Effects of tibolone and continuous combined hormone therapy on mammographic breast density and breast histochemical markers in postmenopausal women

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1 FERTILITY AND STERILITY VOL. 81, NO. 3, MARCH 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Effects of tibolone and continuous combined hormone therapy on mammographic breast density and breast histochemical markers in postmenopausal women Isabel Valdivia, M.D., a Italo Campodónico, M.D., a Augusto Tapia, M.D., a María Capetillo, M.D., b Arturo Espinoza, M.D., b and Pablo Lavín, M.D. a School of Medicine, University of Chile, Santiago, Chile Received January 21, 2003; revised and accepted July 31, Supported in part by an unrestricted research grant from Hormoquímica de Chile, Santiago, Chile (branch of NV Organon, Oss, the Netherlands). Reprint requests: Isabel Valdivia, M.D., Vitacura 5900, Of. 412, Santiago, Chile (FAX: ; imvaldivia@ entelchile.net). a Department of Obstetrics and Gynecology. b Department of Pathology /04/$30.00 doi: /j.fertnstert Objective: To compare changes in mammographic density and the expression of markers of proliferation (Ki67) and apoptosis (Bcl-2) after 1 year of treatment with tibolone and continuous conjugated equine estrogens combined with medroxyprogesterone acetate (CEE-MPA). Design: Comparative, randomized, evaluator-blinded study. Setting: City research hospital. Patient(s): Thirty-seven postmenopausal women. Intervention(s): Tibolone (2.5 mg; n 18) or continuous conjugated estrogens (0.625 mg) combined with medroxyprogesterone acetate (5 mg; n 19) for 1 year. Main Outcome Measure(s): Mammographic density (BI-RADS density score), expression of immunohistochemical markers Ki67 and Bcl-2. Result(s): Mean breast density score decreased significantly from 2.22 to 1.67 in the tibolone group, compared with a significant increase in the CEE-MPA treated group from 1.84 to Ki67 expression decreased in 12 of 15, increased in 2 of 15, and remained unchanged in 1 of 15 subjects in the tibolone group, compared with 1 of 19, 15 of 19, and 3 of 19 subjects, respectively, in the CEE-MPA group. Bcl-2 expression decreased in 12 of 15, increased in 2 of 15, and remained unchanged in 1 of 15 subjects in the tibolone group, compared with 5 of 19, 9 of 19, and 5 of 19 subjects, respectively, in the CEE-MPA group. Conclusion(s): One-year treatment with tibolone induced a decrease in breast density, with a reduction in proliferation and a stimulation of apoptosis, whereas 1-year treatment with CEE-MPA induced an increase in breast density, with stimulation of proliferation and inhibition of apoptosis, indicating that tibolone effects on the breast are different from those of CEE-MPA. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: Mammographic breast density, hormone therapy, tibolone, Ki67, Bcl-2, proliferation, apoptosis Postmenopausal women use various medications to relieve climacteric symptoms and to prevent osteoporosis. Continuous use of estrogens, combined with progestagens (continuous combined E P orcce P), is one of the most commonly used treatments. An alternative to this more conventional hormonal therapy (HT) is tibolone (Livial, Organon, The Netherlands), a unique tissue-specific compound (1). After oral administration, tibolone is rapidly converted by the intestine and the liver, mainly into two estrogenic metabolites (3 - and 3 OH-metabolites), which are responsible for its estrogenic effects on bone, vagina, and climacteric complaints. A third metabolite, the 4-isomer, has progestagenic and androgenic activities and is formed in the liver and intestine as well as locally in the endometrium, thereby preventing endometrial stimulation. One of the most common reasons for withdrawing from long-term use of conventional HT is fear of breast cancer. Although available data are equivocal, according to a meta-analysis of all epidemiological data, breast cancer risk increases by 2.3% for each year of estro- 617

2 gen use (2). In the Women s Health Initiative Randomized Controlled Trial, treatment for 5.2 years with conjugated estrogens medroxyprogesterone acetate (MPA) resulted in a 26% increase in risk of breast cancer compared with placebo, leading to an absolute excess risk of 8 per 10,000 person-years (3). Two recent studies suggest that combined estrogen and progestogen enhances the risk of breast cancer relative to estrogen alone (4, 5). Breast cancers diagnosed during conventional HT use, however, are smaller and less aggressive; most reports indicate no increase in breast cancer mortality (6). Conventional HT has been associated with an increase in breast density on mammography (7, 8), which is reversible after discontinuation of therapy. Increased density of the breast may complicate the detection of small tumors during mammographic screenings. Though high breast density is associated with a fourfold to sixfold increased risk of breast cancer (9, 10), there are several reasons to suspect that the increase in breast density reported with conventional HT may not be identical to the high breast density that is associated with an increased risk of breast cancer (8). The effects of tibolone on the breast differ from those observed with conventional HT. During treatment with tibolone, the incidence of breast tenderness was reported to be lower, and no relevant effect on mammographic density could be observed (7, 11, 12). Estrogens are known to enhance the rate of cell proliferation in the glandular tissue of the breast. Progestogens, which differ in structure and function, show different effects; some show breast tissue proliferation, whereas others show antiproliferation. Though an increase in proliferation per se does not necessarily mean induction of DNA mutations, stimulation of cell proliferation may increase the likelihood of a DNA replication error-escaping repair and could potentially act both as cofactor on the initiation and promotion of breast cancer. Recent studies suggest that some estrogen metabolites could act as carcinogens per se. The final stage in the life cycle of a cell is apoptosis (programmed cell death). The last step of this process is mediated by proapoptotic enzymes, which are regulated by proteins of the Bcl-2 family. Bcl-2 expression displays a strong cyclical variation and has been shown to be hormone dependent (13). A negative effect on apoptosis, for example, an increase in cell survival time, may increase the chance of inducing breast cancer. In this study, the effects of 1-year treatment with tibolone on breast density and on markers of proliferation (Ki67) and apoptosis (Bcl-2) are compared with those of continuous conjugated equine estrogens (CEE) combined with MPA therapy. Ki67 is a monoclonal antibody that reacts with a nuclear antigen that is expressed only in proliferating cells throughout the cell cycle and is absent from quiescent cells. Ki67 is the most widely used proliferation marker, whereas Bcl-2, which counteracts the occurrence of apoptosis, is one of the principal cytoplasmic apoptosis markers (14, 15). MATERIALS AND METHODS Study Design The study was designed as a randomized, single-center, assessor-blind study in postmenopausal women. It was performed at Hospital del Salvador, Santiago de Chile, between August 1998 and November 2001 and was in full compliance with the current version of the Declaration of Helsinki (Republic of South Africa, amendment October 1996) and the ICH Guideline for Good Clinical Practice. The Investigation and Education Board of the Ethical Committee of Hospital del Salvador approved the study protocol. All volunteers had given written informed consent before participating in the study. Thirty-seven volunteers were randomized to receive either tibolone (2.5 mg daily; Livial, Organon, Oss, the Netherlands) or conjugated equine estrogens (0.625 mg) medroxyprogesterone acetate (5 mg daily; Prempak plus; Wyeth-Ayerst Labs) for 12 months. Subjects To be considered for inclusion, women had to [1] have climacteric complaints, [2] be less than 65 years of age and in natural menopause (between 12 and 60 months of amenorrhea), [3] not have used hormone therapy or hormones in the past 6 months, [4] have no contraindications for hormone therapy, [5] have a negative mammography for cancer, and [6] have endometrium thickness (double layer) of 6 mm (by vaginal ultrasound). Subjects were excluded from the trial if they were overweight (body mass index 28) or had uncontrolled arterial hypertension or diabetes. Study Procedures and Methods of Evaluation Mammography was performed at baseline and after completion of 1 year of treatment with Lorad equipment (model Elite). Pretreatment and posttreatment mammographies were randomly coded and blindly assessed for density at the end of the study by a single evaluator. Mammographic density was scored according to the Breast Imaging Reporting and Data System (BI-RADS) proposed by the American College of Radiologists (16). Possible density categories were [1] entirely fatty: involutive, [2] fatty with scattered fibro-glandular tissue, [3] heterogeneously dense, and [4] extremely dense. Mean density score per treatment group was calculated. At baseline and after 1 year of treatment, a breast tissue sample (core biopsy type) was withdrawn under local anesthesia from the upper-external quadrant of the left breast using an automatic pistol system with a 1.2-mm (14G) biopsy needle. Three to five specimens of about 3 cm in length were obtained, fixed in 3.7% neutral buffered formalin, and subsequently embedded in paraffin. The samples 618 Valdivia et al. Effects of tibolone and CEE-MPA on breast tissue Vol. 81, No. 3, March 2004

3 were randomly coded to ensure blinding and assessed at the end of the study. Adjacent 3- m serial sections were placed on slides coated with APES, deparaffinized, and rinsed twice with PBS Buffer Power Block (Casein). Subsequently, the slides were evaluated for expression of Ki67 and Bcl-2 using automatic Optimax Plus equipment, by using the B-SA system method (Biogenex). The slides were placed in either a solution of monoclonal antibody Ki67 (MIB1; dilution of 1:150) for 90 minutes or in a solution of monoclonal antibody Bcl-2 (CLONE (100); dilution of 1:90) for 40 minutes. Subsequently the slides were incubated with secondary antibodies for 20 minutes (biotinylated anti-mouse, anti-rabbit, and anti-goat immunoglobulin), followed by a 20-minute incubation with streptavidin peroxidase (Biogenex) for labeling. The slides were stained using NOVARED Vector (SK 4800), followed by counterstaining with Harris Hematoxiline. The presence of both antibodies was independently quantified by two pathologists, by using an optical microscope, and was expressed as the percentage of Ki67 positive or Bcl-2 positive cells relative to the total number of cells. Statistical analysis was done using EPI-Info 2002 revision 1 software. Effects of treatment were assessed using twosided Student s t-test for mean differences and 2 test for difference in proportions. Differences in the distributions of the percentages of cells with staining for expression of Bcl-2 and Ki67 were assessed with Fisher s exact test, using Stata 7.0 software. TABLE 1 Changes in Ki67 expression from baseline to end of treatment (12 months). Type of change Tibolone, n (%) CEE-MPA, n (%) Increase 2/15 (13.3) 15/19 (78.9) Decrease 12/15 (80.0) 1/19 (5.3) No change 1/15 (6.7) 3/19 (15.8) Note: CEE-MPA conjugated equine estrogens combined with medroxyporgesterone acetate. RESULTS All 37 subjects completed the study as planned. There were no statistically significant differences between the two treatment groups with respect to demographic data. The 18 subjects in the tibolone group had the following mean (SD) values: 51.1 (3.2) years of age; 3.6 (2.2) deliveries; 28.6 (17.3) months since menopause; (6.7) cm in height; kg in weight, and body mass index of 26.2 (2.6) kg/m 2. The 19 subjects in the CEE-MPA group had mean (SD) values of 52.6 (3.9) years of age; 2.9 (2.3) deliveries; 40.3 (28.3) months since menopause; (5.4) cm in height; kg in weight; and body mass index of 26.6 (2.5) kg/m 2. There was no statistically significant difference (0.38 BI-RADS; P.27) in mean (SD) breast density at baseline between groups (2.22 [1.11] for tibolone group and 1.84 [0.96] BI-RADS for CEE-MPA group). After 12 months of treatment with tibolone, mean (SD) breast density score decreased to 1.67 (0.84) BI-RADS, whereas in the CEE- MPA treated group, an increase was observed to 2.63 (1.17) BI-RADS; this group difference of the means of 0.97 BI- RADS was statistically significant (P.007). The net mean difference of in BI-RADS change from baseline between the two groups ( [0.86] and 0789 [0.92], respectively) was statistically significant (P.00053). In the tibolone group, in only 1 (of 18; 5.5%) subject was an increase in breast density observed, whereas in 8 of 18 (45.5%) subjects, breast density score decreased, and in 9 of 18 (50.0%) subjects, no change was observed. In the CEE- MPA group, the majority of subjects (57.9%) showed an increase in breast density (11/19), whereas in only 1 of 19 (5.3%) a decrease was observed and in 7 of 19 (36.8%), breast density score remained unchanged. Of a total of 78 breast biopsies, only 3 specimens were inadequate for evaluation, leading to 15 subjects with adequate biopsies pretreatment and posttreatment in the tibolone group and 19 subjects in the CEE-MPA group. The effects of treatment with tibolone and CEE-MPA on the percentage of cells stained for expression of Ki67, expressed as the direction of change in individual subjects (increase, decrease, or no change), is presented in Table 1. In the tibolone-treated group, most women (80%) had a decrease in Ki67 expression, whereas in the CEE-MPA group, most women (78.9%) presented with an increase. The difference between the two groups in the distribution of changes was statistically significant (P.0013). The distribution of subjects presenting with a specific proportion of cells stained for Ki67 expression in the tibolone-treated group varied from baseline to after 12 months of treatment, with a shift to lower percentages of Ki67 expression (statistically significant; P.035), with, respectively, 8 and 13 cases in the 0 to 5% stained cells, 7 and 1 cases in the 6% to 10% stained cells, and 0 and 1 case in the 11% to 15% stained cells. In the CEE-MPA treated group, a nonstatistically significant shift to higher percentages could be noted (P.016), with, respectively, 13 and 6 cases in the 0 to 5% stained cells, 4 and 6 cases in the 6% to 10% stained cells, 1 to 2 cases in the 11% to 15% stained cells, 1 and 4 cases in the 16% to 20% stained cells, and 0 and 1 cases in the 21% to 25% stained cells. The absence of statistical significance in this last treatment group is probably related to the relatively small number of subjects in each treatment group. Typical examples of slides stained for Ki67 expression, FERTILITY & STERILITY 619

4 FIGURE 1 Breast cells stained for expression of cell proliferation marker Ki67 in breast biopsies taken from women before (left-hand side) and after (right-hand side) 12 months of treatment with (A and B) tibolone or with (C and D) CEE-MPA. Magnification: A, 100; B, 100; C, 100; D, 400. prepared from biopsies taken before and after 12 months of treatment with tibolone (upper quadrants, A and B, respectively) and CEE-MPA (lower quadrants, C and D, respectively), are presented in Figure 1, in which a reduction in the number of samples showing nuclear staining in the group with tibolone treatment and an increase in the number of samples showing nuclear staining in the group with CEE- MPA treatment is clearly seen after 12 months. The effects of treatment with tibolone and CEE-MPA on the percentage of cells stained for expression of Bcl-2, expressed as the direction of change in individual subjects, are presented in Table 2. In the tibolone-treated group, most women (80%) presented with a decrease in Bcl-2 expression, whereas in the CEE-MPA treated group, most women (47.4%) had an increase in Bcl-2 expression. The difference in distribution of changes between treatment groups was statistically significant (P.03). The distribution of subjects presenting with a specific proportion of cells stained for Bcl-2 expression is as follows. For the tibolone group, the number of cases with 0 10, 11 20, 21 30, 31 40, 41 50, 51 60, 61 70, and cells with positive expression before and after 12 months of treatment changed from 1 to 3, from 2 to 6, from 7 to 3, from 2 to 2, from 1 to 0, from 1 to 0, from 1 to 0, and from 0 to 1 subjects, respectively, and for the CEE-MPA treated TABLE 2 Changes in Bcl-2 expression from baseline to end of treatment (12 months). Type of change Tibolone, n (%) CEE-MPA, n (%) Increase 2/15 (13.3) 9/19 (47.4) Decrease 12/15 (80.0) 5/19 (26.3) No change 1/15 (6.7) 5/19 (26.3) Note: CEE-MPA conjugated equine estrogens combined with medroxyprogesterone acetate. 620 Valdivia et al. Effects of tibolone and CEE-MPA on breast tissue Vol. 81, No. 3, March 2004

5 FIGURE 2 Breast cells stained for expression of antiapoptotic protein Bcl-2 in breast biopsies taken from women before (left-hand side) and after (right-hand side) 12 months of treatment with (A and B) tibolone or with (C and D) CEE-MPA. Magnification: A, 400; B, 400; C, 400; D, 400. group, the distribution, respectively, was as follows: 5 to 3, 3 to 2, 5 to 7, 0 to 1, 4 to 1, 1 to 1, 0 to 1, and 1 to 3 subjects. In summary, it can be said that in the tibolone-treated group, a shift to lower percentages of antiapoptotic protein Bcl-2 expression was observed (though not statistically significant; P.248), whereas in the CEE-MPA treated group, the overall effect on the distribution is less obvious (P.612). Typical examples of slides stained for antiapoptotic protein Bcl-2 expression, prepared from biopsies taken before and after 12 months of treatment with tibolone (upper quadrants A and B, respectively) and CEE-MPA (lower quadrants C and D, respectively), are presented in Figure 2, in which a reduction of the cytoplasmic staining in the group with tibolone treatment and an increase of the cytoplasmic staining in the group with CEE-MPA treatment is clearly seen after 12 months. DISCUSSION The relative proportion of fat and fibroglandular tissue in the breast determines breast density on mammograms. High breast density is believed to represent proliferation of fibroglandular tissue. Several methods for classifying breast density are available, including Wolfe s patterns (a visual parenchymal method) (17) and estimation of the proportion (as a percentage) of the dense breast area (the percentagedensity method). In this study we used the BI-RADS density score method, which is the standard system currently used in clinical radiology practice in the United States. After 12 months of treatment with tibolone, the mean BI-RADS score decreased from 2.22 to 1.67, whereas in the CEE-MPA group, breast density increased from 1.84 to 2.63; the difference in change from baseline between the two groups was statistically significant. These results are in line with those reported in other studies. In our previous study, published in 1997 (18), using Wolfe s visual parenchymal method, breast density increased in 3.3% of the tibolone users, as compared with an increase in 66.7% to 26.7% of women using different estrogen-containing HT. The increase in the tibolone group was similar to that observed in the nontreated control group. The evaluation scores in this study FERTILITY & STERILITY 621

6 were highly reliable, with between-radiologist agreement ranging from 70% to 90%. In a recent study published by Lundström etal.(7), the effects of tibolone on breast density were compared with those of continuous combined hormone therapy (E 2 with norethisterone acetate) and placebo. In the women treated with estrogen-progestogen, breast density was increased in 46% 50%, as compared with an increase in 2% 6% in the group receiving tibolone and with an increase of 0 in the placebo group. An increase in breast density with estrogen-containing HT and the absence of a relevant effect with tibolone was also reported by Erel et al. (19, 20), Colacurci et al. (21), Egarter et al. (22), and Sendag et al. (23). In addition to the effect on breast density, we also investigated the effects of tibolone and CEE-MPA on cell proliferation (assessed as expression of Ki67) and apoptosis (assessed as expression of Bcl-2). In the present study, tibolone treatment showed a strong antiproliferative activity; most of the subjects presented decreased Ki67 expression, whereas in the CEE-MPA group, an increase in proliferation was observed. An HT-induced increase in proliferation was also observed in a cross-sectional observational study by Hofseth et al. (15). Compared with no HT, the breast epithelium of women who had received estrogen either alone or combined with progestogen had significantly higher proliferation (as assessed by determination of Ki67 expression and anti-proliferative cell nuclear antigen, anti-pcna), the effect of the combined treatment being greater than that of estrogen alone. Furthermore, with the combined therapy, breast proliferation was localized to the terminal duct-lobular unit of the breast, which is the site of development of most breast cancers. In a recent study by von Schoultz (24), the effects of 6 months treatment with E 2 (2 mg) combined with norethisterone acetate (1 mg), with tibolone, or with placebo on breast cell proliferation in cytological specimens obtained by fine needle aspiration, by using the Ki67 antibody technique, were compared. Treatment with E 2 norethisterone acetate induced an almost threefold increase in proliferation, whereas the effects of tibolone did not differ from those of placebo. The effects of treatment with tibolone and CEE-MPA on Bcl-2 expression differed significantly in the present study. In the tibolone group, the expression of Bcl-2 diminished in 80% of the subjects, whereas in only 13.3% of the subjects was an increase observed. In the CEE-MPA group, a decrease was observed in only 26.3% of the subjects, compared with an increase in 47% of the subjects. There are no published data on the effects of HT on markers of apoptosis (such as Bcl-2) in clinical studies. In cell cultures of normal and transformed breast epithelial cells, E 2 was shown to have an anti-apoptotic effect, whereas progestagens were found to be proapoptotic; tibolone was also found to stimulate apoptosis (15, 25 27). It is interesting to speculate on the causes of the differences in effects observed with tibolone and estrogen-progestogen treatment. Normal breast tissue contains high concentrations of estrogens. Tibolone and its 4 -isomer have been shown to lower estrogen concentrations in breast tissue by inhibiting sulfatases (inhibition of estrone formation from sulfated precursors), with a concomitant stimulation of sulfotransferase enzymes (stimulation of formation of biologically inactive sulfated forms of estrogen). Moreover, tibolone and its 4 - isomer block the 17 -hydroxysteroid dehydrogenase type I and stimulate the 17 -hydroxysteroid dehydrogenase type II, resulting in reduced local levels of E 2. The overall effect is a reduction of estrogenic exposure in the breast (28). The above effects have been observed in normal breast tissue and breast cancer lines (29, 30) but have not been found in other tissues such as bones (31). Estrogens are also synthesized from androgens via the aromatase pathway. However, tibolone and its main metabolites do not act as substrates for aromatase enzymes (32). A consequence of conventional HT treatment is that breast tissue is exposed to far higher concentrations of estrogen than before. Estrogens are known to enhance the rate of cell proliferation in the glandular tissue of the breast. The observation that conventional HT leads to an increase in tissue concentrations of estrogen in the breast, whereas tibolone treatment induces a reduction of such, may explain the differences in effects on breast density, cell proliferation, and apoptosis. An additional explanation of the difference in effect may be the effects of tibolone on free T levels and sex hormone binding globulin. In a study by Dören et al. (33), tibolone treatment was found to increase free T levels and decrease sex hormone binding globulin concentrations, whereas treatment with E 2 norethindrone acetate induced the opposite pattern. Breast cell proliferation has been described to be negatively correlated with free T in women who are using oral contraceptives (34) and to inhibit estrogen-induced breast epithelial proliferation and estrogen receptor expression in rhesus monkeys (35). Increased T levels may add to local actions in the breast to explain the different effects on breast proliferation during treatment with tibolone, as compared with conventional HT. In conclusion, 1-year treatment with tibolone induced a decrease in breast density, with a reduction in proliferation (reduction of Ki67 expression) and a stimulation of apoptosis (decrease in anti-apoptotic protein Bcl-2), whereas 1-year treatment with CEE-MPA induced an increase in breast density, with an increase in proliferation and inhibition of 622 Valdivia et al. Effects of tibolone and CEE-MPA on breast tissue Vol. 81, No. 3, March 2004

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