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Effects of tamoxifen on the breast in the luteal phase of the menstrual cycle
International Journal of Gynecology & Obstetrics 62 Ž1998. 77]82
Article
Effects of tamoxifen on the breast in the luteal phase of the menstrual cycle J. UeharaU , A.C.P. Nazario, ´ G. Rodrigues de Lima, M.J. Simoes, ˜ Y. Juliano, L.H. Gebrim Department of Gynecology, Federal Uni¨ ersity of Sao ˜ Paulo, Escola Paulista de Medicina, Sao ˜ Paulo, Brazil Received 23 October 1997; received in revised form 24 February 1998; accepted 2 March 1998
Abstract Objecti¨ es: The effect of tamoxifen on cyclic mastalgia and on chemoprophylaxis against breast cancer is little known, mainly due to the difficulties in studying the normal human gland. We proposed to evaluate the mitotic index and the nuclear volume of the lobule of women medicated with tamoxifen only during the luteal phase of the menstrual cycle in order to observe the effect of tamoxifen on the normal human mammary gland. Methods: Twenty-four premenopausal women with fibroadenoma diagnosed via biopsy were studied. The phase of the cycle was determined by the date of menstruation and serum progesterone level in the luteal phase ŽG 3 ngrml.. The patients admitted to the study and were given written informed consent to participate in the investigation, which was previously approved of by the hospital Ethics Committee. Patients were divided at random into two groups: Group I consisted of 12 untreated women Žcontrol. and Group II consisted of 12 patients treated with 20 mgrday tamoxifen for 10 consecutive days beginning on the 13th day of the menstrual cycle. In both groups, the patients were submitted to biopsies of the nodule and of a 1-cm3 fragment of adjacent mammary parenchyma between the 23rd and 26th day of the cycle. The mitotic index Žnumber of mitosesr1000 nuclei counted. and mean nuclear volume Žmean of 10 nuclear volumes for each case. were measured. Results: No mitosis was observed in Group II. There was a reduction in the mean nuclear volume in Group II ŽMann]Whitney test.. Conclusions: Tamoxifen, when administered only during the luteal phase of the menstrual cycle, significantly reduces the nuclear volume and mitotic activity of the epithelium. This data demonstrates an antagonistic action of tamoxifen on estrogen even when administered for short periods of time. Q 1998 International Federation of Gynecology and Obstetrics Keywords: Breast; Mitotic index; Tamoxifen; Premenopause
U
Corresponding author. Rua Caripunas 456, 03444-030 Sao ˜ Paulo SP, Brazil. Tel.: q55 11 2173820; fax: q55 11 5739228.
0020-7292r98r$19.00 Q 1998 International Federation of Gynecology and Obstetrics PII S0020-7292Ž98.00050-2
78
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
1. Introduction The human mammary gland, when not in ‘biological repose’, undergoes morphological and functional changes during the menstrual cycle. The structural changes depend on the complex influence of hormones, most importantly the sexual steroids, as well as local peptide growth factors. Control over the endocrine, paracrine and autocrine systems is complex and many facets of mammary histophysiology still remain unclear, impairing the interpretation of pharmacokinetic studies of benign and malignant disease. Tamoxifen, a drug with an anti-estrogenic effect in carcinoma, has been used as a primary prophylaxis against this disease, although its major effects on normal mammary tissue have not been adequately determined w1,2x. The choice of this drug may be justified by the clinical and epidemiological evidence of the reduction of contralateral primary cancer in patients that received adjuvant treatment w3]5x. The difficulty of ethically obtaining material forces researchers to use tissues adjacent to benign tumors or those obtained through reduction mammaplasty. Tamoxifen acts as a partial agonist]antagonist and its main action varies in accordance with the species and target tissue studied, so that understanding its mechanism of action in premenopause is a very controversial and complex process w6,7x. Ricketts et al. w8x, in studies on normal mammary tissue, observed that estradiol receptors are decreased in the luteal phase of the cycle. Although no similar study in which estrogen receptors were studied only during the luteal phase is available for tamoxifen, it is understood that the drug occupies the estrogen receptors, thus acting as an antagonist. The drug, when administered during the periovulatory period and during the luteal phase, interferes less with ovarian steroidogenesis, bringing about a local effect on the mamma. Thus, at present there is no evidence that tamoxifen has a direct action on normal mammary epithelium, principally with respect to cellular proliferation. These facts sparked our interest in observing the action of tamoxifen on the normal human mammary epithelium in
women medicated with tamoxifen only during the luteal phase. 2. Material and methods Material for study was obtained from a group of 24 women with breast fibroadenoma seen at and selected from the out-patient clinic of the Gynecology Department of the Federal University of Sao ˜ Paulo from March 1994 to April 1995. The patients gave written informed consent to participate in the study, which was previously approved by the Ethics Committee of Sao ˜ Paulo Hospital. The study was conducted on young women aged 16]37 years, eumenorrheic for at least 6 months and aware of the exact date of their last menstruation. Patients with endocrine disease, pregnant women, or those who had used any type of hormonal medication within the previous 12 months were excluded from the study as suggested by Master et al. w9x and Meyer w10x. Patients were divided at random into two groups: Group I Žcontrol. consisted of 12 women not treated with tamoxifen and Group II Žtamoxifen. consisted of 12 women who took 20 mg of tamoxifen ŽTecnotax. per day for 10 consecutive days beginning after the 13th day of the menstrual cycle. To better determine the phase of the cycle, the following parameters were measured: date of the last menstruation and serum progesterone level during the luteal phase ŽG 3 ngrml.. The patients were submitted to excisional biopsies of the breast nodule at the outpatient clinic of Sao ˜ Paulo Hospital using a local anesthetic without a vasoconstrictor Ž2% lidocaine. in accordance with Fanger and Ree w11x. The surgeries were performed between the 20th and 29th day of the menstrual cycle between 10.00 and 11.00 h in an effort to minimize the possibility of circadian variation in cellular kinetics w9x. The mammary fragments Žnormal and nodules. were removed, fixed in Bouin solution and later dehydrated in ethyl alcohol, cleared with xylene and impregnated with liquid wax in an incubator set at 598C, according to the methodology of Masson w12x. Next, the blocks were cut into 5-m m sections with a Minot microtome
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
ŽAmerican Optical, model 820.. The sections were mounted on slides previously coated with Mayer’s albumin and kept in an incubator at 378C over 24 h for drying and adhesion. The histological sections were stained with hematoxylin and eosin for morphological description. A Zeiss microscope was used with = 8 objectives whose magnifications varied from 2.5 to 100 times Žtotal magnification from= 20 to = 800.. The histological normality of the parenchyma to be studied and the diagnosis of fibroadenoma in the excised nodules were previously determined. Measures were taken directly with the aid of an = 8 rotating drum measuring Zeiss lens model K, adapted to a Nikon microscope with a final magnification of = 800. Since the nuclei of epithelial cells are ellipsoid, the cells were measured along two perpendicular lines, one corresponding to the largest diameter and the other to the smallest diameter. From these measurements it was possible to calculate the nuclear volume of the cells using the formula below w13x: Vs
d2 =D 1.91
where V is the nuclear volume, D is the largest diameter, d the smallest diameter and 1.91 is a constant. The diameters were measured in each case from 10 nuclei of epithelial cells from the terminal duct, chosen at random. The mean was then calculated from these 10 volumes. In all, 240 nuclei were examined, yielding 24 mean nuclear volumes. A Zeiss microscope with an = 8 magnifying eyepiece and an = 100 objective lens was used, allowing for a total magnification of = 800. The figures of normal mitosis were determined from whole nuclei and counted from a total of 1000 nuclei from epithelial cells in each case. Care was taken to consider only well defined, whole nuclei, with no signs of degeneration and present in the luminal epithelium of the terminal duct. A mitotic index was calculated whose numerator consisted of the number of mitoses and whose denominator consisted of the 1000 nuclei counted for each case.
MI s
79
X 1000
where MI is the mitotic index and X is the number of mitoses. Statistical analysis was performed using the Mann]Whitney and Friedman tests according to the nature of the variable analyzed, with the level of significance set at 0.05 or 5%. Significant values were marked with asterisks w14x. 3. Results and discussion The mean mitotic index for Group I Žcontrol. was significantly higher than that observed in Group II Žtamoxifen. ŽTable 1.. The mean nuclear volume for Group I Žcontrol. was significantly larger than that observed for Group II Žtamoxifen. ŽTable 2.. Friedman’s analysis of variance did not show a significant difference between the 10 nuclear volume means calculated for each case in both groups. The two groups were homogeneous in terms of the main variables that could influence the results, such as age, menarche, number of pregnancies, parity and date of surgery ŽTables 3]5.. The proliferative activity of the mammary epithelium has been studied in an attempt to establish its possible carcinogenic effect. Several authors, studying the cellular kinetics of the mammary gland, observed that the mitotic activity Table 1 Mitotic index Žnumber of mitosesr1000. in Group I Žcontrol. and Group II ŽTamoxifen.
Mean
Group I
Group II
12 11 8 10 9 9 9 8 12 8 11 11
0 0 0 0 0 0 0 0 0 0 0 0
9.8
0
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
80
Table 2 Mean nuclear volume Ž m m3 . in Group I Žcontrol. and Group II Žtamoxifen.
Mean
Group I
Group II
169.1 166.4 153.3 141.9 127.7 156.3 147.6 137.4 119.6 122.5 163.3 166.0
53.9 70.0 64.5 75.6 68.4 59.2 60.6 68.2 51.0 65.8 65.5 49.8
147.6
62.7
Mann]Whitney test: Group I = Group II } U calculated s 0U , U critical s 37, Group I ) Group II.
was more intense in the luteal phase of the menstrual cycle w9,10,15x. We used mammary tissue adjacent to the fibroadenoma with the objective of studying the morphometric aspects of the epithelium under the effects of tamoxifen, because the mammary tissue is from the periphery of a circumscribed lesion whose etiopathogenic factor
practically does not interfere with the adjacent tissue w16x. Based on previous menstruations, tamoxifen was administered on the 13th day of the menstrual cycle, most likely the beginning of the luteal phase. Our data demonstrated that the drug significantly reduced the proliferative activity of the mammary lobule during the luteal phase. There are no studies which demonstrate that tamoxifen has a direct action on normal mammary epithelium. Thus, its mechanism of action is still rather controversial, principally in premenopausal women. A full understanding of this mechanism in normal mammary glands would be very valuable to evaluate the effect of tamoxifen on cyclic mastalgia, fibrocystic anomalies and chemoprophylaxis against cancer and would also permit a reduction in the dose administered and the time of exposure, with a consequent reduction of costs and side-effect. Jordan et al. w17x observed an increase of estrone, estradiol, progesterone and SHBG in premenopausal women treated with tamoxifen, showing that tamoxifen may have a direct action on the ovaries and may block the estrogen receptors along the hypothalamus]pituitary axis w18]21x. This result surprised us, because classi-
Table 3 Distribution of women from Group I Žcontrol. and Group II Žtamoxifen. according to age, menarche and number of pregnancies Age Žyears.
Mean
Menarche Žyears.
Number of pregnancies
Group I
Group II
Group I
Group II
Group I
Group II
22 22 26 33 17 22 21 30 32 16 29 20
37 26 30 33 25 17 19 20 32 29 17 28
14 14 14 10 13 15 12 12 12 13 14 15
14 12 12 16 14 10 14 11 12 12 13 15
0 0 0 3 0 0 0 4 4 0 0 0
6 0 4 4 2 0 0 0 7 2 0 1
24.2
26.1
13.2
12.9
0.9
2.2
Mann]Whitney test: Group I = Group II, U critical s 37.0; age, U calculated s 62.0; menarche, U calculated s 63.5; pregnancies, U calculated s 49.5.
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82 Table 4 Distribution of women from Group I Žcontrol. and Group II Žtamoxifen. according to number of births Žyears. Number of births
Mean
Group I
Group II
0 0 0 3 0 0 0 0 3 3 0 0
4 0 4 4 2 0 0 0 7 1 0 0
0.7
1.8
Mann]Whitney test: Group I = Group II, U critical s 37.0; births, U calculated s 51.0.
cally the anti-estrogenic effect of the drug was described only in carcinoma and in cell cultures after long periods of exposure to tamoxifen w22,23x. Thus, in the present study we sought to use the drug only during the luteal phase in order to reduce its eventual interference with the secondary effects of the interaction between the Table 5 Day of the cycle on which surgery was performed on women from Group I Žcontrol. and Group II Žtamoxifen.
Mean
Group I ŽDay of the cycle.
Group II ŽDay of the cycle.
20 20 27 22 29 23 25 25 26 24 23 25
23 22 25 23 24 29 23 24 27 23 23 24
24.1
24.2
Mann]Whitney test: Group I = Group II, U calculated s 68.5, U critical s 37.
81
hypothalamus]pituitary]ovarian axis and mammary tissue. In our study the patients medicated with tamoxifen showed a significant reduction in mitotic index and nuclear volume. Such results suggest that the drug exerts an anti-estrogenic action on normal mammary epithelium in premenopausal women even when used only during the luteal phase of the menstrual cycle. Facina et al. w24x, studying the effect of tamoxifen in women treated only during the luteal phase, observed a decrease in the number of lysosomes in normal mammary tissue adjacent to fibroadenomas, demonstrating an antagonistic estrogenic action of the drug. Nevertheless, more studies are necessary, using the drug over periods of treatment of three or more cycles and monitoring possible changes in estradiol, estrone, SHBG, progesterone, FSH, LH and prolactin levels. Confirmation of these results may contribute to the reduction of treatment time in premenopausal women submitted to primary chemoprophylaxis. References w1x Walker KJ, Price-Thomas JM, Candlish W, Nicholson RI. Influence of anti-oestrogen tamoxifen on normal breast tissue. Br J Cancer 1991;64:764]768. w2x Jordan VC. Estrogen receptor antagonists. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive endocrinology, surgery and technology. Philadelphia: Lippincott-Raven, 1996:527]545. w3x Nayfield SG, Karp JE, Ford LG, Dorr FA, Kramer BS. Potential role of tamoxifen in prevention of breast cancer. J Natl Cancer Inst 1991;83:1450]1459. w4x Early Breast Cancer Trialists’ Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy: 133 randomised trials involving 31.000 recurrences and 24.000 deaths among 75000 women Žparts 1 and 2.. Lancet 1992; 339Ž1r15.:71]85. w5x Jordan VC. Studies on the estrogen receptor in breast cancer } 20 years as a target for the treatment and prevention of cancer. Breast Cancer Res Treat 1995;36:267]285. w6x Rochefort H. Mechanism of action of high-affinity antiestrogen. Am J Clin Oncol 1991;14:S1]4. w7x Wieb VJ, Osborne CK, Fuqua SAW. Tamoxifen resistance in breast cancer. Crit Rev Oncol Hematol 1993;14:173.
82
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
w8x Ricketts D, Turnbull L, Ryal G. Estrogen and progesterone receptors in the normal breast female. Cancer Res 1991;51:1817. w9x Master JRW, Drife JO, Scarisbrick JJ. Cyclic variation of DNA synthesis in human breast epithelium. J Natl Cancer Inst 1977;58:1263]1265. w10x Meyer JS. Cell proliferation in normal human breast ducts, fibroadenomas and other ductal hyperplasias measured by nuclear labeling with tritiated thymidine. Hum Pathol 1977;8:67]81. w11x Fanger H, Ree HJ. Cyclic changes of human mammary gland epithelium in relation to the menstrual cycle } an ultrastructural study. Cancer 1974;34:571]585. w12x Masson P. Human tumors: histology, diagnosis and technique. 2nd ed. Paris: Libraire Maloine, 1956:1061]1148. w13x Salvatore CA. About the nature of nuclear hypertrophy of uterine muscle cells during pregnancy. An Bras Ginecol 1948;26:453]462. w14x Siegel S. Non-parametric statistics applied to the behavioral sciences. 2nd ed. Mexico: Trilhas, 1975:346. w15x Nazario ´ ACP, de Lima GR, Simies MJ, Novo NF. Cell kinetics of the human mammary lobule during the proliferative and secretory phase of the menstrual cycle. Bull Assoc Anat 1995;79:23]27. w16x Dupont WD, Page DL, Parl FF, Vnencak-Jone CL, Plummer WD, Rados MS, Schuyler PA. Long-term risk of breast cancer in women with fibroadenoma. N Engl J Med 1994;331:10]15. w17x Jordan VC, Fritz NF, Langan-Fahey S. Alteration of
w18x
w19x
w20x
w21x w22x w23x w24x
endocrine parameters in premenopausal women with breast cancer during low-term adjuvant therapy with tamoxifen as the single agent. J Natl Cancer Inst 1991;83:1488]1496. Groom GV, Griffiths K. Effect of the anti-oestrogen tamoxifen on plasma levels of luteinizing hormone, follicle-stimulating hormone, prolactin, oestradiol and progesterone in normal premenopausal women. J Endocrinol 1976;70:421]428. Sherman BM, Chapler FK, Crickard K, Wycoff D. Endocrine consequences of continuous anti-estrogen therapy with tamoxifen in premenopausal women. J Clin Invest 1979;64:398]404. Ravdin PM, Fritz NF, Tormey DC, Jordan VC. Endocrine status of premenopausal node-positive breast cancer patients following adjuvant chemotherapy and long-term tamoxifen. Cancer Res 1988;48:1026]1029. Sunderland MC, Osborne CK. Tamoxifen in the premenopausal patients with metastatic breast cancer: a review. J Clin Oncol 1991;9:1283]1297. Howell A. New endocrine treatments for breast cancer: biological and clinical aspects. Breast 1996;5:170]174. Nicholson RI, Francis AB, Kyme S, Gee JMW. Properties and mode of pure anti-oestrogens in breast cancer in vitro. Breast 1996;5:175]180. Facina G, Rodrigues de Lima G, Simies MJ, Novo NF, Gebrim LH. Estrogenic activity of tamoxifen on normal mammary parenchyma in the luteal phase of the menstrual cycle. Int J Gynecol Obst 1997;56:19]24.
Article
Effects of tamoxifen on the breast in the luteal phase of the menstrual cycle J. UeharaU , A.C.P. Nazario, ´ G. Rodrigues de Lima, M.J. Simoes, ˜ Y. Juliano, L.H. Gebrim Department of Gynecology, Federal Uni¨ ersity of Sao ˜ Paulo, Escola Paulista de Medicina, Sao ˜ Paulo, Brazil Received 23 October 1997; received in revised form 24 February 1998; accepted 2 March 1998
Abstract Objecti¨ es: The effect of tamoxifen on cyclic mastalgia and on chemoprophylaxis against breast cancer is little known, mainly due to the difficulties in studying the normal human gland. We proposed to evaluate the mitotic index and the nuclear volume of the lobule of women medicated with tamoxifen only during the luteal phase of the menstrual cycle in order to observe the effect of tamoxifen on the normal human mammary gland. Methods: Twenty-four premenopausal women with fibroadenoma diagnosed via biopsy were studied. The phase of the cycle was determined by the date of menstruation and serum progesterone level in the luteal phase ŽG 3 ngrml.. The patients admitted to the study and were given written informed consent to participate in the investigation, which was previously approved of by the hospital Ethics Committee. Patients were divided at random into two groups: Group I consisted of 12 untreated women Žcontrol. and Group II consisted of 12 patients treated with 20 mgrday tamoxifen for 10 consecutive days beginning on the 13th day of the menstrual cycle. In both groups, the patients were submitted to biopsies of the nodule and of a 1-cm3 fragment of adjacent mammary parenchyma between the 23rd and 26th day of the cycle. The mitotic index Žnumber of mitosesr1000 nuclei counted. and mean nuclear volume Žmean of 10 nuclear volumes for each case. were measured. Results: No mitosis was observed in Group II. There was a reduction in the mean nuclear volume in Group II ŽMann]Whitney test.. Conclusions: Tamoxifen, when administered only during the luteal phase of the menstrual cycle, significantly reduces the nuclear volume and mitotic activity of the epithelium. This data demonstrates an antagonistic action of tamoxifen on estrogen even when administered for short periods of time. Q 1998 International Federation of Gynecology and Obstetrics Keywords: Breast; Mitotic index; Tamoxifen; Premenopause
U
Corresponding author. Rua Caripunas 456, 03444-030 Sao ˜ Paulo SP, Brazil. Tel.: q55 11 2173820; fax: q55 11 5739228.
0020-7292r98r$19.00 Q 1998 International Federation of Gynecology and Obstetrics PII S0020-7292Ž98.00050-2
78
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
1. Introduction The human mammary gland, when not in ‘biological repose’, undergoes morphological and functional changes during the menstrual cycle. The structural changes depend on the complex influence of hormones, most importantly the sexual steroids, as well as local peptide growth factors. Control over the endocrine, paracrine and autocrine systems is complex and many facets of mammary histophysiology still remain unclear, impairing the interpretation of pharmacokinetic studies of benign and malignant disease. Tamoxifen, a drug with an anti-estrogenic effect in carcinoma, has been used as a primary prophylaxis against this disease, although its major effects on normal mammary tissue have not been adequately determined w1,2x. The choice of this drug may be justified by the clinical and epidemiological evidence of the reduction of contralateral primary cancer in patients that received adjuvant treatment w3]5x. The difficulty of ethically obtaining material forces researchers to use tissues adjacent to benign tumors or those obtained through reduction mammaplasty. Tamoxifen acts as a partial agonist]antagonist and its main action varies in accordance with the species and target tissue studied, so that understanding its mechanism of action in premenopause is a very controversial and complex process w6,7x. Ricketts et al. w8x, in studies on normal mammary tissue, observed that estradiol receptors are decreased in the luteal phase of the cycle. Although no similar study in which estrogen receptors were studied only during the luteal phase is available for tamoxifen, it is understood that the drug occupies the estrogen receptors, thus acting as an antagonist. The drug, when administered during the periovulatory period and during the luteal phase, interferes less with ovarian steroidogenesis, bringing about a local effect on the mamma. Thus, at present there is no evidence that tamoxifen has a direct action on normal mammary epithelium, principally with respect to cellular proliferation. These facts sparked our interest in observing the action of tamoxifen on the normal human mammary epithelium in
women medicated with tamoxifen only during the luteal phase. 2. Material and methods Material for study was obtained from a group of 24 women with breast fibroadenoma seen at and selected from the out-patient clinic of the Gynecology Department of the Federal University of Sao ˜ Paulo from March 1994 to April 1995. The patients gave written informed consent to participate in the study, which was previously approved by the Ethics Committee of Sao ˜ Paulo Hospital. The study was conducted on young women aged 16]37 years, eumenorrheic for at least 6 months and aware of the exact date of their last menstruation. Patients with endocrine disease, pregnant women, or those who had used any type of hormonal medication within the previous 12 months were excluded from the study as suggested by Master et al. w9x and Meyer w10x. Patients were divided at random into two groups: Group I Žcontrol. consisted of 12 women not treated with tamoxifen and Group II Žtamoxifen. consisted of 12 women who took 20 mg of tamoxifen ŽTecnotax. per day for 10 consecutive days beginning after the 13th day of the menstrual cycle. To better determine the phase of the cycle, the following parameters were measured: date of the last menstruation and serum progesterone level during the luteal phase ŽG 3 ngrml.. The patients were submitted to excisional biopsies of the breast nodule at the outpatient clinic of Sao ˜ Paulo Hospital using a local anesthetic without a vasoconstrictor Ž2% lidocaine. in accordance with Fanger and Ree w11x. The surgeries were performed between the 20th and 29th day of the menstrual cycle between 10.00 and 11.00 h in an effort to minimize the possibility of circadian variation in cellular kinetics w9x. The mammary fragments Žnormal and nodules. were removed, fixed in Bouin solution and later dehydrated in ethyl alcohol, cleared with xylene and impregnated with liquid wax in an incubator set at 598C, according to the methodology of Masson w12x. Next, the blocks were cut into 5-m m sections with a Minot microtome
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
ŽAmerican Optical, model 820.. The sections were mounted on slides previously coated with Mayer’s albumin and kept in an incubator at 378C over 24 h for drying and adhesion. The histological sections were stained with hematoxylin and eosin for morphological description. A Zeiss microscope was used with = 8 objectives whose magnifications varied from 2.5 to 100 times Žtotal magnification from= 20 to = 800.. The histological normality of the parenchyma to be studied and the diagnosis of fibroadenoma in the excised nodules were previously determined. Measures were taken directly with the aid of an = 8 rotating drum measuring Zeiss lens model K, adapted to a Nikon microscope with a final magnification of = 800. Since the nuclei of epithelial cells are ellipsoid, the cells were measured along two perpendicular lines, one corresponding to the largest diameter and the other to the smallest diameter. From these measurements it was possible to calculate the nuclear volume of the cells using the formula below w13x: Vs
d2 =D 1.91
where V is the nuclear volume, D is the largest diameter, d the smallest diameter and 1.91 is a constant. The diameters were measured in each case from 10 nuclei of epithelial cells from the terminal duct, chosen at random. The mean was then calculated from these 10 volumes. In all, 240 nuclei were examined, yielding 24 mean nuclear volumes. A Zeiss microscope with an = 8 magnifying eyepiece and an = 100 objective lens was used, allowing for a total magnification of = 800. The figures of normal mitosis were determined from whole nuclei and counted from a total of 1000 nuclei from epithelial cells in each case. Care was taken to consider only well defined, whole nuclei, with no signs of degeneration and present in the luminal epithelium of the terminal duct. A mitotic index was calculated whose numerator consisted of the number of mitoses and whose denominator consisted of the 1000 nuclei counted for each case.
MI s
79
X 1000
where MI is the mitotic index and X is the number of mitoses. Statistical analysis was performed using the Mann]Whitney and Friedman tests according to the nature of the variable analyzed, with the level of significance set at 0.05 or 5%. Significant values were marked with asterisks w14x. 3. Results and discussion The mean mitotic index for Group I Žcontrol. was significantly higher than that observed in Group II Žtamoxifen. ŽTable 1.. The mean nuclear volume for Group I Žcontrol. was significantly larger than that observed for Group II Žtamoxifen. ŽTable 2.. Friedman’s analysis of variance did not show a significant difference between the 10 nuclear volume means calculated for each case in both groups. The two groups were homogeneous in terms of the main variables that could influence the results, such as age, menarche, number of pregnancies, parity and date of surgery ŽTables 3]5.. The proliferative activity of the mammary epithelium has been studied in an attempt to establish its possible carcinogenic effect. Several authors, studying the cellular kinetics of the mammary gland, observed that the mitotic activity Table 1 Mitotic index Žnumber of mitosesr1000. in Group I Žcontrol. and Group II ŽTamoxifen.
Mean
Group I
Group II
12 11 8 10 9 9 9 8 12 8 11 11
0 0 0 0 0 0 0 0 0 0 0 0
9.8
0
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82
80
Table 2 Mean nuclear volume Ž m m3 . in Group I Žcontrol. and Group II Žtamoxifen.
Mean
Group I
Group II
169.1 166.4 153.3 141.9 127.7 156.3 147.6 137.4 119.6 122.5 163.3 166.0
53.9 70.0 64.5 75.6 68.4 59.2 60.6 68.2 51.0 65.8 65.5 49.8
147.6
62.7
Mann]Whitney test: Group I = Group II } U calculated s 0U , U critical s 37, Group I ) Group II.
was more intense in the luteal phase of the menstrual cycle w9,10,15x. We used mammary tissue adjacent to the fibroadenoma with the objective of studying the morphometric aspects of the epithelium under the effects of tamoxifen, because the mammary tissue is from the periphery of a circumscribed lesion whose etiopathogenic factor
practically does not interfere with the adjacent tissue w16x. Based on previous menstruations, tamoxifen was administered on the 13th day of the menstrual cycle, most likely the beginning of the luteal phase. Our data demonstrated that the drug significantly reduced the proliferative activity of the mammary lobule during the luteal phase. There are no studies which demonstrate that tamoxifen has a direct action on normal mammary epithelium. Thus, its mechanism of action is still rather controversial, principally in premenopausal women. A full understanding of this mechanism in normal mammary glands would be very valuable to evaluate the effect of tamoxifen on cyclic mastalgia, fibrocystic anomalies and chemoprophylaxis against cancer and would also permit a reduction in the dose administered and the time of exposure, with a consequent reduction of costs and side-effect. Jordan et al. w17x observed an increase of estrone, estradiol, progesterone and SHBG in premenopausal women treated with tamoxifen, showing that tamoxifen may have a direct action on the ovaries and may block the estrogen receptors along the hypothalamus]pituitary axis w18]21x. This result surprised us, because classi-
Table 3 Distribution of women from Group I Žcontrol. and Group II Žtamoxifen. according to age, menarche and number of pregnancies Age Žyears.
Mean
Menarche Žyears.
Number of pregnancies
Group I
Group II
Group I
Group II
Group I
Group II
22 22 26 33 17 22 21 30 32 16 29 20
37 26 30 33 25 17 19 20 32 29 17 28
14 14 14 10 13 15 12 12 12 13 14 15
14 12 12 16 14 10 14 11 12 12 13 15
0 0 0 3 0 0 0 4 4 0 0 0
6 0 4 4 2 0 0 0 7 2 0 1
24.2
26.1
13.2
12.9
0.9
2.2
Mann]Whitney test: Group I = Group II, U critical s 37.0; age, U calculated s 62.0; menarche, U calculated s 63.5; pregnancies, U calculated s 49.5.
J. Uehara et al. r International Journal of Gynecology & Obstetrics 62 (1998) 77]82 Table 4 Distribution of women from Group I Žcontrol. and Group II Žtamoxifen. according to number of births Žyears. Number of births
Mean
Group I
Group II
0 0 0 3 0 0 0 0 3 3 0 0
4 0 4 4 2 0 0 0 7 1 0 0
0.7
1.8
Mann]Whitney test: Group I = Group II, U critical s 37.0; births, U calculated s 51.0.
cally the anti-estrogenic effect of the drug was described only in carcinoma and in cell cultures after long periods of exposure to tamoxifen w22,23x. Thus, in the present study we sought to use the drug only during the luteal phase in order to reduce its eventual interference with the secondary effects of the interaction between the Table 5 Day of the cycle on which surgery was performed on women from Group I Žcontrol. and Group II Žtamoxifen.
Mean
Group I ŽDay of the cycle.
Group II ŽDay of the cycle.
20 20 27 22 29 23 25 25 26 24 23 25
23 22 25 23 24 29 23 24 27 23 23 24
24.1
24.2
Mann]Whitney test: Group I = Group II, U calculated s 68.5, U critical s 37.
81
hypothalamus]pituitary]ovarian axis and mammary tissue. In our study the patients medicated with tamoxifen showed a significant reduction in mitotic index and nuclear volume. Such results suggest that the drug exerts an anti-estrogenic action on normal mammary epithelium in premenopausal women even when used only during the luteal phase of the menstrual cycle. Facina et al. w24x, studying the effect of tamoxifen in women treated only during the luteal phase, observed a decrease in the number of lysosomes in normal mammary tissue adjacent to fibroadenomas, demonstrating an antagonistic estrogenic action of the drug. Nevertheless, more studies are necessary, using the drug over periods of treatment of three or more cycles and monitoring possible changes in estradiol, estrone, SHBG, progesterone, FSH, LH and prolactin levels. Confirmation of these results may contribute to the reduction of treatment time in premenopausal women submitted to primary chemoprophylaxis. References w1x Walker KJ, Price-Thomas JM, Candlish W, Nicholson RI. Influence of anti-oestrogen tamoxifen on normal breast tissue. Br J Cancer 1991;64:764]768. w2x Jordan VC. Estrogen receptor antagonists. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive endocrinology, surgery and technology. Philadelphia: Lippincott-Raven, 1996:527]545. w3x Nayfield SG, Karp JE, Ford LG, Dorr FA, Kramer BS. Potential role of tamoxifen in prevention of breast cancer. J Natl Cancer Inst 1991;83:1450]1459. w4x Early Breast Cancer Trialists’ Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy: 133 randomised trials involving 31.000 recurrences and 24.000 deaths among 75000 women Žparts 1 and 2.. Lancet 1992; 339Ž1r15.:71]85. w5x Jordan VC. Studies on the estrogen receptor in breast cancer } 20 years as a target for the treatment and prevention of cancer. Breast Cancer Res Treat 1995;36:267]285. w6x Rochefort H. Mechanism of action of high-affinity antiestrogen. Am J Clin Oncol 1991;14:S1]4. w7x Wieb VJ, Osborne CK, Fuqua SAW. Tamoxifen resistance in breast cancer. Crit Rev Oncol Hematol 1993;14:173.
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