specimens were obtained on c/p ratios are summarized in Table 2. Examination of the cyst sequential time intervals, it is clear that these high concentra fluid levels of these compounds in 946 patients showed a wide tions of steroids can accumulate in as little time as 3 to 4 range of values ranging up to many thousand-fold the normal months. female plasma range (Table 3). Transport studies in progress in this laboratory demonstrate When multiple cysts were aspirated on a single visit, the that only dehydroisoandrosterone sulfate can enter cyst fluid percentage of variation among the various cysts was in fairly from the peripheral circulation. None of a wide variety of other close agreement for these hormones (Table 4). When cysts aspirated on sequential visits were analyzed, there was a much Table 2 greater percentage of variation in the cyst fluid steroid levels, C- / 9 steroid ratio in cyst fluid and plasma samples obtained from the same particularly for dehydroisoandrosterone and its sulfate. subjects In Table 4, the percentage of change in cysts aspirated on ± 6.32a a single visit and that on sequential visits in the same patient Dehydroisoandrosterone are compared. The same differences between single-visit and Dehydroisoandrosterone sulfate 4.11 ±46.3 1.4-236.8 76 multiple-visit ratios were observed in these individual subjects. Androsterone7476a 8.48 ± 6.32 0.57-266.3 Androsterone sulfateRatio8.48 110.6 ±81.8Range0.057-23.642.83-360.3Pairs76 From the repeated cyst studies, it can also be demonstrated Mean ±S.D. that high levels of steroids can accumulate in these cysts within 3 to 4 months. The total amounts of these steroids (concentra Table 3 tions x ml) in cyst fluid do not show any such relationship. Levels of 17-ketosteroids in 946 breast cyst fluid specimens Samples with the same volume may have widely differing fe cyst fluid male plasma concentrations. levels"20-50 concentrations925 Estrone and estradiol levels in cyst fluid were comparable to ±1087b Androsterone (ng/dl) those in plasma while estrone sulfate, and, in particular estra Dehydroisoandrosterone 2569±10104020 0-200.00086-160,00040-100025-50 diol sulfate, were greatly elevated in cyst fluid (Table 5). The (ng/dl) latter has indeed never been reported in plasma in significant Androsterone sulfate (/ig/ ±4510 dl) amounts. Previous studies have also shown elevated values for Dehydroisoandrosterone 3800 ±3860Range0-95020-30,500Normal 60-100 estriol and estriol sulfate in cyst fluid (14). sulfate (mi ill)Mean Using the method of Trapp and West (19), corticosteroid- Normal plasma range as determined in our laboratory. 6 Mean ±S.D. binding globulin was essentially undetectable in cyst fluid sam Comparison of mean cyst fluid level of ketosteroids and the normal plasma ples. Low concentrations of steroid hormone-binding globulin range for these compounds.
Table 1 c/p Ratio for hormones not present in elevated concentrations Only dihydrotestosterone shows a significant difference in the mean concentrations; p < 0.05. The mean ratios are the average of the individual ratio value.
Ofsam cyst fluid plasma ratio0.98 level10.6± level12.9± ples72 ±0.06a 3.5b 9.96 Cortisol -2.41 514 ±645b 553 ±7406 Progesterone 2.66 ±0.35 0.95 -29.3 70 47.3 ± 45. 9C 40.9 ± 21. 1C Testosterone 1.53 ±0.15 0.069-6.60 70 151.0± 20.1°Mean49.1 ± 35.0CNo. DihydrotestosteroneMean 5.41 ±0.70Range0.300.80 -35.85Mean 71 " Mean ±S.D. 0 Values presented in /ig/dl. c Values presented in ng/dl.
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Table 4 Percentage of concentration change of steroids in multiple cyst fluid samples aspirated on a single visit or on repeated visits population"Repeated subjects"P<0.0001 of single
visit visits visit visits (126)1.79 (62)5.81 (76)1.65 (42)6.63 Dehydroisoandrosterone ±0.15 ±0.74 ±0.83 ±7.20 Dehydroisoandrosterone sulfate 2.81 ±0.29 8.59 ±0.97 <0.002 2.86 ±3.37 5.74 ±7.73 <0.001 Androsterone 2.25 ±1.01 5.67 ±8.74 <0.001 2.21 ±1.86 4.43 ±3.73 <0.0001 Androsterone sulfateSingle 1.98 ±2.00Total 3.23 ±3.41Analysis <0.002Single 1.97 ±1.79Repeated2.98 ±2.39p<0.00001<0.002 a When more than 2 cysts were aspirated on a single visit or cysts were aspirated on more than 2 occasions, the ratio of the highest and lowest values was used. 6 Paired analysis of single visit and multiple visits in the same subject. When more than one single visit result was available, each was compared with the multiple- visit ratio.
Table 5 cyst fluid samples from their patients. We are also indebted to Mrs. Delia Breast cyst fluid concentration of estrone and estradiol and their sulfates Schwartz for her skillful assistance in collection of demographic data and speci mens. Cyst fluid concentration (pg/ml)
Range of female No. of Mean Range plasma samples REFERENCES EstroneEstradiolEstrone 1. Black, M., Barclay. T., Cutler, B.. Hankey, B., and Asiré.A. Association of follicular"890 sulfateEstradiol atypical characteristics of benign breast lesions in the subsequent risk of luteal40403535 breast cancer. Cancer (Phila.), 29. 338-343. 1973. sulfate6696962027408-20437-2402240-21001060-459030-30036-710470 2. Boyar, R. M., Rosenfeld, R. S.. Kapen, S., Finkelstein. J. W., Roffwarg, H "Ref. 17. P., Weitzman, E. D., and Hellman, L. Human puberty. Simultaneous aug mented secretion of luteinizing hormone and testosterone during sleep. J. Clin. Invest., 54: 609-618, 1974. Table 6 3. Bradlow. H. L.. Fukushima. D K.. Rosenfeld. R. S., Boyar, R M., Kream, J . Concentration of 17-ketosteroids in cysts other than breast Fleisher. M., and Schwartz, M. K. Hormone levels in breast cyst fluid. Clin. All samples were analyzed by the same procedure described for breast fluids. Chem., 22. 213, 1976. 4. Bradlow. H. L.. Schwartz, M. K.. Fleisher, M.. Nisselbaum, J. S., Boyar. R., O'Connor, J., and Fukushima, D. K. Accumulation of hormones in breast isoan- isoan- droste- cyst fluid. J. Clin. Endocrinol. Metab., 49: 778-782, 1979. droste- rone sul rone sul 5. Davis, H.. Simons, M.. and Davis, J. Cystic disease of the breast: relationship to carcinoma. Cancer (Phila.), 17: 957-978. 1964. rone rone fate fate (ng/dl)13816614439NDaND63524850164152NDND3070Dehydro-(ng/dl)1943153132211127189552111NDND2511355417Androste
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H. Leon Bradlow, Robert S. Rosenfeld, Jacob Kream, et al.
Cancer Res 1981;41:105-107.
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