0021-972X/79/4905-0778$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 49, No. 5
Printed in U.S.A.
Comments
Accumulation of Hormones in Breast Cyst Fluid* H. LEON BRADLOW, MORTON K. SCHWARTZ, MARTIN FLEISHER, JEROME S. NISSELBAUM, ROBERT BOYAR, JOHN O'CONNOR, AND DAVID K. FUKUSHIMA The Rockefeller University (H.L.B.), New York, 10021; the Memorial Sloan-Kettering Cancer Center (M.K.S., M.F., J.N.), New York, 10021; and the Institute for Steroid Research, Montefiore Hospital and Medical Center (R.B., J.O., D.K.F.), Bronx, New York 10467
ABSTRACT. The concentration of some immunoreactive peptide hormones in breast cyst fluid and serum from a series of patients with cystic breast disease were compared. The concentrations of the two pituitary gonadotropins, LH and FSH, were consistently higher in serum than in cyst fluid, confirming earlier more limited observations. The mean PRL concentration, on the other hand, was elevated in cyst fluid though the variability was great. The cyst fluid serum ratio of hCG was elevated in the patient group as a whole. However, the results suggested the presence of two subpopulations; group I, in which the cyst fluid
and serum hCG levels were essentially indistinguishable and in the range found in normal serum, and group II, in which the cyst fluid hCG concentration was consistently greater than the
normal serum range. Analysis of hormone concentrations in multiple cysts aspirated at the same time showed a remarkable degree of agreement in the cyst hormone levels in most of the patients. The greatest variability in the levels was seen with hCG, where a small number of subjects showed a very large spread in the values. (J Clin Endocrinol Metab 49: 778,1979)
C
YSTIC breast disease is a common condition in premenopausal patients, appearing after age 25 yr and disappearing at menopause. While cystic lesions are not considered to be precancerous per se, many studies dating back over the last 40 yr (1-7) have indicated that these patients have a 2- to 4 fold greater risk of developing breast cancer than matched subjects without breast disease. It has been suggested that benign and malignant breast disease could have some common factor, such as a unique pattern of hormone or protein concentrations (8). Evidence for such unusual patterns in patients with breast cysts has come from several sources. Haagensen and colleagues (9, 10) have described the isolation and characterization of an unusual glycoprotein from breast cyst fluid; this substance has also been found in high concentrations in the serum of breast cancer patients. An unusual protein with high binding affinity for progesterone and pregnenolone has been isolated from cyst fluid in large amounts (10,11), while Schwartz et al. (12) have reported the presence therein of high concentrations of glutamyl transpeptidase, /?-glucuronidase, and carcinoembryonic antigen. Steroid concentrations in cyst fluid were first determined by Fleisher et al. in 1973 (13); they
reported large amounts of 17-ketosteroids in these samples. Subsequent studies by other workers demonstrated the selective concentrations of androsterone and dehydroisoandrosterone and their sulfates in these fluids (14). High concentrations of estriol conjugates have also been described in this fluid (15). Srivastava and colleagues (16) have recently reported measurements of several pituitary peptides [LH, FSH, human PRL (hPRL) and TSH] in a small number of breast cyst fluid samples. We have also recently measured the concentration of these peptides in cyst fluid; our findings agree only in part with those of Srivastava et al. (16). Materials and Methods LH, FSH, hPRL, and hCG were all determined by standard RIA procedures. LH, FSH, and hPRL were determined as previously described by this laboratory (17,18). Anti-LH (batch 2), anti-FSH (batch 14), anti-hPRL, anti-/?-hCG [batch SB-6 LER-907; standards for human LH (hLH) and human FSH), and hPRL-VLS 4 (standard for hPRL) were obtained from the National Pituitary Agency, NIAMDD. hCG was analyzed by the method of Vaitukaitis et al. (19) using CR-115 as standard. This assay measures both the subunit /?-hCG and the intact parent hormone. The sensitivity of this assay procedure for degradation fragments of /?-hCG is unknown. It should be emphasized that in all cases these assays measure immunoreactive peptides. We have not yet established the biological reactivity of any of the peptides measured in this study. Serial dilution studies to validate the LH, FSH, and hPRL assays as applied to cyst fluid were carried out with and without the addition of known amounts of standards. Less systematic
Received October 12, 1978. Address requests for reprints to: Dr. H. Leon Bradlow, The Rockefeller University, 1230 York Avenue, New York, New York 10021. * This work was supported by Contract NCI-CB 53853-37, Biochemical Analysis of Human Breast Cyst Fluid and Its Correlation with Development of Breast Cancer awarded by the National Cancer Institute, DHEW, and Grant CA-22795 from the National Cancer Institute.
778
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 14:02 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS dilution studies were carried out on the hCG assay, primarily because this assay is carried out in a matrix of normal male plasma resulting in an essentially plasma-like environment for all of the samples in the assay. The absence of any interference with the hCG assay by other components of cyst fluid is illustrated in Table I. Gel chromatography on Sephadex G-75 superfine of 125I-labeled hCG carried through the assay procedure except for the final precipitation with the second antibody demonstrated the absence of any significant proteolytic cleavage of the labeled ligand, a possible source of error in the assay. Serum samples from four postmenopausal women containing >400 ng/ml LH and >2000 ng/ml FSH were assayed for hCG. The immunoreactive hCG levels of these specimens were 1.5, 2.6, 1.7, and 1.8 ng/ml, averaging 26 ng/ml) represent a minimum of three assays, the original and the two carried out subsequently at 1:10 and 1:100 dilutions in phosphate buffered saline. Consequently, the concentration of proteases or other factors that might give falsely elevated values for hCG which might be present in these dilute samples is probably negligible. Were such proteases to play a role then the diluted samples should show lower values. Blood and cyst fluid were obtained simultaneously from each subject when they presented for treatment and were stored frozen until the time of analysis. TABLE 1. Recovery studies on the measurement of immunoreactive hCG in breast cyst fluid
1
2 3 4 5 6 7
Mean
Theoretical
Actual 18.0 16.5
Calculated
%
18.8 15.5 10.0
95.7 106.5 92.0 105.8 112.8 90.6 81.2 97.8 ± 11.0
24.4 18.0 6.9 0.6 3.8 11.7
11.6
12.8
3.1
6.5
8.0
9.2 7.3 9.5
6.9 8.5
TABLE 2. Concentrations of immunoreactive peptide hormones in serum and breast cyst fluids of patients with cystic breast disease Hormone LH (mlU/ml) FSH (mlU/ml) hPRL (ng/ml) hCG (ng/ml) Group I Group II
Range of cone.
No. of subjects
Plasma
Cyst
47 42 130 173 113 60
7-125 7-300* 2.8-37.0" 0.1-5.4* 0.1-2.5 0.2-5.4
2.0-125" 0-15c 7.2-29.0 0.1-340' 0.1-2.0 2.1-340
P
Vol. 49, No. 5
Printed in U.S.A.
Comments
Accumulation of Hormones in Breast Cyst Fluid* H. LEON BRADLOW, MORTON K. SCHWARTZ, MARTIN FLEISHER, JEROME S. NISSELBAUM, ROBERT BOYAR, JOHN O'CONNOR, AND DAVID K. FUKUSHIMA The Rockefeller University (H.L.B.), New York, 10021; the Memorial Sloan-Kettering Cancer Center (M.K.S., M.F., J.N.), New York, 10021; and the Institute for Steroid Research, Montefiore Hospital and Medical Center (R.B., J.O., D.K.F.), Bronx, New York 10467
ABSTRACT. The concentration of some immunoreactive peptide hormones in breast cyst fluid and serum from a series of patients with cystic breast disease were compared. The concentrations of the two pituitary gonadotropins, LH and FSH, were consistently higher in serum than in cyst fluid, confirming earlier more limited observations. The mean PRL concentration, on the other hand, was elevated in cyst fluid though the variability was great. The cyst fluid serum ratio of hCG was elevated in the patient group as a whole. However, the results suggested the presence of two subpopulations; group I, in which the cyst fluid
and serum hCG levels were essentially indistinguishable and in the range found in normal serum, and group II, in which the cyst fluid hCG concentration was consistently greater than the
normal serum range. Analysis of hormone concentrations in multiple cysts aspirated at the same time showed a remarkable degree of agreement in the cyst hormone levels in most of the patients. The greatest variability in the levels was seen with hCG, where a small number of subjects showed a very large spread in the values. (J Clin Endocrinol Metab 49: 778,1979)
C
YSTIC breast disease is a common condition in premenopausal patients, appearing after age 25 yr and disappearing at menopause. While cystic lesions are not considered to be precancerous per se, many studies dating back over the last 40 yr (1-7) have indicated that these patients have a 2- to 4 fold greater risk of developing breast cancer than matched subjects without breast disease. It has been suggested that benign and malignant breast disease could have some common factor, such as a unique pattern of hormone or protein concentrations (8). Evidence for such unusual patterns in patients with breast cysts has come from several sources. Haagensen and colleagues (9, 10) have described the isolation and characterization of an unusual glycoprotein from breast cyst fluid; this substance has also been found in high concentrations in the serum of breast cancer patients. An unusual protein with high binding affinity for progesterone and pregnenolone has been isolated from cyst fluid in large amounts (10,11), while Schwartz et al. (12) have reported the presence therein of high concentrations of glutamyl transpeptidase, /?-glucuronidase, and carcinoembryonic antigen. Steroid concentrations in cyst fluid were first determined by Fleisher et al. in 1973 (13); they
reported large amounts of 17-ketosteroids in these samples. Subsequent studies by other workers demonstrated the selective concentrations of androsterone and dehydroisoandrosterone and their sulfates in these fluids (14). High concentrations of estriol conjugates have also been described in this fluid (15). Srivastava and colleagues (16) have recently reported measurements of several pituitary peptides [LH, FSH, human PRL (hPRL) and TSH] in a small number of breast cyst fluid samples. We have also recently measured the concentration of these peptides in cyst fluid; our findings agree only in part with those of Srivastava et al. (16). Materials and Methods LH, FSH, hPRL, and hCG were all determined by standard RIA procedures. LH, FSH, and hPRL were determined as previously described by this laboratory (17,18). Anti-LH (batch 2), anti-FSH (batch 14), anti-hPRL, anti-/?-hCG [batch SB-6 LER-907; standards for human LH (hLH) and human FSH), and hPRL-VLS 4 (standard for hPRL) were obtained from the National Pituitary Agency, NIAMDD. hCG was analyzed by the method of Vaitukaitis et al. (19) using CR-115 as standard. This assay measures both the subunit /?-hCG and the intact parent hormone. The sensitivity of this assay procedure for degradation fragments of /?-hCG is unknown. It should be emphasized that in all cases these assays measure immunoreactive peptides. We have not yet established the biological reactivity of any of the peptides measured in this study. Serial dilution studies to validate the LH, FSH, and hPRL assays as applied to cyst fluid were carried out with and without the addition of known amounts of standards. Less systematic
Received October 12, 1978. Address requests for reprints to: Dr. H. Leon Bradlow, The Rockefeller University, 1230 York Avenue, New York, New York 10021. * This work was supported by Contract NCI-CB 53853-37, Biochemical Analysis of Human Breast Cyst Fluid and Its Correlation with Development of Breast Cancer awarded by the National Cancer Institute, DHEW, and Grant CA-22795 from the National Cancer Institute.
778
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 14:02 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS dilution studies were carried out on the hCG assay, primarily because this assay is carried out in a matrix of normal male plasma resulting in an essentially plasma-like environment for all of the samples in the assay. The absence of any interference with the hCG assay by other components of cyst fluid is illustrated in Table I. Gel chromatography on Sephadex G-75 superfine of 125I-labeled hCG carried through the assay procedure except for the final precipitation with the second antibody demonstrated the absence of any significant proteolytic cleavage of the labeled ligand, a possible source of error in the assay. Serum samples from four postmenopausal women containing >400 ng/ml LH and >2000 ng/ml FSH were assayed for hCG. The immunoreactive hCG levels of these specimens were 1.5, 2.6, 1.7, and 1.8 ng/ml, averaging 26 ng/ml) represent a minimum of three assays, the original and the two carried out subsequently at 1:10 and 1:100 dilutions in phosphate buffered saline. Consequently, the concentration of proteases or other factors that might give falsely elevated values for hCG which might be present in these dilute samples is probably negligible. Were such proteases to play a role then the diluted samples should show lower values. Blood and cyst fluid were obtained simultaneously from each subject when they presented for treatment and were stored frozen until the time of analysis. TABLE 1. Recovery studies on the measurement of immunoreactive hCG in breast cyst fluid
1
2 3 4 5 6 7
Mean
Theoretical
Actual 18.0 16.5
Calculated
%
18.8 15.5 10.0
95.7 106.5 92.0 105.8 112.8 90.6 81.2 97.8 ± 11.0
24.4 18.0 6.9 0.6 3.8 11.7
11.6
12.8
3.1
6.5
8.0
9.2 7.3 9.5
6.9 8.5
TABLE 2. Concentrations of immunoreactive peptide hormones in serum and breast cyst fluids of patients with cystic breast disease Hormone LH (mlU/ml) FSH (mlU/ml) hPRL (ng/ml) hCG (ng/ml) Group I Group II
Range of cone.
No. of subjects
Plasma
Cyst
47 42 130 173 113 60
7-125 7-300* 2.8-37.0" 0.1-5.4* 0.1-2.5 0.2-5.4
2.0-125" 0-15c 7.2-29.0 0.1-340' 0.1-2.0 2.1-340
P
