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Breast Cancer Chemoprevention Phase I Evaluation of Biomarker Modulation by Arzoxifene, a Third Generation Selective Estrogen Receptor Modulator

¹ University of Kansas Medical Center, Kansas City, Kansas; ² Ohio State University, Columbus, Ohio; ³ US Oncology, Inc., Dallas, Texas; ⁴ Loyola University Medical Center, Maywood, Illinois; ⁵ Desert Comprehensive Cancer Center, Palm Springs, California; ⁶ Cleveland Clinic Foundation, Cleveland, Ohio; ⁷ University of California Los Angeles, Los Angeles, California; ⁸ University of Missouri-Columbia, Columbia, Missouri; ⁹ University of Alabama-Birmingham, Birmingham, Alabama; ¹⁰ Oncotech, Inc., Irvine, California; ¹¹ Midwest Research Institute, Kansas City, Missouri; ¹² Bacus Laboratories, Inc., Elmhurst, Illinois; ¹³ St. Mary’s Hospital, San Francisco, California; and ¹⁴ Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland

	ABSTRACT

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Purpose: Arzoxifene, a new selective estrogen receptor modulator with strong breast antiestrogen activity and absence of uterine agonist activity, was explored as a potential chemoprevention agent. We performed a multi-institutional evaluation of arzoxifene in women with newly diagnosed ductal carcinoma in situ or T1/T2 invasive cancer.

Experimental Design: In a Phase IA trial, 50 pre- or postmenopausal women were randomized to 10, 20, or 50 mg of arzoxifene daily in the interval between biopsy and re-excision or were enrolled as no-treatment controls. In a Phase IB trial, 76 postmenopausal women were randomized to 20 mg of arzoxifene versus matched placebo. Serum specimens collected at entry and at re-excision were assayed for various hormones and growth factors. Tissue from biopsies (estrogen receptor + and/or progesterone receptor +) and re-excision specimens was evaluated immunohistochemically for proliferation (Ki-67 by MIB-1 and proliferating cell nuclear antigen) and other biomarkers.

Results: In both trials, increases in serum sex hormone binding globulin were noted, as were decreases in insulin-like growth factor (IGF)-I and the IGF-I:IGF binding protein-3 ratio (P < 0.007 versus control/placebo). For 45 evaluable women in Phase IA, decreases in proliferation indices were more prevalent for arzoxifene (particularly 20 mg) than for controls. For 58 evaluable women in Phase IB, a decrease in estrogen receptor expression for arzoxifene was observed compared with no change with placebo (P = 0.0068). However, decreases in proliferation indices for arzoxifene were not statistically different from placebo, perhaps due to a confounding effect of stopping hormone replacement therapy before entry.

Conclusion: Given the favorable side effect profile and the biomarker modulations reported here, arzoxifene remains a reasonable candidate for additional study as a breast cancer chemoprevention agent.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Arzoxifene (LY353381) is a third-generation selective estrogen receptor modulator similar to raloxifene but modified to improve bioavailability as well as estrogen antagonist potency in the breast and uterus (1) . In vitro and xenograft studies have shown arzoxifene to be more potent than tamoxifen, 4-OH tamoxifen, or raloxifene in inhibiting the growth of tamoxifen-sensitive, estradiol-stimulated MCF-7 cells (2) . Arzoxifene also was able to suppress the growth of T47D tamoxifen-resistant tumors but not MCF-7 tamoxifen-resistant tumors in xenograft models (3) . The two tamoxifen-resistant models differ in that the resistant MCF-7 cells have reduced levels of the estrogen receptor corepressor NCOR, whereas resistant T47D cells have increased levels of estrogen receptor coactivators such as AIB1 (3, 4, 5) . Arzoxifene was found to be more effective than equimolar doses of raloxifene and equivalent to tamoxifen in the nitrosomethylurea rat breast cancer prevention model (2) . Preclinical studies in ovariectomized rats indicate favorable effects on cholesterol, bone mineral density, and uterine weight, which support the potential utility of arzoxifene as a prevention agent. Indeed, in preclinical studies arzoxifene was more potent than raloxifene in the prevention of bone loss (1 , 6) . Low-dose arzoxifene and a novel rexinoid have been demonstrated to be synergistic in the rat model of breast cancer (7) .

Animal models predicted antitumor activity at equivalent human doses of 10 mg/day (2) . Studies in healthy postmenopausal volunteers and in women with metastatic disease indicated that the pharmacokinetics of arzoxifene were linear over a wide dose range (8 , 9) . Pharmacodynamic changes were observed starting at the 10 mg per day dose and included reduced levels of low-density lipoprotein cholesterol, serum fibrinogen, antithrombin III activity, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and the bone turnover biomarkers osteocalcin and urine type I collagen fragments, whereas an increase was observed for sex hormone binding globulin (9) .

Significant antitumor activity has been reported for tamoxifen-naive or -sensitive subjects in Phase II trials of women with metastatic disease. Baselga et al. (10) reported a complete and partial response rate of 43% for arzoxifene at 20 mg/day and 27% for arzoxifene at 50 mg/day in a cohort with a median age of 70 and in whom only 9% had received prior tamoxifen. Only 10% of tamoxifen refractory subjects responded to 20–50 mg daily arzoxifene (11) . No uterine agonist effects were reported in women who had not been exposed to tamoxifen previously (11) .

Despite preclinical and clinical evidence of partial cross-resistance with tamoxifen, the demonstrated efficacy of arzoxifene in advanced breast cancer, lack of uterine agonist activity, and favorable pharmacodynamic effects on bone and lipids make it an attractive selective estrogen receptor modulator for prevention.

A number of studies have demonstrated that selective estrogen receptor modulators likely to be effective in breast cancer treatment and prevention will show a reduction in proliferation in estrogen receptor-positive tumors after 2–4 weeks of treatment (12, 13, 14, 15, 16) .

Therefore, before beginning large-scale prevention studies with arzoxifene, a short-term Phase IA/IB preoperative biomarker modulation study in women with newly diagnosed breast cancer was initiated by a multi-institutional group as part of a Division of Cancer Prevention, National Cancer Institute-sponsored contract. In the Phase IA portion of the trial, women with core biopsy evidence of ductal carcinoma in situ and/or invasive cancer were randomized to one of several doses of arzoxifene in the interval between core biopsy and re-excision. The purpose of the Phase IA trial was to select the lowest dose associated with a reduction in proliferation as well as identify other modulated biomarkers consistent with the presumed mechanism of action (17 , 18) . In the Phase IB trial, the dose selected from the Phase IA trial was compared in a randomized double-blind fashion to placebo with the primary end point of reduction in proliferation (17) .

We report the results of Phase IA and Phase IB biomarker modulation trials of arzoxifene administered to women with hormone receptor-positive ductal carcinoma in situ and/or T1-T2 invasive breast cancer in the interval between diagnostic biopsy and definitive surgical treatment.

	MATERIALS AND METHODS

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Study Design.
In the Phase IA trial, eligible pre- and postmenopausal women were randomized to one of three doses of arzoxifene administered daily in the planned interval between biopsy and re-excision. The planned interval was not altered by participation in the trial, but subjects were not eligible if the interval was anticipated to be <2 weeks or >6 weeks. Women not wishing to receive drug but who were otherwise eligible and willing to participate served as nonrandomized no-treatment controls. Women on hormone replacement therapy discontinued this medication before study entry. Doses selected for the Phase IA trial were 10, 20, and 50 mg/day (10-mg and 50-mg tablets supplied by Eli Lilly via the National Cancer Institute). Dose selection was based on the favorable pharmacodynamic effects of arzoxifene in doses as low as 10 mg/day (9) and reported responses at 20 and 50 mg/day in advanced breast cancer. Arzoxifene has a terminal elimination half-life of 30–35 h (9) . On the basis of the pharmacokinetic profile of arzoxifene, steady state was not predicted to be reliably reached during a 2–4 week trial using once daily dosing; therefore, a loading dose consisting of twice the assigned daily dose was used on day 1 and day 2 for both the Phase IA and Phase IB trials. Subjects were instructed to take their study drug with a meal. Once a subject had been randomly assigned a drug dose, the dose a subject was receiving was known to investigators.

The Phase IB trial as originally activated was a double-blind, three-way, 1:1:1 randomization among arzoxifene 20 mg/day, tamoxifen 20 mg/day, and matching placebos for both drugs (supplied by McKesson Laboratories under contract to the National Cancer Institute). The trial was restricted to postmenopausal women who had not received hormone replacement therapy within 30 days of biopsy. However, accrual was extremely slow (14 subjects in 8 months) due in part to the exclusion of women who had received hormone replacement therapy within 30 days of biopsy and the difficulty of explaining a three-way randomization to potential subjects. Consequently, the protocol was amended to allow women who had received hormone replacement therapy in the peribiopsy period to enter the study providing hormone replacement therapy was discontinued before study entry (subjects were stratified according to hormone replacement therapy use within 30 days of biopsy). The protocol was also changed to a design of two sequential phases: 60 subjects in a 2:1 randomization between arzoxifene and matching placebo to be followed by 60 subjects in a 2:1 randomization between tamoxifen and matching placebo. Subsequently, a decision was made to omit the tamoxifen phase and to instead continue subject accrual with a target accrual of 60 subjects on the arzoxifene arm. The blinded assignment between arzoxifene and placebo was maintained throughout the study and subsequent analysis. Only the biostatistician at University of Kansas Medical Center not otherwise associated with this trial who prepared the randomization assignments and the investigational pharmacists at each participating institution were aware of the study agent assignments. For analysis, the study agent assignments were revealed to the University of Kansas Medical Center biostatisticians conducting the analyses.

Table 1 summarizes study design, eligibility, and biomarker endpoints for the Phase IA and Phase IB trials.

Serum Biomarkers.
An average of 15 ml of serum was obtained before entry and again at the off-study point for estradiol, estrone, FSH, LH, insulin-like growth factor (IGF-I), insulin-like growth factor binding protein type 3 (IGFBP-3), progesterone, sex hormone binding globulin, and thyroxine-binding globulin for the Phase IA trial. The same assays were performed for the Phase IB trial, with the exception that progesterone and thyroxine-binding globulin were omitted. Serum was stored at –70 to –80°C until analysis, with pre- and poststudy specimens assayed in the same run. Selective estrogen receptor modulators such as tamoxifen had been described previously as modulating the risk biomarkers IGF-I, IGFBP-3, and sex hormone binding globulin (19, 20, 21) . Modulation of thyroxine-binding globulin and sex hormone binding globulin were also considered potential markers of selective estrogen receptor modulator biological activity without regard to efficacy. Hormone assays were performed to determine whether the systemic hormone levels were substantially altered between the study entry time point and the re-excision time point. For the Phase IA trial, serum assays were conducted at Midwest Research Institute (Kansas City, MO) using radioimmunoassays (for estradiol, estrone, and progesterone) or immunoradiometric assays (for FSH, IGF-1, IGFBP-3, sex hormone binding globulin, and thyroxine-binding globulin). For the Phase IB trial, serum assays were conducted at the Center for Reproductive Endocrinology Laboratories, University of Kansas Medical Center (Dr. Paul Terranova) using enzyme immunoassays from Diagnostic Systems Laboratories, Inc. (Webster, TX; estradiol, estrone, FSH, LH, IGF-I, and IGFBP-3) and Diagnostic Biochemicals (London, Ontario, Canada; sex hormone binding globulin).

Tissue Assays.
The tissue biomarkers assessed in Phase IA and IB are listed in Table 2 . In addition to the primary study end points, a number of other tissue biomarkers were assessed that might be predictive and/or reflective of successful selective estrogen receptor modulator treatment, including predictive markers such as estrogen receptor, progesterone receptor, and Her-2/neu (22 , 23) ; markers of angiogenesis such as thrombospondin, CD31, and p53 incorporated into an angiogenesis index, vascular endothelial growth factor, and microvessel density (24, 25, 26) ; and growth and survival signaling markers such as bcl-2, phosphorylated extracellular regulated kinase, tumor necrosis factor , and insulin-like growth factor receptor (27, 28, 29, 30) Tissue was fixed in 10% neutral-buffered formalin at the individual sites for 6 h but 24 h and then processed to paraffin blocks. After sufficient tissue had been used at the site for diagnostic purposes, blocks were sent to University of Kansas Medical Center for central sectioning, staining and interpretation at the University of Kansas Medical Center Pathology Research Laboratories by O. W. T. Institutions were allowed to send unstained tissue sections on slides provided these were processed and mailed such that slides could be stained within 72 h of sectioning of the block at the central laboratory at University of Kansas Medical Center. Staining and assessment of pre- and poststudy tissue assays were performed together. Sectioning of the block for all of the assays was performed at the same time. Automated stainers (Dako, Ventana) were used for staining all of the specimens, and known positive and negative controls were included with each run. Assessments were done with the CAS 200 image analyzer for Ki-67 (MIB-1 antibody), proliferating cell nuclear antigen (PCNA), estrogen receptor, progesterone receptor, and p53 and produced values for the percentage of cells staining positively. Manual assessment was used for other biomarkers using an immunochemistry weighted index score (range, 0–4) developed by our immunochemistry consultant (W. E. G.; Ref. 31 ). Before beginning the study, all of the immunochemistry procedures were validated and approved. The antibodies used are listed in Table 2 . For the Phase IA trial, assessment of nuclear morphometry was also performed by O. W. T, B. F. K., and J. W. B. using an automated image analysis system (BLISS; Bacus Laboratories, Inc., Elmhurst, IL). Fuelgen-stained specimens were scanned, areas of interest marked, and a z-score computed that incorporated a number of parameters and represents their deviation from normalcy (32) . For the Phase IA trial, a subset of subjects was evaluated by J. P. F. for change in angiogenesis index (24) .

Accrual Goals.
Accrual goals for the Phase IA trial were 10 subjects in each of the three (10, 20, and 50 mg/day) arzoxifene arms, and the nonrandomized no-treatment control group, for whom prestudy and poststudy specimens were available and change in the primary end point (proliferation) could be evaluated. With this number of subjects, if a reduction in proliferation (Ki-67 or PCNA) was seen in at least 8 subjects, we would be 89% confident that the probability of favorable modulation is at least 50%.

The accrual goal for the amended Phase IB trial was 80 subjects from which 60 subjects would be evaluable for the primary end point biomarkers. This accrual goal was based on the number of subjects needed to detect a large effect size of 0.8 SDs in change in the proportion of cells expressing a proliferation maker between the control and the treatment groups with an 80% power and type I error rate of 5%. From the Phase IA results, this would project to an absolute decrease from 10% at baseline to 4% or 5% at re-excision in the proportion of cells expressing Ki-67 or PCNA, respectively.

Data Capture and Entry.
Clinical data were entered onto case report forms at individual sites, and audited data were entered into a computerized database at CCS Associates (Mountain View, CA). The clinical data were down loaded electronically into a joint biomarker and clinical database housed in the Biostatistical Unit of the University of Kansas Medical Center. Tissue biomarker and serum biomarker data were downloaded electronically into this database as well.

Statistical Analyses.
For the Phase IA and Phase IB trials, categorical variables were summarized by frequencies and percentages, and quantitative variables were summarized by medians and ranges. For the Phase IA trial, quantitative variables were compared among the four groups using the Kruskal-Wallis test. The Wilcoxon rank-sum test was used to perform pair-wise comparisons on quantitative variables that were globally different among the four groups. Fisher’s exact test was used to compare categorical variables among the four groups. For the Phase IB trial, except for the evaluation of the primary endpoints (modulation of proliferation index and frequency of adverse events), all of the analyses were considered as exploratory. Thus, no corrections for multiple comparisons were made. Quantitative variables were compared between the two groups using the Wilcoxon rank-sum test. The two-sample t test was also performed as per protocol. Whereas the conclusions are identical for each procedure, given the skewness of the distributions for many variables, the Wilcoxon rank-sum test was the preferred method of analysis. Categorical variables were compared using Fisher’s exact test. A two-way analysis of variance with interaction was modeled to assess the effect of treatment and hormone replacement therapy use within 30 days of biopsy on the quantitative biomarkers. A significant interaction term would indicate a differing effect of treatment depending on hormone replacement therapy status.

	RESULTS

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Results of the Phase IA Trial
Accrual and Participating Institutions.
From November 1998 to December 1999, 50 subjects were entered into the Phase IA trial after signing appropriate informed consents as approved by the site Institutional Review Board. At least 315 potential subjects were screened to accrue this number of subjects (the uncertainty resulted from variations in the definition and reporting of screening between sites). Accrual distribution included four from University of Kansas Medical Center, 15 from Loyola University Medical Center, 14 from US Oncology, 8 from Ohio State University, and 3 each from Cleveland Clinic Foundation, University of California Los Angeles, and Desert Comprehensive Cancer Center.

Forty women with newly diagnosed ductal carcinoma in situ, T1, or T2 tumors were randomized to 10, 20, and 50 mg of arzoxifene daily (13, 13, and 14 subjects, respectively). In addition, 10 subjects were registered as nonrandomized, no-treatment controls. Five subjects were not evaluable for tissue based biomarkers, because there was no residual tumor available for analysis in the re-excision specimen (3 subjects) or because the biopsy was both estrogen-receptor and progesterone-receptor negative (2 subjects) by immunohistochemistry performed at University of Kansas Medical Center. Thus, 45 Phase IA subjects were evaluable for the primary biomarker endpoint including 8 controls, 11 on the 10-mg arm, 13 on the 20-mg arm, and 13 on the 50-mg arm. Characteristics of these subjects are listed in Table 3 . Median age was 61; 84% were postmenopausal. Fifteen of 37 (41%) subjects on study drug and 3 of 8 (38%) no-treatment controls had received hormone replacement therapy within 30 days of biopsy. Eighty percent of biopsy specimens were grade I or II. Seventy-eight percent of women had invasive cancer in both their original biopsies and re-excision specimens, whereas 13% had ductal carcinoma in situ in both specimens. For 9% of subjects, the submitted biopsy and re-excision specimens differed in that one contained only invasive cancer and the other only ductal carcinoma in situ. Median on study interval was 15 days (range, 10–42 days).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Number of subjects on Phase IA trial who exhibit a change (prestudy to poststudy) in serum hormone or growth factor levels. The number above the bars indicate the number of subjects in whom the change was an "improvement" (in the direction that would reduce breast cancer risk); the numbers below the bars indicate subjects in whom no change was observed or the change was in an unfavorable direction. , controls; , arzoxifene. IGFBP, IGF, binding protein; SHBG, sex hormone binding globulin; TBG, thyroxine-binding globulin.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Number of subjects on Phase IA trial who exhibit a change (biopsy to re-excision) in tissue biomarkers. The numbers above the bars indicate the number of subjects in whom the change was an "improvement" (in the direction that would reduce breast cancer risk or would be consistent with the presumed mechanism of action of arzoxifene); the numbers below the bars indicate subjects in whom no change was observed or the change was in an unfavorable direction. , controls; , arzoxifene. ER, estrogen receptor; PR, progesterone receptor; AI, angiogenesis index; EGFR, epidermal growth factor receptor.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Changes in PCNA expression in control subjects and subjects receiving 20 mg of arzoxifene on the Phase IA trial. Two subjects on arzoxifene had high baseline values, which are plotted on a different scale.

On the basis of these Phase IA results, the 20-mg dose of arzoxifene was selected for the subsequent randomized, placebo-controlled Phase IB trial.

Results of the Phase IB Trial
Accrual and Participating Institutions.
From June 5, 2000 to July 19, 2002, 81 subjects were entered onto the Phase IB trial after signing appropriate informed consents as approved by the site Institutional Review Board. At least 1400 potential subjects were screened to accrue this number. Accrual distribution included four from University of Kansas Medical Center, 26 from Ohio State University, 16 from US Oncology, 15 from Desert Comprehensive Cancer Center, 11 from Loyola University Medical Center, 4 from Thomas Jefferson University (Philadelphia, PA), 2 each from Cleveland Clinic Foundation and University of Alabama, and 1 from University of California Los Angeles. The trial was initiated with a three-way randomization to placebo, tamoxifen 20 mg/day, or arzoxifene 20 mg/day. After entry of the first 14 subjects, the protocol was amended due to slow accrual. The tamoxifen arm was discontinued, and a weighted randomization scheme was used such that 2 subjects were randomized to arzoxifene for every 1 randomized to placebo. Of the 81 subjects accrued to the study, 5 were accrued to tamoxifen in the preamendment portion, and the remaining 76 were randomized between arzoxifene and placebo. Seven subjects were excluded from the toxicity and pharmacodynamic analysis due to randomization to tamoxifen (5) , withdrawal from study before receipt of any drug (1) , or receipt of both arzoxifene and tamoxifen (1) . Thus, 74 subjects were evaluable for toxicity analysis and serum pharmacodynamic analysis for the placebo and arzoxifene arms. The median age of the 74 subjects evaluable for toxicity and serum pharmacodynamic parameters was 63 years. The median baseline estradiol level was 28 pg/ml, and 46% of subjects had been taking hormone replacement therapy within 30 days of biopsy, including 23 of 47 (49%) subjects randomized to study drug and 11 of 27 (41%) subjects randomized to placebo. Median time from biopsy to study entry was 10 days (range, 0–45), and median on study interval was 20 days (range, 9–55). There were no significant differences for demographics, tumor characteristics, or intervals between various study events (Table 9) between the arzoxifene and placebo groups.

Change in Serum Pharmacodynamic Parameters for the Phase IB Trial.
Baseline values for serum pharmacodynamic parameters for the 74 placebo and arzoxifene subjects are summarized in Table 11 . There was no significant difference in baseline values between the two groups. Changes in estradiol, estrone, FSH, LH, IGF-I, IGFBP-3, IGF-1:IGFBP-3 molar ratio, and sex hormone binding globulin for placebo and arzoxifene groups are given in Table 12 . Compared with placebo, arzoxifene-treated subjects had significantly greater increases in sex hormone binding globulin (P = 0.0015); and greater decreases in LH (P = 0.03), IGF-I (P = 0.0004), and IGF-I:IGFBP-3 molar ratio (P = 0.0066).

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reduction in the proportion of tumor cells expressing Ki-67 after 2–3 weeks of tamoxifen has been correlated with later clinical response in women with breast cancer (12 , 15) . Several European studies have used preoperative models similar to our Phase IA/IB trial and found reductions in Ki-67 after 2–4 weeks of treatment with a selective estrogen receptor modulator (14 , 16) .

In the trial reported by Decensi et al., a 15% (95% confidence interval, 0–24%) median relative reduction from baseline in the proportion of cells expressing Ki-67 was observed for 120 estrogen receptor + subjects randomized to one of three doses of tamoxifen for 4 weeks, whereas a relative increase of 13% (95% confidence interval, –3.3–33%) was observed in 29 estrogen receptor + nonrandomized controls (16) . Dowsett et al. (14) reported a 21% median relative reduction from baseline in the proportion of cells expressing Ki-67 for 42 subjects randomized to 2 weeks of 60 mg/day raloxifene versus a median relative increase of 7% in 36 subjects randomized to placebo. Dowsett et al. (13) also reported a 35% mean relative reduction from baseline in the proportion of cells expressing Ki-67 for 30 subjects randomized to 2–3 weeks of idoxifene versus a 6% mean relative increase in 27 subjects randomized to placebo. The trial reported by Decensi et al. (16) differed from ours in that three times as many subjects were entered into the study and were treated for twice as long. The two studies reported by Dowsett et al. (13 , 14) differed from ours in that eligible women could not have received hormone replacement therapy for at least 3 months before initial biopsy.

In our IA arzoxifene study, a decrease in Ki-67 from baseline was observed in only 54% of subjects, whereas PCNA was decreased in >80% of subjects at the higher dose levels. PCNA is more likely than Ki-67 to be expressed in cells in early G₁ and/or in cells that have traversed the cell cycle recently but are currently in G₀ (38, 39, 40) . Thus, when proliferation is low, as observed in the predominately well-differentiated tumors in this clinical model, PCNA may be a more sensitive indicator of proliferation than Ki-67 (41 , 42) . Unfortunately, PCNA detection varies with length of fixation and processing conditions. Moreover, assessment of positive staining is more susceptible to interpretive variance than Ki-67 (43) . Although the arzoxifene dose for our Phase IB trial was selected on the basis of PCNA, Ki-67 is considered to be more reproducible and consistent in its pattern of staining (42 , 44 , 45) and, thus, both were retained as measures of proliferation. In the Phase IB trial, the baseline expression of Ki-67 and PCNA were similar, both in the placebo group and in the arzoxifene group.

For women randomized to arzoxifene in the Phase IB trial, we observed a median relative reduction of 25% in the proportion of tumor cells expressing Ki-67, similar to that reported for tamoxifen, raloxifene, and idoxifene, as well as a 46% reduction in PCNA. However, unlike the trials reported by Dowsett et al. (13 , 14) , a median relative reduction of 20% in Ki-67 and 23% in PCNA was also observed in the randomized placebo group. Thus, for our primary endpoint proliferation, there was no significant change between 20 mg/day arzoxifene and placebo. Subgroup analysis indicates that the lack of significant reduction in proliferation index in women randomized to arzoxifene versus those randomized to placebo may have been in part secondary to the confounding effects of discontinuance of hormone replacement therapy in the peribiopsy period before re-excision.

Prasad et al. (46) have reported recently on the effects of discontinuing hormone replacement therapy in the interval between core needle biopsy and definitive surgery (re-excision, lumpectomy, or mastectomy) for invasive breast cancer. They found that there was a decrease in Ki-67 expression associated with discontinuation of hormone replacement therapy that was statistically significant when compared with patients that had either not been using hormone replacement therapy before biopsy or who had continued to use hormone replacement therapy until definitive surgery. This effect was observed for tumors that were estrogen receptor + but not for tumors that were estrogen receptor –.

For those women in our study not on hormone replacement therapy previously, the 3% (Ki-67) or 17% (PCNA) median relative increase for placebo and the 8% (Ki-67) or 30% (PCNA) median relative decrease for arzoxifene are similar to proliferation effects observed in the United Kingdom trials of raloxifene and idoxifene. On the other hand, subjects who had received hormone replacement therapy within 30 days of biopsy exhibited a dramatic reduction in the proportion of cells expressing Ki-67 or PCNA, regardless of whether randomized to placebo or to arzoxifene. This implies that future studies using the preoperative model should not enroll women with estrogen receptor + tumors who have been taking hormone replacement therapy before biopsy if proliferation is to be used as an endpoint and if the hormone replacement therapy is to be discontinued between biopsy and re-excision. Although our Phase IB trial was initially designed to exclude both premenopausal women as well as postmenopausal women who had received hormone replacement therapy recently, accrual problems prompted us to amend the study to allow entry of postmenopausal women on hormone replacement therapy at the time of biopsy.

Another factor that may contribute variability and, thus, affect the evaluation of modulation due to a drug effect is the duration of exposure to study agent. This variation is inherent in this clinical model where the scheduling of re-excision surgery could not be dictated by the study. Rather, the timing of surgery was at the discretion of the treating physician and was planned before study entry. To participate, this planned interval could be no shorter than 2 weeks and no longer than 6 weeks. The actual time on study agent ranged from 9 to 55 days for the 58 subjects evaluable for Ki-67. Three subjects received agent for <15 days and 5 subjects received agent for >30 days. There was no difference for the time on study agent between the placebo (median 21 days) and arzoxifene (median 20 days) groups (Table 13) . Whereas it might seem desirable to have a standard, longer interval on study agent, there was no evidence to suggest that a longer exposure to arzoxifene resulted in any greater modulation of proliferation.

Serum IGF-I:IGFBP-3 ratio and non-sex hormone binding globulin bound levels of estradiol, as well as free testosterone have been reported as risk factors for breast cancer (19 , 47) . LH, which stimulates the conversion of cholesterol to pregnenolone as the first step in steroidogenesis, has been found to be elevated in breast cancers (48) . High levels of sex hormone binding globulin, which may result in decreased levels of bioavailable estradiol and testosterone, are associated with reduced breast cancer risk (20) . The reductions in serum LH, IGF-I, and IGF-I:IGFBP-3 molar ratio and the increase in sex hormone binding globulin observed with arzoxifene are similar to those reported for tamoxifen (21 , 49) and are probably the result of partial estrogen agonist properties of arzoxifene on the central nervous system and liver metabolism (50 , 51) . Change in serum hormones in our trial of women with ductal carcinoma in situ or small invasive cancers are similar to those reported in Phase I and II treatment trials of arzoxifene in women with metastatic disease (9 , 11) .

Both intraepithelial neoplasia and breast cancer are often associated with an increase in the proportion of cells expressing estrogen receptor (52, 53, 54) . The significant (P = 0.0068) reduction in estrogen receptor expression that we observed for arzoxifene has also been reported for idoxifene (13) but not consistently for tamoxifen (12 , 55 , 56) . We also observed that for those subjects not on hormone replacement therapy in the peribiopsy period, there were significant (P = 0.009) differences in both absolute change and relative change for placebo (medians of 0%) versus arzoxifene (median 5% decreases). For subjects on hormone replacement therapy in the peribiopsy period, the differences in change in estrogen receptor expression between placebo and arzoxifene were not statistically significant, again underscoring the powerful effect that stopping hormone replacement therapy has upon breast tissue biomarker expression. Importantly, unlike what has been reported for tamoxifen (57) , no increase was observed for progesterone receptor expression after arzoxifene administration, which might indicate less likelihood of a partial estrogen agonist affect on breast tissue.

In summary, we did not demonstrate a significant reduction in tumor cell proliferation with arzoxifene relative to placebo in our randomized, double-blind Phase IB trial. Failure to demonstrate significant reduction in proliferation relative to placebo may be secondary to the small sample size, the confounding effects of discontinuing hormone replacement therapy in a high proportion of study subjects, or other factors. We were able to demonstrate favorable modulation of tumor estrogen receptor expression and serum LH, IGF-I:IGFBP-3 ratio, and sex hormone binding globulin relative to placebo. Favorable modulation of these biomarkers and a favorable toxicity profile in Phase II treatment trials continues to make this agent an excellent candidate for additional study in the prevention setting, either alone or in combination with other agents. A Phase II chemoprevention trial in 200 women at high risk for development of breast cancer will be completed in 2004. The primary endpoint is modulation of cytomorphology in specimens obtained by random periareolar fine needle aspirations at baseline and after 6 months of study agent.

	FOOTNOTES

 
Grant support: NO1-CN-85035 from the Chemoprevention Branch, Cancer Prevention Research Program-Cancer Control, Division of Cancer Prevention of the National Cancer Institute, NIH.

Notes: C. Fabian and B. Kimler contributed equally to this work.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Carol J. Fabian, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. Phone: (913) 588-7791; Fax: (913) 588-3679; E-mail: cfabian{at}kumc.edu

Received 1/28/04; revised 5/ 7/04; accepted 5/17/04.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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