Neoadjuvant Percutaneous 4-Hydroxytamoxifen Decreases Breast Tumoral Cell Proliferation: A Prospective Controlled Randomized Study Comparing
http://www.100md.com
《临床肿瘤学》
the Centre Val d'Aurelle
U 450 INSERM, CHU, Montpellier
Centre rene Gauducheau, Nantes
Laboratoires Besins-International
Lab Pharmacologie, IFR Saint-Louis, Paris
ABSTRACT
PURPOSE: Two chemoprevention randomized studies using tamoxifen showed drug efficacy; however, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain a problem. 4 hydroxytamoxifen (4-OHT) is a very active metabolite of tamoxifen. This randomized study was designed to analyze if 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen.
PATIENTS AND METHODS: Fifty-five postmenopausal women with an invasive estrogen receptor–positive breast cancer were randomly assigned to receive (for 2 to 3 weeks) either 4-OHT gel (0.5, 1, or 2 mg/d) or oral tamoxifen (20 mg/d) or no treatment. Response was evaluated using proliferation markers (Ki-67, proliferating cell nuclear antigen) and apoptosis markers in tissue samples obtained by Tru-cut biopsy before treatment, and at surgery after treatment.
RESULTS: Administration of 4-OHT gel resulted in reductions in tumor tissue proliferation indexes (Ki-67 and PCNA), with approximate equivalence between the1.0 mg/d or 2.0 mg/d 4-OHT dose, and oral tamoxifen, but had no effect on apoptotic markers. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the gel groups. No dose-related pattern was shown for estrogen or progesterone receptor levels, and topical 4-OHT gel appeared to be generally well tolerated. Hot flushes are as common in the two higher gel doses as with tamoxifen.
CONCLUSION: Percutaneous 4-OHT gel has a local impact on tumor proliferation. It could be tested in future propective trials of chemoprevention or ductal carcinoma in situ adjuvant hormonotherapy.
INTRODUCTION
On the basis of the National Adjuvant Breast and Bowel Project (NSABP) –P1 prevention trial, in which tamoxifen resulted in a 49% reduction in the incidence of invasive breast cancer and in a 50% reduction of noninvasive cancer, the drug has been approved in the United States as a breast cancer–preventive treatment.1 However, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain an utmost problem. The ideal therapeutic approach might be to obtain the protective mammary effect of tamoxifen without affecting other target tissues.
4 hydroxytamoxifen (4-OHT) is the most active metabolite of tamoxifen, binding the estrogen receptors with higher affinity than its precursor,2 and is an effective antiproliferative agent on normal human breast cells in culture.3 In 1986, Mauvais-Jarvis et al4 obtained effective mammary concentration of 4-OHT by means of topical application over the breast, which yielded a local antiestrogen effect without the systemic effects observed with oral administration of tamoxifen. Pujol et al5 recently reported the results of a randomized study of percutaneous 4-OHT gel versus oral tamoxifen in women with breast cancer. They found that application of gel to the skin of the breast produced consistent tumor concentrations of 4-OHT, with much lower plasma levels. In the group receiving oral tamoxifen, its classic effects on coagulation and lipid metabolism were observed when pre- and post-treatment values of these biological variables were compared, whereas no differences in these parameters were observed in the patients randomized to 4-OHT gel.
This randomized study was designed to analyze whether 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen. Several doses were tested to determine the best ratio of efficacy/safety.
PATIENTS AND METHODS
Patients
Participants in this study were postmenopausal women who had attended the Montpellier or Nantes Cancer Institutes Outpatient Clinics from August 1998 to July 2000. Women who had a Tru-cut biopsy because of a suspected breast mass larger than 1 cm were screened for study participation. Criteria for enrollment included age older than 50 years, histologically confirmed T1 or T2 estrogen receptor–positive primary breast cancer, and being fit for surgery within 1 month of the biopsy. Prestudy assessments included medical history, concomitant therapy, demographics, current medical conditions, hematology, and biochemistry screening. Each woman gave written informed consent, and the study was approved by the Ethics Committees of both centers.
Patients with inflammatory cancer, known metastasis or lymph node involvement were excluded, as were patients with previous radiotherapy or chemotherapy and ongoing hormone replacement therapy (a minimum washout period of 8 days was observed before the Tru-cut biopsy). Other exclusion criteria were a history of thrombophlebitis requiring anticoagulant treatment, retinopathy, cutaneous allergy to alcohol, or mammary dermatitis contraindicating the application of a gel.
Patients were randomly assigned to one of five treatment groups: 0.5 mg/d 4-OHT (0.5 mL gel [0.25 mg 4-OHT] applied all over each breast daily); 1.0 mg/d 4-OHT (1 mL gel [0.5 mg 4-OHT] applied all over each breast daily); 2.0 mg/d 4-OHT (2 mL gel [1.0 mg 4-OHT] applied all over each breast daily); oral tamoxifen (20 mg/d as a single dose); or no treatment (untreated control). Patients were scheduled for tumor resection surgery with curative intent between day 15 and day 22 after the start of treatment. On the day before surgery, a blood sample was obtained for determination of 4-OHT concentrations. On the day of surgery, patients were reassessed for concomitant therapy, concomitant conditions, hematology, biochemistry, plasma 4-OHT, and estradiol (E2) concentrations.
Tumor Sampling
The Tru-cut/core biopsy taken at the first clinic attendance for diagnostic purposes was used as the pre–random assignment tumor sample. The post-treatment specimen was obtained at definitive surgical resection. All of the tissue samples were fixed in 3.7% formalin immediately after removal, then embedded in paraffin wax for sectioning and subsequent analysis of biologic markers. Tumor extracts (before and after treatment) were also stored in liquid nitrogen for subsequent assay of estrogen and progesterone receptors, as well as levels of 4-OHT.
Analysis of Tumor Marker Expression
Proliferation-associated antigen expression. Ki-676,7 and proliferating cell nuclear antigen (PCNA)8 labeling indices (LIs) were assessed on paraffin embedded sections of the pre- and post-treatment tissue specimens. The Ki-67 antigen was assessed using the monoclonal antibody MIB 1 (DAKO, Copenhagen, Denmark)9 as used in routine conditions in pathology. Anti-PCNA (DAKO) completed the panel of antibodies used to analyze proliferation in fixed tissues.10 Detection was made without heat denaturation.11 Six serial sections were analyzed per case to standardize sampling. Analysis was performed with a computer-assisted system (Système Microphotometrique à Balayage Automatique; Samba-Alcatel, Grenoble, France). For each preparation, optical density (OD) thresholds were determined using real microscopic images of the analyzed field as reference. Measurements of immunostaining were performed at x25. Twenty fields were analyzed for each section. Stained nuclear surface was determined (segmentation and thresholding), and an LI scored (stained cells/counterstained elements). OD immunostaining was expressed in arbitrary units. Control of immunostaining quantitative analysis and reproducibility of the procedure were carried out by comparison to iterative measurements done on the same preparations, and by comparison to measurements compiled on six sequential sections of the same specimen. Evaluation of tissue variations in immunostaining quantitative analysis was carried out by comparing the OD immunostaining measurements of 20 fields taken from nonconsecutive sections from each specimen. The intensity of PCNA immunoreactivity displayed by actively proliferating cells in lymph nodes (centroblasts) served as a reference for thresholds of positive staining in breast tissues. Results were expressed as an LI as previously described, in arbitrary units.
Extent of apoptosis. The terminal deoxynucleotidyl transferase–mediated deoxyuracil triphosphate nick-end labeling (TUNEL) technique was used for the detection and quantification of apoptosis in histological tissue sections, as reported by Nemani et al.12 The antiapoptotic protein BCL-2 was detected by a specific commercially available antibody (clone 124; DAKO), and cytoplasmic staining semiquantitated by three different observers. Results were expressed as the percentage of apoptotic cells among all tumor cells found (2 x 103 to 5 x 103; scanning whole sections in each slide).
Estrogen and progesterone receptor expression. Estrogen (ER) and progesterone (PgR) receptor concentrations were assayed in tumor tissue before and after treatment with either ligand binding assay using the Dextran-Coated Charcoal method13 and/or by immunohistochemical (ICH) method on paraffin-embedded sections. The cutoff for ER positivity was more than 10 fmol/mg measured using radioimmunoassay or more than 10% of tumoral cells labeled by an immunoenzymatic assay.14
4-OHT assay. Concentrations of 4-OHT in plasma, tumor tissue, and normal tissue were performed using gas chromatography (GC) combined with mass spectrometry (MS) used in the negative ion chemical ionization mode (NICI).15 The quantification limits of the method were 5 pg/mL for the plasma and 50 pg/g for tissue samples, respectively. Tumor extracts and normal breast tissue obtained at the time of surgery (after treatment) were stored in liquid nitrogen until subsequent assay of 4-OHT.
Adverse Events Monitoring
Safety parameters included concomitant medication use before and after treatment, adverse events reported during treatment, and change from before treatment to after treatment in clinical laboratory parameters. All treated patients were evaluated for safety.
Statistical Analysis
Based on the planned sample size of 14 patients in the no treatment (control) group and in the oral tamoxifen group and 42 patients (three groups) assigned to 4-OHT gel, this study had good power (90%) to detect a hypothesized 50% or greater relative decrease in Ki-67 labeling index in the 4-OHT group than in the no treatment group using a two-sided test with 5% type I error. The primary efficacy end point was change in tumor proliferation marker expression. Other study variables were considered secondary end points. This "per protocol" analysis included only those who received at least 13 days of treatment, completed the end of treatment assessment for the primary end point, and had no significant protocol deviations or violations.
Treatment group comparisons for continuous variables were made using the nonparametric Kruskal-Wallis test due to the small sample size. Categorical variables were analyzed using Fisher's exact test. Hypothesis testing was conducted at the = .05 significance level adjusted for multiple comparisons. Inferences fell into the following categories for the variables measured testing for differences among the five treatment groups, examining differences among the three dose levels of 4-OHT, and examining differences between each dose level of 4-OHT and oral tamoxifen.
RESULTS
Patient Characteristics
A total of 55 patients were enrolled in the trial, 49 in Montpellier and six in Nantes. One other center did not include their planned 15 patients, three in each group. Two patients were excluded from the safety analysis—one in the 4-OHT 1.0-mg/d group withdrew her consent, and one in the 4-OHT 0.5-mg/d group was lost to follow-up. Of the remaining 53 patients, four were excluded from the efficacy analysis population due to protocol violations as follows: one patient was ER–negative at the start of the study in the 4-OHT 2.0-mg/d group, one received hormone replacement therapy on study (4-OHT 0.5-mg/d group), and two discontinued their treatment (4-OHT 0.5- and 1.0-mg/d groups) after only 6 and 12 days, respectively. Therefore, a total of 49 patients were assessable for efficacy, and 53, for safety. Groups were well balanced with respect to tumor size, duration of amenorrhea, and tumor grade at surgery. Patients in the untreated control group were older than those in the active treatment groups (Table 1) .
Proliferation-Associated Antigen Expression
The change in tumor tissue Ki-67 LI levels after treatment from baseline differed significantly among the five groups (Table 2). Treated patients (4-OHT [all doses] and oral tamoxifen) had a significantly lower change in median Ki-67 LI scores as compared with the untreated group (P = .0055). We did not find a statistically significant dose-dependence difference between the three 4-OHT groups; however, a dose-response relationship with approximate equivalence between the 4-O HT 1.0-mg/d and 2.0-mg/d groups and the oral tamoxifen group is demonstrated in Table 3, which shows the percentage of patients who had decreases in Ki-67 LI of 1, 2, or 3 arbitrary units (%). The tumor tissue response to the various treatments measured by PCNA LI paralleled that assessed by the Ki-67 LI (Tables 2 and 3; Fig 1). After treatment, tumor tissue PCNA LI for the four treated groups differed significantly, from the untreated control (P = .002). As was seen with Ki-67 LIs, the percentage of patients who had decreases in PCNA index of 1, 2, or 3 units (%) demonstrated a strong treatment effect of 4-OHT, especially for the 4-OHT 1- and 2-mg/d groups relative to untreated controls, and showed approximate equivalence with oral tamoxifen (Table 3). There were no significant differences in PCNA labeling between tamoxifen and any dose of 4-OHT. Response defined as a tumor having a PCNA LI or Ki-67 LI decrease of 3 arbitrary units demonstrated a tendency to increase with 4-OHT dosage. However, sample size was not sufficient to demonstrate significance for the relationship. Previous hormone replacement therapy did not seem to affect results for tumor tissue Ki-67 LI or PCNA LI (data not shown).
Apoptosis Markers
Apoptosis as measured by percentage tumor tissue TUNEL and Bcl-2 did not show any effect of treatment or dose of 4-OHT (data not shown).
Hormone Receptors: ER Expression
Using radioimmunoassay (RIA), pretreatment ER concentrations were statistically similar across the groups, with the mean group values ranging from 5 to 56 fmol/mg. Individual values demonstrated a wide range (9 to 321 fmol/mg). Treatment of ER-positive tumors with tamoxifen or 4-OHT resulted in a significant decrease (P = .012) in ER concentration relative to the untreated control group (Table 2). Using ICH, ER concentration (% of labeled cells) demonstrated the same median value at baseline (70% to 85%) across groups and showed no statistically significant increasing trend after treatment in all groups.
PgR Expression
RIA measurements of PgR were widespread at baseline and increased after treatment in all groups, with no statistical significance or dose-related-pattern. PgR results with ICH measurements did not indicate any consistent effect of treatment or dose-related changes.
4-OHT Tissue and Plasma Concentrations
Tumor 4-OHT median concentration was roughly two-fold higher in the oral tamoxifen group (4,237 pg/g) compared with that in the 2.0 mg/d 4-OHT group (1,698 pg/g). Despite an increase of median tissue 4-OHT concentrations (pg/g) according to the percutaneous dose delivered (687, 1,377, and 1,698 pg/g for 0.5, 1, and 2 mg/d, respectively), the difference between the three groups was not statistically significant (P = .13). Nontumoral tissue 4-OHT concentration was about half those seen in tumor tissue in all except the 0.5 mg/d group. For 4-OHT median plasma concentration, there was a significant difference between the four treatments groups (P = .0015), with a higher level for the oral tamoxifen group versus the 4-OHT gel groups (1,495 pg/mL v 31, 35, and 164 pg/mL respectively). Furthermore, there was a significant (P = .035) increase of plasma 4-OHT with ascending 4-OHT percutaneous dose (Table 4, Fig 2).
Tolerability
No serious adverse events (AEs) were reported during this study. AEs were reported for three patients in the oral tamoxifen group (27%); no patients in the untreated group; five patients in the 1.0-mg/d 4-OHT group, including one unrelated to treatment (50%); and five patients in the 2.0-mg/d 4-OHT group, including one unrelated to treatment (45%). No AEs were reported for patients in the 0.5-mg/d 4-OHT group. The most common AE was vasodilatation (hot flushes), reported for two patients (18%) in the oral tamoxifen group, three patients (30%) in the 1.0-mg/d group, and two patients (18%) in the 2.0-mg/d group. AEs associated with topical application of the gel (ie, rash or pruritus of the breast) were reported for two patients in the 2.0 mg/d group. Three severe events were reported in two patients. One patient in the oral tamoxifen group reported "abdominal pains" and "hot flushes," both of which resolved. Another patient in the 1.0-mg/d 4-OHT group reported nighttime hot flushes and headaches; both resolved spontaneously. These two patients prematurely discontinued treatment.
DISCUSSION
This study provides the first direct comparison of 4-OHT gel with tamoxifen and untreated controls on the biologic end points of breast tumor Ki-67 labeling, ER and PgR expression in primary breast cancer. The results show that percutaneous administration of 4-OHT gel for 2 to 3 weeks produced reductions in tumor tissue proliferation indexes (Ki-67 and PCNA labeling) in postmenopausal women scheduled to undergo surgery for breast cancer, with approximate equivalence between the 1.0- and 2.0-mg/d 4-OHT doses and 20 mg/d oral tamoxifen. ER and PgR levels did not show dose-related reductions with 4-OHT gel treatment. The antiproliferative effect of 4-OHT was not associated with significant proapoptotic activity. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the 4-OHT gel groups, and tumor tissue concentrations of 4-OHT increased with increasing dose of 4-OHT gel.
Data from the present study also provide information on the biologic processes underlying the effects of tamoxifen. The group receiving 2 mg of percutaneous 4-OHT showed the same cytostatic effect on breast cancer cell proliferation as the oral tamoxifen group, despite markedly lower plasma 4-OHT concentrations. This finding suggests that a major effect of tamoxifen is locally mediated by ERs in breast tumor tissue. In contrast, several investigators have suggested a systemic endocrine role for tamoxifen acting to decrease plasma IGF-1.
In the present study, neither 4-OHT nor oral tamoxifen had a discernable effect on apoptosis. These observations are consistent with the findings of Robertson et al,16 who reported an absence of mammary tumor apoptotic index increase with fulvestrant or tamoxifen treatment in postmenopausal women. A plausible explanation for the lack of an increase in apoptosis is that 4-OHT tumor concentrations subsequent to oral tamoxifen or topical 4-OHT treatments were largely below the levels required for the two described pathways of programmed cell death.17,18 The duration of treatment in this study as well as in that of Robertson et al16 was probably too short to achieve a stable tamoxifen or 4-OHT tissue concentration since more than 4 weeks would be necessary to obtain stable plasma tamoxifen concentration during oral tamoxifen treatment. Nevertheless, sufficient 4-OHT was able to reach the tumor to produce significant biologic effects on Ki-67 LI and PCNA LI. 4-OHT gel can be an efficient method to control mammary cell proliferation, with no systemic effect.
Both oral tamoxifen and percutaneous 4-OHT were well tolerated, but our sample size is insufficient for drawing definitive conclusions. Untreated controls and the 4-OHT 0.5-mg/d group had no adverse effects. Patients receiving 4-OHT 1 mg and 2 mg/d reported hot flushes, suggesting that even a low plasma level of 4-OHT might be enough to produce a systemic effect. However, these women were postmenopausal, and hot flushes are clinically similar, whether driven by physiologic estrogenic deprivation or due to tamoxifen effect.
The ultimate clinical goal of chemoprevention studies is to reduce cancer incidence and the recent clinical data of breast carcinoma chemoprevention trials and hormonal therapy of ductal carcinoma in situ emphasize interest for a local hormonal therapy, especially for premenopausal women. Two of four chemopreventive trials using tamoxifen showed a significantly decreased incidence.1,19-21 NSABP-P1 showed a highly statistically significant reduction in breast cancer incidence both for invasive (49%) or noninvasive (50%) breast cancer in women treated with tamoxifen.1 This decreased risk occurred in women of all ages and in all risk groups, but only for ER-positive breast cancer (69%). Side effects of tamoxifen included hot flushes (81%), vaginal discharge (29%), increased risk of endometrial cancer (relative risk [RR], 2.5 which increased to 4.01 in women 50 years or older), deep vein thrombosis (RR, 1.71), and pulmonary emboli (RR, 3), especially for women 50 years or older. The International Breast Cancer Intervention Study (IBIS) I trial showed in the same way a reduction of 32% in the odds of invasive breast cancer and ductal carcinoma in situ in the tamoxifen group.21 Adverse effects also included an increased rate of thromboembolic events (RR, 3), endometrial cancer (RR, 2.5), and hot flushes. Furthermore, the IBIS I study showed a significant increase in the total number of deaths in the tamoxifen group. NSABP-B24 is one of the largest trials that randomized tamoxifen versus control for ductal carcinoma in situ treated by lumpectomy and radiation therapy.22 With a median follow-up of 74 months, women in the tamoxifen group had fewer breast cancer events than did those on placebo (8.2% v 13.4%; P = .0009), a lower rate of ipsilateral-breast tumors (44% reduction) with a relative risk of controlateral breast tumors of 0.48. Tamoxifen administration decreased by 38% ipsilateral-breast tumors in women younger than 50 years and by 22% after 50 years, brought back the local recurrence rate of positive margin to those of negative margins without tamoxifen (17%), and lowered the recurrence rate of women who had no comedonecrosis (23%). But there was also an excess of endometrial cancer (1.5 per 1,000 v 0.45; P = .02) and thromboembolic events in the treated group compared with controls.
A theoretical optimal strategy would be to administer tamoxifen to patients with an increased risk of breast cancer in chemoprevention or for ductal carcinoma in situ adjuvant hormonotherapy. In clinical practice, the systemic side effects of oral/systemic tamoxifen largely preclude this therapeutic strategy. 4-OHT administered topically, if effective and demonstrating an acceptable safety profile, might be a potential agent for such studies, even in younger premenopausal women.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Served as an officer or member of the Board of a company: Elisabeth Le Nestour, Besins International.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Fisher B, Costantino JP, Wickerham DL, et al: Tamoxifen for prevention of breast cancer: Report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 90:1371-1388, 1998
Jordan VC, Collins MM, Rowsny L, et al: A monohydroxylated metabolite of tamoxifen with potent antioestrogenic activity. J Endocrinol 75:305-316, 1977
Malet C, Gompel A, Spritzer P, et al: Tamoxifen and hydroxytamoxifen isomers versus estradiol effects on normal human breast cells in culture. Cancer Res 48:7193-7199, 1988
Mauvais-Jarvis P, Baudot N, Castaigne D, et al: Trans-4-Hydroxytamoxifen concentration and metabolism after local percutaneous administration to human breast. Cancer Res 46:1521-1525, 1986
Pujol H, Girault J, Rouanet P, et al: Phase I study of percutaneous 4-hydroxy-tamoxifen with analyses of 4-hydroxy-tamoxifen concentrations in breast cancer and normal breast tissue. Cancer Chemother Pharmacol 36:493-498, 1995
Gerdes J, Lemke H, Baisch, et al: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715, 1984
Schlüter C, Duchrow M, Wohlensberg C, et al: The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements representing a new kind of cell maintaining proteins. Cell Biol 123:513-522, 1993
Waseem NH, Lane DP: Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA): Structural conservation and the detection of the nucleolar form. J Cell Sci 96:121-129, 1990
Cattoretti G, Becker MHG, Key G, et al: Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 168:357-363, 1992
Chang K, Lee T, Linares-Cruz G, et al: Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril 63:785-791, 1995
Linares-Cruz G, Bruzzoni-Giovanelli H, Alvaro V, et al: p21WAF-1 reorganizes the nucleus in tumor suppression. Proc Natl Acad Sci U S A 95:1131-1135, 1998
Nemani M, Linares-Cruz G, Bruzzoni-Giovanelli H, et al: Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression . Proc Natl Acad Sci U S A 93:9039-9042, 1996
Korenman SG, Stevens RH, Carpenter LA, et al: Estradiol radioimmunoassay without chromatography: Procedure, validation and normal values. J Clin Endocrinol Metab 38:718-720, 1974
Bevitt D, Milton ID, Piggot N, et al: New monoclonal antibodies to estrogen and progesterone receptors effective for paraffine section immunohistochemistry. J Pathol 183:228-232, 1997
Girault J, Istin B, Fourtillan JB: Quantitative measurement of 4-hydroxytamoxifen in human plasma and mammary tumours by combined gas chromatography/negative chemical ionization mass spectrometry. Biol Mass Spectrom 22:395-402, 1993
Robertson JF, Nicholson RI, Bundred NJ, et al: Comparaison of the short-term biological effects of 7-[9-(4,4,5,5,5-pentafluoropentysulfinyl)-nonyl]estra-1,3,5(10)-triene-3,17-diol (Faslodex) versus Tamoxifen in postmenopausal women with primary breast cancer. Cancer Res 61:6739-6746, 2001
Mandlekar S, Yu R, Tan TH, et al: Activation of caspase-3 and c-jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 60:5995-6000, 2000
Obrero M, Yu DV, Shapiro DJ: Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem 277:45695-45703, 2002
Powles T, Eeles R, Ashley S, et al: Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 352:98-101, 1998
Veronesi U, Maisonneuve P, Sacchini V, et al: Italian Tamoxifen Study Group: Tamoxifen for breast cancer among hysterectomised women. Lancet 359:1122-1124, 2002
IBIS investigators. First results from the international breast cancer intervention study (IBIS-I): A randomised prevention trial. Lancet 360:817-824, 2002
Fisher B, Dignam J, Wolmark N, et al: Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomised controlled trial. Lancet 353:1993-2000, 1999(Philippe Rouanet, Gustavo)
U 450 INSERM, CHU, Montpellier
Centre rene Gauducheau, Nantes
Laboratoires Besins-International
Lab Pharmacologie, IFR Saint-Louis, Paris
ABSTRACT
PURPOSE: Two chemoprevention randomized studies using tamoxifen showed drug efficacy; however, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain a problem. 4 hydroxytamoxifen (4-OHT) is a very active metabolite of tamoxifen. This randomized study was designed to analyze if 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen.
PATIENTS AND METHODS: Fifty-five postmenopausal women with an invasive estrogen receptor–positive breast cancer were randomly assigned to receive (for 2 to 3 weeks) either 4-OHT gel (0.5, 1, or 2 mg/d) or oral tamoxifen (20 mg/d) or no treatment. Response was evaluated using proliferation markers (Ki-67, proliferating cell nuclear antigen) and apoptosis markers in tissue samples obtained by Tru-cut biopsy before treatment, and at surgery after treatment.
RESULTS: Administration of 4-OHT gel resulted in reductions in tumor tissue proliferation indexes (Ki-67 and PCNA), with approximate equivalence between the1.0 mg/d or 2.0 mg/d 4-OHT dose, and oral tamoxifen, but had no effect on apoptotic markers. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the gel groups. No dose-related pattern was shown for estrogen or progesterone receptor levels, and topical 4-OHT gel appeared to be generally well tolerated. Hot flushes are as common in the two higher gel doses as with tamoxifen.
CONCLUSION: Percutaneous 4-OHT gel has a local impact on tumor proliferation. It could be tested in future propective trials of chemoprevention or ductal carcinoma in situ adjuvant hormonotherapy.
INTRODUCTION
On the basis of the National Adjuvant Breast and Bowel Project (NSABP) –P1 prevention trial, in which tamoxifen resulted in a 49% reduction in the incidence of invasive breast cancer and in a 50% reduction of noninvasive cancer, the drug has been approved in the United States as a breast cancer–preventive treatment.1 However, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain an utmost problem. The ideal therapeutic approach might be to obtain the protective mammary effect of tamoxifen without affecting other target tissues.
4 hydroxytamoxifen (4-OHT) is the most active metabolite of tamoxifen, binding the estrogen receptors with higher affinity than its precursor,2 and is an effective antiproliferative agent on normal human breast cells in culture.3 In 1986, Mauvais-Jarvis et al4 obtained effective mammary concentration of 4-OHT by means of topical application over the breast, which yielded a local antiestrogen effect without the systemic effects observed with oral administration of tamoxifen. Pujol et al5 recently reported the results of a randomized study of percutaneous 4-OHT gel versus oral tamoxifen in women with breast cancer. They found that application of gel to the skin of the breast produced consistent tumor concentrations of 4-OHT, with much lower plasma levels. In the group receiving oral tamoxifen, its classic effects on coagulation and lipid metabolism were observed when pre- and post-treatment values of these biological variables were compared, whereas no differences in these parameters were observed in the patients randomized to 4-OHT gel.
This randomized study was designed to analyze whether 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen. Several doses were tested to determine the best ratio of efficacy/safety.
PATIENTS AND METHODS
Patients
Participants in this study were postmenopausal women who had attended the Montpellier or Nantes Cancer Institutes Outpatient Clinics from August 1998 to July 2000. Women who had a Tru-cut biopsy because of a suspected breast mass larger than 1 cm were screened for study participation. Criteria for enrollment included age older than 50 years, histologically confirmed T1 or T2 estrogen receptor–positive primary breast cancer, and being fit for surgery within 1 month of the biopsy. Prestudy assessments included medical history, concomitant therapy, demographics, current medical conditions, hematology, and biochemistry screening. Each woman gave written informed consent, and the study was approved by the Ethics Committees of both centers.
Patients with inflammatory cancer, known metastasis or lymph node involvement were excluded, as were patients with previous radiotherapy or chemotherapy and ongoing hormone replacement therapy (a minimum washout period of 8 days was observed before the Tru-cut biopsy). Other exclusion criteria were a history of thrombophlebitis requiring anticoagulant treatment, retinopathy, cutaneous allergy to alcohol, or mammary dermatitis contraindicating the application of a gel.
Patients were randomly assigned to one of five treatment groups: 0.5 mg/d 4-OHT (0.5 mL gel [0.25 mg 4-OHT] applied all over each breast daily); 1.0 mg/d 4-OHT (1 mL gel [0.5 mg 4-OHT] applied all over each breast daily); 2.0 mg/d 4-OHT (2 mL gel [1.0 mg 4-OHT] applied all over each breast daily); oral tamoxifen (20 mg/d as a single dose); or no treatment (untreated control). Patients were scheduled for tumor resection surgery with curative intent between day 15 and day 22 after the start of treatment. On the day before surgery, a blood sample was obtained for determination of 4-OHT concentrations. On the day of surgery, patients were reassessed for concomitant therapy, concomitant conditions, hematology, biochemistry, plasma 4-OHT, and estradiol (E2) concentrations.
Tumor Sampling
The Tru-cut/core biopsy taken at the first clinic attendance for diagnostic purposes was used as the pre–random assignment tumor sample. The post-treatment specimen was obtained at definitive surgical resection. All of the tissue samples were fixed in 3.7% formalin immediately after removal, then embedded in paraffin wax for sectioning and subsequent analysis of biologic markers. Tumor extracts (before and after treatment) were also stored in liquid nitrogen for subsequent assay of estrogen and progesterone receptors, as well as levels of 4-OHT.
Analysis of Tumor Marker Expression
Proliferation-associated antigen expression. Ki-676,7 and proliferating cell nuclear antigen (PCNA)8 labeling indices (LIs) were assessed on paraffin embedded sections of the pre- and post-treatment tissue specimens. The Ki-67 antigen was assessed using the monoclonal antibody MIB 1 (DAKO, Copenhagen, Denmark)9 as used in routine conditions in pathology. Anti-PCNA (DAKO) completed the panel of antibodies used to analyze proliferation in fixed tissues.10 Detection was made without heat denaturation.11 Six serial sections were analyzed per case to standardize sampling. Analysis was performed with a computer-assisted system (Système Microphotometrique à Balayage Automatique; Samba-Alcatel, Grenoble, France). For each preparation, optical density (OD) thresholds were determined using real microscopic images of the analyzed field as reference. Measurements of immunostaining were performed at x25. Twenty fields were analyzed for each section. Stained nuclear surface was determined (segmentation and thresholding), and an LI scored (stained cells/counterstained elements). OD immunostaining was expressed in arbitrary units. Control of immunostaining quantitative analysis and reproducibility of the procedure were carried out by comparison to iterative measurements done on the same preparations, and by comparison to measurements compiled on six sequential sections of the same specimen. Evaluation of tissue variations in immunostaining quantitative analysis was carried out by comparing the OD immunostaining measurements of 20 fields taken from nonconsecutive sections from each specimen. The intensity of PCNA immunoreactivity displayed by actively proliferating cells in lymph nodes (centroblasts) served as a reference for thresholds of positive staining in breast tissues. Results were expressed as an LI as previously described, in arbitrary units.
Extent of apoptosis. The terminal deoxynucleotidyl transferase–mediated deoxyuracil triphosphate nick-end labeling (TUNEL) technique was used for the detection and quantification of apoptosis in histological tissue sections, as reported by Nemani et al.12 The antiapoptotic protein BCL-2 was detected by a specific commercially available antibody (clone 124; DAKO), and cytoplasmic staining semiquantitated by three different observers. Results were expressed as the percentage of apoptotic cells among all tumor cells found (2 x 103 to 5 x 103; scanning whole sections in each slide).
Estrogen and progesterone receptor expression. Estrogen (ER) and progesterone (PgR) receptor concentrations were assayed in tumor tissue before and after treatment with either ligand binding assay using the Dextran-Coated Charcoal method13 and/or by immunohistochemical (ICH) method on paraffin-embedded sections. The cutoff for ER positivity was more than 10 fmol/mg measured using radioimmunoassay or more than 10% of tumoral cells labeled by an immunoenzymatic assay.14
4-OHT assay. Concentrations of 4-OHT in plasma, tumor tissue, and normal tissue were performed using gas chromatography (GC) combined with mass spectrometry (MS) used in the negative ion chemical ionization mode (NICI).15 The quantification limits of the method were 5 pg/mL for the plasma and 50 pg/g for tissue samples, respectively. Tumor extracts and normal breast tissue obtained at the time of surgery (after treatment) were stored in liquid nitrogen until subsequent assay of 4-OHT.
Adverse Events Monitoring
Safety parameters included concomitant medication use before and after treatment, adverse events reported during treatment, and change from before treatment to after treatment in clinical laboratory parameters. All treated patients were evaluated for safety.
Statistical Analysis
Based on the planned sample size of 14 patients in the no treatment (control) group and in the oral tamoxifen group and 42 patients (three groups) assigned to 4-OHT gel, this study had good power (90%) to detect a hypothesized 50% or greater relative decrease in Ki-67 labeling index in the 4-OHT group than in the no treatment group using a two-sided test with 5% type I error. The primary efficacy end point was change in tumor proliferation marker expression. Other study variables were considered secondary end points. This "per protocol" analysis included only those who received at least 13 days of treatment, completed the end of treatment assessment for the primary end point, and had no significant protocol deviations or violations.
Treatment group comparisons for continuous variables were made using the nonparametric Kruskal-Wallis test due to the small sample size. Categorical variables were analyzed using Fisher's exact test. Hypothesis testing was conducted at the = .05 significance level adjusted for multiple comparisons. Inferences fell into the following categories for the variables measured testing for differences among the five treatment groups, examining differences among the three dose levels of 4-OHT, and examining differences between each dose level of 4-OHT and oral tamoxifen.
RESULTS
Patient Characteristics
A total of 55 patients were enrolled in the trial, 49 in Montpellier and six in Nantes. One other center did not include their planned 15 patients, three in each group. Two patients were excluded from the safety analysis—one in the 4-OHT 1.0-mg/d group withdrew her consent, and one in the 4-OHT 0.5-mg/d group was lost to follow-up. Of the remaining 53 patients, four were excluded from the efficacy analysis population due to protocol violations as follows: one patient was ER–negative at the start of the study in the 4-OHT 2.0-mg/d group, one received hormone replacement therapy on study (4-OHT 0.5-mg/d group), and two discontinued their treatment (4-OHT 0.5- and 1.0-mg/d groups) after only 6 and 12 days, respectively. Therefore, a total of 49 patients were assessable for efficacy, and 53, for safety. Groups were well balanced with respect to tumor size, duration of amenorrhea, and tumor grade at surgery. Patients in the untreated control group were older than those in the active treatment groups (Table 1) .
Proliferation-Associated Antigen Expression
The change in tumor tissue Ki-67 LI levels after treatment from baseline differed significantly among the five groups (Table 2). Treated patients (4-OHT [all doses] and oral tamoxifen) had a significantly lower change in median Ki-67 LI scores as compared with the untreated group (P = .0055). We did not find a statistically significant dose-dependence difference between the three 4-OHT groups; however, a dose-response relationship with approximate equivalence between the 4-O HT 1.0-mg/d and 2.0-mg/d groups and the oral tamoxifen group is demonstrated in Table 3, which shows the percentage of patients who had decreases in Ki-67 LI of 1, 2, or 3 arbitrary units (%). The tumor tissue response to the various treatments measured by PCNA LI paralleled that assessed by the Ki-67 LI (Tables 2 and 3; Fig 1). After treatment, tumor tissue PCNA LI for the four treated groups differed significantly, from the untreated control (P = .002). As was seen with Ki-67 LIs, the percentage of patients who had decreases in PCNA index of 1, 2, or 3 units (%) demonstrated a strong treatment effect of 4-OHT, especially for the 4-OHT 1- and 2-mg/d groups relative to untreated controls, and showed approximate equivalence with oral tamoxifen (Table 3). There were no significant differences in PCNA labeling between tamoxifen and any dose of 4-OHT. Response defined as a tumor having a PCNA LI or Ki-67 LI decrease of 3 arbitrary units demonstrated a tendency to increase with 4-OHT dosage. However, sample size was not sufficient to demonstrate significance for the relationship. Previous hormone replacement therapy did not seem to affect results for tumor tissue Ki-67 LI or PCNA LI (data not shown).
Apoptosis Markers
Apoptosis as measured by percentage tumor tissue TUNEL and Bcl-2 did not show any effect of treatment or dose of 4-OHT (data not shown).
Hormone Receptors: ER Expression
Using radioimmunoassay (RIA), pretreatment ER concentrations were statistically similar across the groups, with the mean group values ranging from 5 to 56 fmol/mg. Individual values demonstrated a wide range (9 to 321 fmol/mg). Treatment of ER-positive tumors with tamoxifen or 4-OHT resulted in a significant decrease (P = .012) in ER concentration relative to the untreated control group (Table 2). Using ICH, ER concentration (% of labeled cells) demonstrated the same median value at baseline (70% to 85%) across groups and showed no statistically significant increasing trend after treatment in all groups.
PgR Expression
RIA measurements of PgR were widespread at baseline and increased after treatment in all groups, with no statistical significance or dose-related-pattern. PgR results with ICH measurements did not indicate any consistent effect of treatment or dose-related changes.
4-OHT Tissue and Plasma Concentrations
Tumor 4-OHT median concentration was roughly two-fold higher in the oral tamoxifen group (4,237 pg/g) compared with that in the 2.0 mg/d 4-OHT group (1,698 pg/g). Despite an increase of median tissue 4-OHT concentrations (pg/g) according to the percutaneous dose delivered (687, 1,377, and 1,698 pg/g for 0.5, 1, and 2 mg/d, respectively), the difference between the three groups was not statistically significant (P = .13). Nontumoral tissue 4-OHT concentration was about half those seen in tumor tissue in all except the 0.5 mg/d group. For 4-OHT median plasma concentration, there was a significant difference between the four treatments groups (P = .0015), with a higher level for the oral tamoxifen group versus the 4-OHT gel groups (1,495 pg/mL v 31, 35, and 164 pg/mL respectively). Furthermore, there was a significant (P = .035) increase of plasma 4-OHT with ascending 4-OHT percutaneous dose (Table 4, Fig 2).
Tolerability
No serious adverse events (AEs) were reported during this study. AEs were reported for three patients in the oral tamoxifen group (27%); no patients in the untreated group; five patients in the 1.0-mg/d 4-OHT group, including one unrelated to treatment (50%); and five patients in the 2.0-mg/d 4-OHT group, including one unrelated to treatment (45%). No AEs were reported for patients in the 0.5-mg/d 4-OHT group. The most common AE was vasodilatation (hot flushes), reported for two patients (18%) in the oral tamoxifen group, three patients (30%) in the 1.0-mg/d group, and two patients (18%) in the 2.0-mg/d group. AEs associated with topical application of the gel (ie, rash or pruritus of the breast) were reported for two patients in the 2.0 mg/d group. Three severe events were reported in two patients. One patient in the oral tamoxifen group reported "abdominal pains" and "hot flushes," both of which resolved. Another patient in the 1.0-mg/d 4-OHT group reported nighttime hot flushes and headaches; both resolved spontaneously. These two patients prematurely discontinued treatment.
DISCUSSION
This study provides the first direct comparison of 4-OHT gel with tamoxifen and untreated controls on the biologic end points of breast tumor Ki-67 labeling, ER and PgR expression in primary breast cancer. The results show that percutaneous administration of 4-OHT gel for 2 to 3 weeks produced reductions in tumor tissue proliferation indexes (Ki-67 and PCNA labeling) in postmenopausal women scheduled to undergo surgery for breast cancer, with approximate equivalence between the 1.0- and 2.0-mg/d 4-OHT doses and 20 mg/d oral tamoxifen. ER and PgR levels did not show dose-related reductions with 4-OHT gel treatment. The antiproliferative effect of 4-OHT was not associated with significant proapoptotic activity. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the 4-OHT gel groups, and tumor tissue concentrations of 4-OHT increased with increasing dose of 4-OHT gel.
Data from the present study also provide information on the biologic processes underlying the effects of tamoxifen. The group receiving 2 mg of percutaneous 4-OHT showed the same cytostatic effect on breast cancer cell proliferation as the oral tamoxifen group, despite markedly lower plasma 4-OHT concentrations. This finding suggests that a major effect of tamoxifen is locally mediated by ERs in breast tumor tissue. In contrast, several investigators have suggested a systemic endocrine role for tamoxifen acting to decrease plasma IGF-1.
In the present study, neither 4-OHT nor oral tamoxifen had a discernable effect on apoptosis. These observations are consistent with the findings of Robertson et al,16 who reported an absence of mammary tumor apoptotic index increase with fulvestrant or tamoxifen treatment in postmenopausal women. A plausible explanation for the lack of an increase in apoptosis is that 4-OHT tumor concentrations subsequent to oral tamoxifen or topical 4-OHT treatments were largely below the levels required for the two described pathways of programmed cell death.17,18 The duration of treatment in this study as well as in that of Robertson et al16 was probably too short to achieve a stable tamoxifen or 4-OHT tissue concentration since more than 4 weeks would be necessary to obtain stable plasma tamoxifen concentration during oral tamoxifen treatment. Nevertheless, sufficient 4-OHT was able to reach the tumor to produce significant biologic effects on Ki-67 LI and PCNA LI. 4-OHT gel can be an efficient method to control mammary cell proliferation, with no systemic effect.
Both oral tamoxifen and percutaneous 4-OHT were well tolerated, but our sample size is insufficient for drawing definitive conclusions. Untreated controls and the 4-OHT 0.5-mg/d group had no adverse effects. Patients receiving 4-OHT 1 mg and 2 mg/d reported hot flushes, suggesting that even a low plasma level of 4-OHT might be enough to produce a systemic effect. However, these women were postmenopausal, and hot flushes are clinically similar, whether driven by physiologic estrogenic deprivation or due to tamoxifen effect.
The ultimate clinical goal of chemoprevention studies is to reduce cancer incidence and the recent clinical data of breast carcinoma chemoprevention trials and hormonal therapy of ductal carcinoma in situ emphasize interest for a local hormonal therapy, especially for premenopausal women. Two of four chemopreventive trials using tamoxifen showed a significantly decreased incidence.1,19-21 NSABP-P1 showed a highly statistically significant reduction in breast cancer incidence both for invasive (49%) or noninvasive (50%) breast cancer in women treated with tamoxifen.1 This decreased risk occurred in women of all ages and in all risk groups, but only for ER-positive breast cancer (69%). Side effects of tamoxifen included hot flushes (81%), vaginal discharge (29%), increased risk of endometrial cancer (relative risk [RR], 2.5 which increased to 4.01 in women 50 years or older), deep vein thrombosis (RR, 1.71), and pulmonary emboli (RR, 3), especially for women 50 years or older. The International Breast Cancer Intervention Study (IBIS) I trial showed in the same way a reduction of 32% in the odds of invasive breast cancer and ductal carcinoma in situ in the tamoxifen group.21 Adverse effects also included an increased rate of thromboembolic events (RR, 3), endometrial cancer (RR, 2.5), and hot flushes. Furthermore, the IBIS I study showed a significant increase in the total number of deaths in the tamoxifen group. NSABP-B24 is one of the largest trials that randomized tamoxifen versus control for ductal carcinoma in situ treated by lumpectomy and radiation therapy.22 With a median follow-up of 74 months, women in the tamoxifen group had fewer breast cancer events than did those on placebo (8.2% v 13.4%; P = .0009), a lower rate of ipsilateral-breast tumors (44% reduction) with a relative risk of controlateral breast tumors of 0.48. Tamoxifen administration decreased by 38% ipsilateral-breast tumors in women younger than 50 years and by 22% after 50 years, brought back the local recurrence rate of positive margin to those of negative margins without tamoxifen (17%), and lowered the recurrence rate of women who had no comedonecrosis (23%). But there was also an excess of endometrial cancer (1.5 per 1,000 v 0.45; P = .02) and thromboembolic events in the treated group compared with controls.
A theoretical optimal strategy would be to administer tamoxifen to patients with an increased risk of breast cancer in chemoprevention or for ductal carcinoma in situ adjuvant hormonotherapy. In clinical practice, the systemic side effects of oral/systemic tamoxifen largely preclude this therapeutic strategy. 4-OHT administered topically, if effective and demonstrating an acceptable safety profile, might be a potential agent for such studies, even in younger premenopausal women.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Served as an officer or member of the Board of a company: Elisabeth Le Nestour, Besins International.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Fisher B, Costantino JP, Wickerham DL, et al: Tamoxifen for prevention of breast cancer: Report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 90:1371-1388, 1998
Jordan VC, Collins MM, Rowsny L, et al: A monohydroxylated metabolite of tamoxifen with potent antioestrogenic activity. J Endocrinol 75:305-316, 1977
Malet C, Gompel A, Spritzer P, et al: Tamoxifen and hydroxytamoxifen isomers versus estradiol effects on normal human breast cells in culture. Cancer Res 48:7193-7199, 1988
Mauvais-Jarvis P, Baudot N, Castaigne D, et al: Trans-4-Hydroxytamoxifen concentration and metabolism after local percutaneous administration to human breast. Cancer Res 46:1521-1525, 1986
Pujol H, Girault J, Rouanet P, et al: Phase I study of percutaneous 4-hydroxy-tamoxifen with analyses of 4-hydroxy-tamoxifen concentrations in breast cancer and normal breast tissue. Cancer Chemother Pharmacol 36:493-498, 1995
Gerdes J, Lemke H, Baisch, et al: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715, 1984
Schlüter C, Duchrow M, Wohlensberg C, et al: The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements representing a new kind of cell maintaining proteins. Cell Biol 123:513-522, 1993
Waseem NH, Lane DP: Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA): Structural conservation and the detection of the nucleolar form. J Cell Sci 96:121-129, 1990
Cattoretti G, Becker MHG, Key G, et al: Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 168:357-363, 1992
Chang K, Lee T, Linares-Cruz G, et al: Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril 63:785-791, 1995
Linares-Cruz G, Bruzzoni-Giovanelli H, Alvaro V, et al: p21WAF-1 reorganizes the nucleus in tumor suppression. Proc Natl Acad Sci U S A 95:1131-1135, 1998
Nemani M, Linares-Cruz G, Bruzzoni-Giovanelli H, et al: Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression . Proc Natl Acad Sci U S A 93:9039-9042, 1996
Korenman SG, Stevens RH, Carpenter LA, et al: Estradiol radioimmunoassay without chromatography: Procedure, validation and normal values. J Clin Endocrinol Metab 38:718-720, 1974
Bevitt D, Milton ID, Piggot N, et al: New monoclonal antibodies to estrogen and progesterone receptors effective for paraffine section immunohistochemistry. J Pathol 183:228-232, 1997
Girault J, Istin B, Fourtillan JB: Quantitative measurement of 4-hydroxytamoxifen in human plasma and mammary tumours by combined gas chromatography/negative chemical ionization mass spectrometry. Biol Mass Spectrom 22:395-402, 1993
Robertson JF, Nicholson RI, Bundred NJ, et al: Comparaison of the short-term biological effects of 7-[9-(4,4,5,5,5-pentafluoropentysulfinyl)-nonyl]estra-1,3,5(10)-triene-3,17-diol (Faslodex) versus Tamoxifen in postmenopausal women with primary breast cancer. Cancer Res 61:6739-6746, 2001
Mandlekar S, Yu R, Tan TH, et al: Activation of caspase-3 and c-jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 60:5995-6000, 2000
Obrero M, Yu DV, Shapiro DJ: Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem 277:45695-45703, 2002
Powles T, Eeles R, Ashley S, et al: Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 352:98-101, 1998
Veronesi U, Maisonneuve P, Sacchini V, et al: Italian Tamoxifen Study Group: Tamoxifen for breast cancer among hysterectomised women. Lancet 359:1122-1124, 2002
IBIS investigators. First results from the international breast cancer intervention study (IBIS-I): A randomised prevention trial. Lancet 360:817-824, 2002
Fisher B, Dignam J, Wolmark N, et al: Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomised controlled trial. Lancet 353:1993-2000, 1999(Philippe Rouanet, Gustavo)