Published OnlineFirst November 3, 2009; DOI: 10.1158/1078-0432.CCR-09-1354

Randomized Biomarker Trial of Anastrozole or Low-Dose or Their Combination in Subjects with Breast Intraepithelial Neoplasia Bernardo Bonanni,1 Davide Serrano,1 Sara Gandini,2 Aliana Guerrieri-Gonzaga,1 Harriet Johansson,1 Debora Macis,1 Massimiliano Cazzaniga,1 Alberto Luini,3 Enrico Cassano,4 Sabina Oldani,5 Ernst A. Lien,7 Giuseppe Pelosi,6 and Andrea Decensi1,8

Abstract Purpose: In the Anastrozole, Tamoxifen Alone or in Combination trial, the combination arm was inferior to anastrozole alone in terms of disease-free survival possibly due to an adverse pharmacokinetic interaction or a predominant estrogenic effect of tamoxi- fen under deprivation. We assessed whether the addition of a lower dose of tamoxifen influenced anastrozole bioavailability and favorably modulated biomarkers of bone fracture, , cardiovascular disease, and endometrial cancer risk. The influence of CYP2D6 genotype on tamoxifen effects was also determined. Experimental Design: Seventy-five postmenopausal women with breast intraepithelial neoplasia were randomly allocated to either 1 mg/d anastrozole or 10 mg/wk tamoxifen or their combination for 12 months. Study endpoints were plasma drug concentrations and changes of C-telopeptide, osteocalcin, / binding globulin (SHBG) ratio, sulfate, insulin-like growth factor-I (IGF-I)/insulin-like growth fac- tor binding protein-3 (IGFBP-3), C-reactive protein, antithrombin-III, endometrial Ki-67 expression, and thickness. Results: Anastrozole concentrations were not affected by the combination with low- dose tamoxifen, whereas endoxifen levels were lower in poor CYP2D6 metabolizers. C-telopeptide increased by 20% with anastrozole and decreased by 16% with tamoxifen and by 7% with their combination (P < 0.001); osteocalcin showed similar changes. Compared with anastrozole, the combination arm showed lower IGF-I/IGFBP-3 levels (-17% versus -9%; P = 0.004) and lower estradiol/SHBG and reductions (-15% versus -29% and -30% versus 38%, respectively). However, IGF-I/IGFBP-3 and es- tradiol/SHBG did not decrease in poor CYP2D6 metabolizers. Endometrial thickness was not greater in the combination than in the anastrozole arm. Conclusions: The addition of a weekly tamoxifen administration did not impair anastro- zole bioavailability and modulated favorably its safety profile, providing the rationale for further studies. (Clin Cancer Res 2009;15(22):7053–60)

Aromatase inhibitors are a standard treatment of estrogen crine resistance with the emergence of estrogen hypersensitive receptor–positive breast cancer in postmenopausal patients cell clones (6). One potential way to counteract these phenom- (1–4) and are being tested as chemopreventive agents in at-risk ena is to add tamoxifen to exploit its partial estrogenicity. In the women (5). However, these agents are associated with in- Anastrozole, Tamoxifen Alone or in Combination study, a creased bone fracture rate, joint and tendon disorders, and phase III adjuvant trial with three arms (1 mg anastrozole, possibly increased cardiovascular risk due to their profound 20 mg tamoxifen, and their combination), anastrozole signifi- estrogen-suppressive effect rising concern on their long-term cantly prolonged disease-free survival versus tamoxifen. More- safety, especially in the prevention setting. Moreover, prolonged over, the combination of anastrozole and tamoxifen at the dose treatment may lead to the onset of endo- of 20 mg/d was inferior to anastrozole alone and slightly, albeit

Authors' Affiliations: Divisions of 1Cancer Prevention and Genetics, The costs of publication of this article were defrayed in part by the payment 2Epidemiology and Biostatistics, 3Breast Surgery, 4Breast Diagnostics, of page charges. This article must therefore be hereby marked advertisement 5Gynaecology, 6Pathology, European Institute of Oncology, Milan, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. University of Milan School of Medicine, Milan, Italy; 7Hormone Laboratory, Requests for reprints: Andrea Decensi, Division of Medical Oncology, E.O. Haukeland University Hospital, Bergen; Section for Endocrinology, Institute Ospedali Galliera, Mura delle Cappuccine 14, 16128 Genoa, Italy. Phone: 39- of Medicine, University of Bergen, Bergen, Norway; and 8Division of Medical 105634501; Fax: 39-1057481090; E-mail: [email protected]. Oncology, E.O. Ospedali Galliera, Genoa, Italy F 2009 American Association for Cancer Research. Received 5/28/09; revised 8/11/09; accepted 8/24/09; published OnlineFirst 11/3/09. doi:10.1158/1078-0432.CCR-09-1354

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kers over anastrozole alone? (d) Does the addition of low- Translational Relevance dose tamoxifen negatively influence anastrozole effect on endometrial tissue? (e) Does anastrozole negatively affect Primary and secondary breast cancer chemopre- low-dose tamoxifen bioavailability? and (f)DoesCYP2D6 vention strategies are becoming a predominant is- genotype, which may influence tamoxifen efficacy in both sue for social health. The spread of the screening adjuvant (13) and preventive (14) settings, affect tamoxifen programs has increased diagnosis of intraepithelial metabolite concentrations and, in turn, the biological effects lesions, and until today, there is no consensus on of tamoxifen-containing treatments? how to treat such lesions. In the cardiovascular set- ting, treatment of risk factors has been confirmed to Patients and Methods be a very successful strategy, and recent protocols indicate that control of multiple pathways gives bet- Study participants. This randomized, open-label phase IIb trial was ter results and attenuates adverse events. To further approved by the Institutional Review Board of the European Institute of investigate the possible role of low-dose tamoxifen, Oncology, where it was conducted as a mono-institutional study (study anastrozole, and their combination in breast cancer S109). A total of 75 postmenopausal women with excised estrogen re- prevention, we conducted this randomized trial. This ceptor–positive and/or progesterone receptor–positive or unknown study provides a rationale to further study the com- ductal carcinoma in situ (DIN 2-3) or lobular carcinoma in situ (LIN bination of the two drugs because, with these do- 2-3) were enrolled. Postmenopausal status was defined as amenorrhea sages, there is not a negative pharmacokinetic for 12 months and FSH levels >40 IU/L. Written informed consent was obtained from each subject. Women were excluded if they had previous interaction. Furthermore, the combination maintains malignancy, grade 2 or greater hematologic and biochemical tests (Na- a reduction of breast cancer risk biomarkers and may tional Cancer Institute Common Toxicity Criteria version 2), retinal dis- have a safer toxicity profile on bone orders, history of thromboembolic events, untreated osteoporosis, and on the cardiocirculatory system. treatment with selective modulators or aromatase in- hibitors in the previous 6 months. Study procedures. Women were randomly assigned to one of the n not significantly, worse than tamoxifen alone in terms of dis- following three treatment arms: 1 mg/d anastrozole ( = 25), 10 mg/ wk tamoxifen (n = 25), or their combination (n = 25) for 12 months. ease-free survival, thus leading to the early discontinuation of Blood tests and clinical examination were done at baseline and at 3-, 6-, the combination arm (1, 7, 8). Different explanations have and 12-month visits. At baseline and 12 months, women underwent been advocated, including a documented adverse pharmacoki- transvaginal ultrasound (with endometrial biopsy at 12 months) and netic interaction leading to low anastrozole concentrations, al- mammography. though estradiol suppression was similar between anastrozole Pathologists and laboratory researchers remained blinded to treat- and combination arms (9) and a predominant estrogenic activ- ment arm until study completion. Study drugs were purchased by the ity of tamoxifen under anastrozole-induced estrogen-deprived Institute's Pharmacy using commercially available tamoxifen (10 mg milieu (1). tablets; Nomafen; Fidia) and anastrozole (1 mg tablets; Arimidex; As- Previous studies have shown that tamoxifen doses as low as traZeneca) specifically labeled and repackaged for the trial. Patients re- 5 or 1 mg/d exert an equivalent antiproliferative effect on ceived 7-month drug supply at baseline and at 6-month visit and were instructed to take either 1 tablet/d anastrozole or 1 tablet/wk tamoxifen breast cancer cells compared with the standard dose of 20 or both for 1 year. Compliance was evaluated by self- mg/d (10). In contrast, a linear dose-response relationship reporting calendar, pill count, and blood drug concentrations. was observed on the change in circulating biomarkers of breast Study endpoints. The main study endpoint was the trough/peak cancer and cardiovascular disease known to reflect tamoxifen plasma concentration of anastrozole, tamoxifen, and its metabolites estrogenicity, including insulin-like growth factor-I (IGF-I), at 3, 6, and 12 months. Secondary endpoints were the changes induced sex hormone binding globulin (SHBG), low-density lipopro- by treatment on biomarkers of bone fracture risk, such as C-telopeptide tein-cholesterol, C-reactive protein (CRP), fibrinogen, and anti- and osteocalcin (15); breast cancer risk, including SHBG, estradiol, es- thrombin-III, suggesting that a dose reduction of tamoxifen trone, estrone sulfate, IGF-I, and its ratio over insulin-like growth factor – retains a significant antiproliferative effect while reducing its es- binding protein-3 (IGFBP-3; refs. 16 18); and cardiovascular risk, in- trogenic effects. Moreover, the long half-life of tamoxifen and cluding lipids, coagulation, and ultrasensitive CRP (19, 20). Addition- ally, endometrial thickness and Ki-67 expression after a 12-month its most potent metabolites, endoxifen and 4-hydroxytamoxi- treatment were used as biomarkers of endometrial cancer risk (21). fen, provides the rationale for an alternative schedule using a Assay methods. Fasting blood samples were collected and stored at single weekly dose in an attempt to reduce toxicity while retain- -80°C until centrally assayed. Anastrozole concentrations were mea- ing efficacy (11, 12). sured on sodium heparin-treated samples and analyzed using liquid In this study, we compared the biological and pharmaco- chromatography with tandem mass spectrometric detection. The stan- kinetic effect of the combination of 1 mg/d anastrozole and dard curve range is from 1.00 to 100 ng/mL for anastrozole using a 10 mg/wk tamoxifen with either agent alone in postmeno- plasma sample volume of 0.100 mL (Covance Laboratories). Tamoxi- pausal patients with estrogen receptor–positive or progester- fen serum concentrations and metabolites (N-desmethyltamoxifen, one receptor–positive breast intraepithelial neoplasia. The 4-hydroxytamoxifen, and endoxifen) were measured according to trial was designed to answer the following questions: (a) previously described methods (22). Serum IGF-I, IGFBP-3, and high-sensitivity CRP were determined as Does the addition of 10 mg/wk tamoxifen still impair ana- b described previously (21). Serum SHBG was measured by means of en- strozole bioavailability as 20 mg/d? ( ) Can the addition of zyme-labeled chemiluminescent immunometric assays (Diagnostic low-dose tamoxifen counteract anastrozole bone resorption Products) designed for Immulite. Serum C-telopeptide and osteocalcin effect? (c) Is the addition of low-dose tamoxifen beneficial were measured with a chemiluminescent immunometric assay (Elecsys to breast carcinogenesis and cardiovascular disease biomar- 1010 analyzer; Roche Diagnostics). Fibrinogen and antithrombin-III

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Anastrozole and Low-Dose Tamoxifen were measured on citrate plasma by ACL 10000 (Beckman Coulter). with P values adjusted for multiple comparisons (26), with significant Estradiol and estrone sulfate serum concentrations were determined P values in bold. All statistical tests were two-sided. Analyses were done as described previously (23). using SAS statistical software version 9.0 (SAS Institute). Expression of Ki-67 antigen (clone Mib-1; Immunotech) was de- tected in superficial glands and stromal cells scanning on 10 high- power field at ×400 using Pipelle biopsies as described previously (21) Results based on the different proliferative capacity of these endometrial layers (24) and tamoxifen preferential effect on the stromal component (25). Of 158 subjects assessed for eligibility, 41 (26%) refused to Genomic DNA was extracted from whole-blood specimens by the participate and 42 (26.5%) were not eligible. Of the 75 ran- use of QIAamp DNA blood kit (Qiagen). We employed the INFINITI domized participants, 68 completed the 12-month treatment analyzer (AutoGenomics) for CYP2D6T® genotyping according to the and 7 dropped out. A total of 74 patients were included in protocol of the manufacturer. The test screens for the following the final analysis. One subject was excluded from the analysis CYP2D6 allele variants: *2 (2850C>T), normal activity; *3 (2549delA), *4 (1846G>A), *5 (CYP2D6 deleted), and *6 (1707 delT), no activity; for protocol deviation (chronic hepatitis C at baseline). The and *9 (2613_2615delAGA), *29 (1659G>A), and *41A (2988G>A), participant flow diagram is illustrated in Appendix A. reduced activity. We classified the resulting phenotype as poor metabo- The main subject characteristics and biomarker levels at base- lizers when there were nonfunctional variants on both alleles, whereas line by allocated treatment group are summarized in Table 1. all other combinations were grouped in a single category and consid- There were no statistically significant differences among arms, ered as extensive metabolizers. including and HER-2 receptor expression level. Statistical methods. The sample size was calculated to have 90% Compliance was very high, with all the subjects on actual power to assess equivalence in anastrozole plasma concentrations treatment taking ≥90% expected drug . Most ad- through 12 months between anastrozole and combination arms, as- verse events were grade 1, with no significant difference suming one-tailed 5% significance and normal distribution of the log among arms (data not shown). There were three serious ad- anastrozole [c], with SD = 0.41. Drug concentrations and quantitative biomarker measurements were analyzed by means of the repeated- verse events, one breast cancer and one colorectal cancer in measures ANOVA model at 3, 6, and 12 months, adjusted for baseline the combination arm and one grade 3 sideropenic anemia in values. Treatment and time effects, with their interaction taken as fixed the anastrozole arm. factors and patient effect as random factor, were evaluated in a mixed- The results of repeated-measures analysis of circulating effect model (PROC MIXED, SAS). As exploratory investigation, we concentrations of anastrozole, tamoxifen, and its metabolites evaluated the effect of CYP2D6 phenotype as a factor in the mixed- at 6 and 12 months are illustrated in Table 2. Mean circu- effect models. The analysis on endometrial thickness and Ki-67 expres- lating levels of anastrozole and endoxifen at 3, 6, and 12 sion was carried out with simple ANOVA models at 12 months using months by treatment arm are illustrated in Fig. 1. There available data. Log and square-root transformations were used when was no difference in anastrozole concentration in the com- necessary to achieve normality. bination arm compared with the anastrozole arm through Two orthogonal contrasts were used to compare biomarker changes among treatment groups: anastrozole versus combination and tamoxi- month 12. Conversely, a trend to lower concentrations of N fen versus anastrozole or combination. These contrasts were specified a endoxifen and -desmethyltamoxifen was observed at 12 priori and are consistent with the aims of the study. Least square means months in the combination arm compared with the tamox- for the three treatment groups, with P values for contrasts and for time ifen arm (P = 0.049 and 0.075, respectively). In a further effect, are presented. P values for all the biomarkers were compared analysis based on the CYP2D6 genotype, the differences

Table 1. Main subject characteristics and biomarker levels (median and interquartile range) at baseline according to treatment arm

Ana (n = 25) Tam (n = 25) Ana + Tam (n = 25)

Age (y; mean ± SD) 60.6 ± 5.7 61.1 ± 6.7 59.1 ± 5.7 Age at menopause (y; mean ± SD) 50.0 ± 4.6 51.5 ± 2.9 50.2 ± 4.1 Body mass index (kg/m2; mean ± SD) 24.9 ± 5.0 26.3 ± 5.1 24.7 ± 4.4 Ever use of menopause hormone therapy (%) 36 44 48 Lobular/ductal intraepithelial neoplasia (n) 1/24 2/23 2/23 Radiotherapy (%) 16 20 28 Estrogen receptor expression level* 90 (70-90) 90 (90-95) 90 (80-90) Progesterone receptor expression level* 28 (1-75) 20 (10-80) 30 (1-70) HER-2 overexpression (2/3+; %)* 46 39 48 C-telopeptide (ng/mL) 0.51 (0.39-0.67) 047 (0.27-0.64) 0.46 (0.39-0.57) Osteocalcin (ng/mL) 29 (24-39) 26 (19-35) 28 (23-32) IGF-I/IGFBP-3 80 (61-93) 63 (57-94) 80 (67-94) Estrone sulfate (ng/mL) 0.85 (0.7-1.03) 0.89 (0.7-1.01) 0.86 (0.7-0.97) Estradiol/SHBG 0.21 (0.16-0.34) 0.24 (0.16-0.34) 0.18 (0.13-0.23) CRP (mg/dL) 0.19 (0.10-0.37) 0.28 (0.10-0.52) 0.11 (0.07-0.29) Fibrinogen (mg/dL) 293 (262-318) 287 (249-324) 295 (266-328) Antithrombin-III (%) 96 (89-101) 98 (91-105) 97 (92-103)

Abbreviations: Ana, anastrozole; Tam, tamoxifen.Biomarker levels are expressed as median and interquartile range. *Values referred to ductal intraepithelial neoplasia patients only.

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Table 2. Repeated-measures analysis of drug and metabolite concentrations (ng/mL) at 6 and 12 months by allocated arm (mean and 95% confidence interval)

Drug Month Arm Least square mean (95% confidence interval) Contrast P

Anastrozole 6 Ana 31 (25-37) Ana vs Ana + Tam 0.794 Ana + Tam 32 (26-39) 12 Ana 27 (22-32) Ana vs Ana + Tam 0.224 Ana + Tam 32 (26-40) Tamoxifen 6 Tam 9.4 (6.75-12.34) Tam vs Ana + Tam 0.301 Ana + Tam 7.35 (5.08-10.04) 12 Tam 9.03 (6.49-11.99) Tam vs Ana + Tam 0.293 Ana + Tam 6.98 (4.75-9.65) 4-Hydroxytamoxifen 6 Tam 0.46 (0.34-0.60) Tam vs Ana + Tam 0.290 Ana + Tam 0.36 (0.25-0.49) 12 Tam 0.43 (0.31-0.57) Tam vs Ana + Tam 0.295 Ana + Tam 0.34 (0.23-0.47) N-desmethyltamoxifen 6 Tam 20.86 (15.62-26.86) Tam vs Ana + Tam 0.224 Ana + Tam 16.25 (11.67-21.59) 12 Tam 22.25 (16.83-28.44) Tam vs Ana + Tam 0.075 Ana + Tam 15.40 (10.90-20.66) Endoxifen 6 Tam 4.69 (3.52-6.02) Tam vs Ana + Tam 0.281 Ana + Tam 3.77 (2.73-4.97) 12 Tam 4.83 (3.65-6.19) Tam vs Ana + Tam 0.049 Ana + Tam 3.18 (2.22-4.32)

for endoxifen were no longer significant after excluding poor zole + combination P = 0.025). The number of evaluable sub- metabolizers [median (ng/mL; interquartile range), 4.65 jects for Ki-67 expression was smaller given the inability to (2.07-6.49) and 4.6 (3.98-6.85) for the combination and ta- obtain sufficient tissue due to atrophic endometrium in sever- moxifen arm, respectively; P = 0.19, Wilcoxon test]. al subjects. However, the combination arm induced a higher The results of repeated-measures analysis of circulating bio- proliferation of both the stroma and the superficial gland markers at 12 months are summarized in Table 3 with P values compartments compared with anastrozole alone (Ki-67 for the two contrasts: anastrozole alone versus the combination 1.21% versus 0.21% in the stroma compartment and 3.85% and tamoxifen versus anastrozole alone or their combination. versus 0.91% in the superficial gland P contrast 0.034 and Six-month data are not shown because they are similar to the 0.017, respectively). The contrast tamoxifen versus anastrozole 12 month results. C-telopeptide levels increased by 20% in the plus the combination was not significant. anastrozole arm relative to baseline, whereas they decreased by Based on CYP2D6 phenotype, there were 5 poor metabolizers 16% in the tamoxifen arm and by 7% in the combination arm and 20 extensive metabolizers in the combination arm and (P < 0.001 for both contrasts). Likewise, the addition of low- dose tamoxifen blunted the 22% increase of osteocalcin associ- ated with anastrozole treatment (P < 0.001 for both; Fig. 2). As for breast cancer risk biomarkers, the addition of tamoxi- fen to anastrozole significantly lowered IGF-I/IGFBP-3 levels compared with anastrozole alone (-17% versus -9%; P = 0.004), whereas the reduction of estradiol/SHBG and estrone sulfate was only lowered, albeit not significantly, in the combi- nation arm relative to the anastrozole arm (-15% versus -29% and -30% versus 38%, respectively; Fig. 2; Table 3). As expected, estrogen levels were not decreased in the tamoxifen arm. Cardiovascular disease biomarkers, fibrinogen, antithrombin- III, and CRP, were nonsignificantly lowered in the combi- nation arm relative to the anastrozole arm, especially at 6 months. Total cholesterol/high-density lipoprotein ratio and triglycerides did not significantly change during any treat- ment (not shown). The endometrial effects are shown in Appendix B. Endome- trial thickness was evaluated by transvaginal ultrasound in 60 patients, the addition of low-dose tamoxifen to anastrozole was not associated with an increased thickness, whereas ta- moxifen alone was associated with a greater thickness (least square mean, 2.19, 2.30, and 2.9 mm for anastrozole, combi- nation, and tamoxifen respectively; contrast anastrozole versus Fig. 1. Circulating concentrations of anastrozole and endoxifen at 3, 6, and combination P = 0.728 and contrast tamoxifen versus anastro- 12 mo according to treatment group.

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Anastrozole and Low-Dose Tamoxifen

Table 3. Repeated-measures analysis of biomarkers at 12 months by allocated arm (mean and 95% confidence interval) and median percentage change from baseline

Biomarker Treatment arm Least square mean % Change Contrasts P (95% confidence interval)

C-telopeptide* (ng/mL) Ana 0.67 (0.62-0.71) 20 Ana vs Ana + Tam <0.001 Tam 0.43 (0.38-0.48) -16 Tam vs (Ana or Ana + Tam) <0.001 Ana + Tam 0.5 (0.45-0.55) -7 Osteocalcin* (ng/mL) Ana 36 (34-38) 22 Ana vs Ana + Tam <0.001 Tam 25 (23-28) -22 Tam vs (Ana or Ana + Tam) <0.001 Ana + Tam 27 (25-30) -10 IGF-I/IGFBP-3 Ana 76 (71-81) -9 Ana vs Ana + Tam 0.004 Tam 64 (59-69) -17 Tam vs (Ana or Ana + Tam) 0.037 Ana + Tam 66 (61-71) -17 Estradiol/SHBG Ana 0.18 (0.14-0.21) -29 Ana vs Ana + Tam 0.44 Tam 0.24 (0.2-0.27) -14 Tam vs (Ana or Ana + Tam) 0.029 Ana + Tam 0.2 (0.16-0.23) -15 Estrone sulfate (ng/mL) Ana 0.64 (0.57-0.71) -38 Ana vs Ana + Tam 0.942 Tam 0.89 (0.8-0.99) 6 Tam vs (Ana or Ana + Tam) <0.001 Ana + Tam 0.64 (0.57-0.71) -30 CRP (mg/dL) Ana 0.14 (0.11-0.2) -13 Ana vs Ana + Tam 0.674 Tam 0.18 (0.13-0.24) -25 Tam vs (Ana or Ana + Tam) 0.429 Ana + Tam 0.16 (0.12-0.22) -18 Fibrinogen (mg/dL) Ana 299 (281-316) 1 Ana vs Ana + Tam 0.383 Tam 276 (259-293) -5 Tam vs (Ana or Ana + Tam) 0.111 Ana + Tam 288 (270-305) -3 Antithrombin-III (%) Ana 96 (92, 99) -3 Ana vs Ana + Tam 0.757 Tam 92 (89-95) -3 Tam vs (Ana or Ana + Tam) 0.089 Ana + Tam 95 (92-98) -5

NOTE: Bold P values are statistically significant after adjustment for multiple comparisons. *P < 0.01 for the time by treatment interaction.

2 poor metabolizers and 23 extensive metabolizers in the ta- 12 months was no more significant after excluding CYP2D6 moxifen arm (data not shown). Table 4 shows median and in- poor metabolizers. Although the frequency of poor metaboli- terquartile range of endoxifen and biomarkers of tamoxifen zers in our study was comparable with the general population activity in a combined analysis of the subjects on tamoxifen (ta- and therefore the power of the analysis is low, these results in- moxifen alone and the combination arm) according to CYP2D6 dicate that the reduction of endoxifen levels may be related to phenotype. Endoxifen levels were lower in poor metabolizers the patient phenotype and not to the drug combination. relative to extensive metabolizers (median concentration at Importantly, the addition of low-dose tamoxifen to ana- 12 months, 4.63 ng/mL in extensive metabolizers versus strozole provided a significant anti-resorption effect on bone 3.67 ng/mL in poor metabolizers). Notably, in tamoxifen users, as shown by the blunting effect on C-telopeptide modulation of IGF-I/IGFBP-3 and estradiol/SHBG ratio showed and osteocalcin increase induced by anastrozole alone. As ob- a different trend based on the genotype of the subjects, with no served previously (27), an increase in C-telopeptide levels by evidence of reduction of IGF-I/IGFBP-3 and estradiol/SHBG ra- ∼20% after 1 year of anastrozole treatment was associated tio in poor metabolizers at 12 months. No difference in endo- with ∼50% higher bone fracture risk in the Anastrozole, Ta- metrial thickness between the two phenotypes was noted moxifen Alone or in Combination trial, where the annual (Table 4). bone fracture rate in the intervention period was 2.93% in the anastrozole arm versus 1.90% in the tamoxifen arm Discussion (28). Also, a decrease in osteocalcin during different drug in- terventions is associated with a bone formation effect in sev- Aromatase inhibitors have become a standard endocrine eral prospective studies (29–31). treatment of postmenopausal women with breast cancer (2– Tamoxifen added to anastrozole induced a significant decline 4) and are being assessed in at-risk individuals, but there is con- of free IGF-I and did not significantly affect the estrogen drop cern on their long-term safety profile, particularly at bone level. induced by anastrozole alone, whereas cardiovascular risk bio- Our proof-of-principle trial aimed at determining whether, in markers were not differently modulated by the addition of low- contrast to the standard dose of 20 mg/d, the addition of a dose tamoxifen. The 8% decline of IGF-I in the combination weekly low dose of tamoxifen might not impair anastrozole arm may be clinically relevant, because several prospective stud- availability but, in fact, improve its safety profile. ies have shown an association between higher IGF-I and IGF-I/ Our results indicate that the addition of 10 mg/wk tamoxifen IGFBP-3 levels and breast cancer risk (18, 32). Interestingly, we does not affect anastrozole bioavailability as measured by serial found a clear association between biomarker modulation by ta- plasma concentrations up to 12 months. Conversely, endoxifen moxifen and patient CYP2D6 phenotype, with no evidence of a levels were lower in the combination arm, but the difference at decrease of IGF-I/IGFBP-3 and estradiol/SHBG ratio in poor

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Fig. 2. Serum levels of C-telopeptide (CTX; A), osteocalcin (B), IGF-I/IGFBP-3 (C), and estradiol/SHBG (D) according to treatment group. P values for contrasts between arms at 12 mo.

metabolizers. In a recent model applied to an adjuvant endo- dometrial cancer. CYP2D6 genotype did not influence endome- crine trial (33), it was shown that tamoxifen could be as effec- trial thickness changes under tamoxifen, but the power of this tive as letrozole if poor metabolizers were excluded. observation is too limited to draw reliable conclusions. Notably, no increase in endometrial thickness with the addi- Our findings confirm the ability of a weekly administration tion of tamoxifen to anastrozole alone was detected, whereas of 10 mg tamoxifen to exert significant biological effects. In a tamoxifen alone was associated with a higher thickness. In a previous dose-ranging trial in healthy hormone replacement small subset of women with available endometrial tissue, how- therapy users (21), 10 mg/wk tamoxifen mainly exerted anti- ever, tamoxifen addition induced a higher proliferation of both estrogenic effects on biomarkers of breast carcinogenesis and glandular and stromal compartments relative to anastrozole no increase in endometrial proliferation and menopausal alone. Yet, the expression of Ki-67 in the endometrial gland symptoms. Conversely, in the present study, tamoxifen exhib- was as low as 2% to 3%, much lower than that observed in nor- ited mild estrogenic effects in an estrogen-deprived environ- mal endometrium adjacent to endometrial cancer (34), or un- ment such as that attained by anastrozole alone. Likewise, at der treatment with 20 mg/d tamoxifen in hyperplastic or bone level, the drug has some antiestrogenic effects in premen- premalignant tissue (35, 36), suggesting that the addition of opausal women and estrogenic effects in postmenopausal low-dose tamoxifen should not lead to an increased risk of en- women, including a significant bone fracture risk reduction

Table 4. Endoxifen and biomarker levels (median and interquartile range) according to CYP2D6 phenotypes in all tamoxifen users

Months Phenotype n Median Q1 Q3

Endoxifen (ng/mL) 6 Extensive metabolizers 43 4.62 3.52 5.62 12 Extensive metabolizers 42 4.63 2.98 6.62 6 Poor metabolizers 7 5.00 4.18 6.79 12 Poor metabolizers 7 3.67 3.03 5.13 IGF-I/IGFBP-3 0 Extensive metabolizers 43 72.11 58.37 98.69 12 Extensive metabolizers 42 58.80 47.47 79.47 0 Poor metabolizers 7 77.91 52.20 94.53 12 Poor metabolizers 7 71.53 50.38 80.06 Estradiol/SHBG 0 Extensive metabolizers 43 0.19 0.13 0.28 12 Extensive metabolizers 42 0.15 0.11 0.24 0 Poor metabolizers 7 0.14 0.11 0.21 12 Poor metabolizers 7 0.15 0.12 0.16 Endometrial thickness (mm) 0 Extensive metabolizers 37 2.3 1.6 3 12 Extensive metabolizers 35 2.4 1.8 3.7 0 Poor metabolizers 5 2 1.9 3.6 12 Poor metabolizers 5 3.1 1.8 4.1

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Anastrozole and Low-Dose Tamoxifen

(37, 38). Moreover, endometrial changes during tamoxifen dif- association with biphosphonates and this may lead to a benefit fer significantly in premenopausal versus postmenopausal also in terms of breast cancer outcome (44, 45). In the preven- women, with an increased endometrial cancer risk observed tion setting, the use of biphosphonates to compensate bone loss predominantly after the menopause (39, 40). Another factor in- by aromatase inhibitor is also being investigated with initial fluencing tamoxifen estrogenicity is the dosage, where a daily promising results (46). Our results provide an alternative strat- dose reduction from 20 to 1 mg tends to attenuate its estrogenic egy, the addition of a very low dose of tamoxifen that, based on effects in postmenopausal women without impairing its anti- surrogate endpoints, counteracts the accelerated bone resorp- proliferative effects on breast cancer cells (10). tion induced by anastrozole and maintains a favorable profile The results of the present study may help to interpret the for breast cancer risk biomarkers. In line with this approach, a complex results of the Anastrozole, Tamoxifen Alone or in further option may contemplate the use of a lower aromatase Combination trial, where the combination arm was inferior inhibitor dose or the addition of low-dose estrogen to aroma- to anastrozole alone and possibly slightly worse than the ta- tase inhibitors (47). In the prevention setting, where safety is moxifen arm alone in terms of disease-free survival, underlying as important as efficacy, a drug combination approach may be the clinical relevance of the hormonal environment and the more reasonable as largely proven in cardiovascular medicine dose in determining tamoxifen efficacy, an issue that has not where control on multiple pathways reaches better results and sufficiently been addressed thus far. At a 20 mg/d dose, tamox- attenuates adverse events (48). The lesson from selective COX-2 ifen was shown to lower anastrozole concentrations by 30%, inhibitors has shown that a superselective approach, albeit very although this effect did not impair the estrogenic drop induced effective, may even become harmful, especially in cancer pre- by anastrozole (9) and has not been considered important to vention where long-term intervention is required (49). explain the lower efficacy of the combination arm. A second, Our study has several limitations, including lack of blinding most likely, explanation encompasses the shift to a predomi- and of more definitive breast cancer biomarkers such as mam- nant estrogenic effect of tamoxifen when administered at the mographic density or tissue samples. Moreover, we were unable dose of 20 mg/d in an estrogen-deprived environment, thus ex- to measure bone mineral density changes pretreatment and plaining why the combination arm (weak estrogen) was not post-treatment. Despite these limitations, our proof-of-principle only inferior to the anastrozole arm (pure ) but study shows that the combination of a standard dose of ana- also to the tamoxifen arm (partial antiestrogen) as regards strozole with a low, weekly dose of tamoxifen does not affect disease-free survival. Our findings indicate that a dose reduc- anastrozole blood concentrations, is safe and well tolerated, tion of tamoxifen by ∼15 times still retains some desirable es- and can diminish anastrozole adverse effects at bone level in trogenic properties when added to anastrozole alone, such as postmenopausal women. These findings provide the rationale decreased bone turnover and increase SHBG levels, suggesting for larger clinical studies in both the prevention and the adju- that the combination may have a more favorable pharmacody- vant setting. namic profile over anastrozole alone. The right balance between estrogenic and antiestrogenic ef- fects in both pharmacologic treatment and prevention of breast Disclosure of Potential Conflicts of Interest cancer is the subject of intense debate. In the advanced setting, No potential conflicts of interest were disclosed. prolonged tamoxifen use leads to resistant tumors in animal models and in humans mostly because of the emergence of tamoxifen-dependent clones, known to respond to aromatase Acknowledgments inhibitor treatment (41). However, a prolonged estrogen depri- vation, such as during anastrozole treatment, may induce resis- We thank Astra Zeneca for providing at no cost anastrozole tablets and tance for the emergence of estrogen hypersensitive clones (42), its plasma level measurements; Medical Systems for providing instruments for CYP analysis; Lega Italiana per la Lotta contro i Tumori, Italian Ministry which may paradoxically respond to estrogen restoration (43). of Health (Ricerca Finalizzata); and Fondazione Italiana per la Ricerca sul In the adjuvant setting, the results of a recent trial indicate that Cancro for financial support; and Giorgia Bollani for excellent contribution the bone loss induced by anastrozole can be prevented by the as data manager.

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Randomized Biomarker Trial of Anastrozole or Low-Dose Tamoxifen or Their Combination in Subjects with Breast Intraepithelial Neoplasia

Bernardo Bonanni, Davide Serrano, Sara Gandini, et al.

Clin Cancer Res 2009;15:7053-7060. Published OnlineFirst November 3, 2009.

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