Citations

Abstract Endoxifen (END), the active metabolite of tamoxifen (TAM), is currently being developed as a drug for the treatment of estrogen receptor positive breast cancer based on recent in vitro and clinical data demonstrating that TAM drug effect is substantially related to circulating plasma concentrations of END, a secondary metabolite produced by human CYP2D6 metabolism. Women with deficient CYP2D6 metabolism have lower END concentrations and a higher risk of breast cancer recurrence. We characterized the pharmacokinetics and oral bioavailability of END in female rats and dogs. We further compared END exposure following equivalent doses of END and TAM. Rats were administered 2 mg/kg i.v. END, 20 − 200 mg/kg oral END or 80 mg/kg oral TAM. Fasted dogs were administered 0.5 mg/kg i.v. END, 15 − 100 mg/kg oral END or 30 mg/kg oral TAM. Plasma END concentrations were also measured in rats or dogs after oral dosing once per day for 4 consecutive days. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. In the single-dose administration study, terminal elimination half-life and plasma clearance values in rats given 2 mg/kg i.v. END were 6.3 h and 2.4 L/h/kg, respectively. Plasma concentrations above 0.1 µM and 1 µM END were achieved with 20 mg/kg and 200 mg/kg doses, respectively, and those concentrations were maintained for longer than 24 h. Oral absorption of END was linear over the range of 20 − 140 mg/kg with a bioavailability greater than 67%. END exposure in rats following 80 mg/kg oral END was 100-fold greater than END exposure following 80-mg/kg oral TAM. Following 0.5 mg/kg i.v. END to dogs, terminal elimination half-life and plasma clearance values were 9.2 h and 0.4 L/h/kg, respectively. Plasma concentrations above 1 µM and 10 µM were achieved with the 15 mg/kg dose and 100 mg/kg, respectively, and those concentrations were maintained for longer than 24 h. Absorption was linear over the range of 15 − 100 mg/kg with a bioavailability greater than 50%. END exposure in dogs following 30 mg/kg oral END was 10-fold greater than END exposure following 30-mg/kg oral TAM. In the repeated-dose administration study, the day 4 END peak plasma concentration in rats increased from 1 to 9 µM as the daily END dose was increased from 20 to 200 mg/kg. The day 4 END peak plasma concentration in dogs exceeded 20 µM and plasma concentrations remained above 2 µM for 24 hours following 4 daily doses of 30 mg/kg END. Based on these data, we conclude that END has high oral bioavailability; high concentrations may be maintained after repeated dosing in dogs and rats; and substantially higher END concentrations are achievable following an oral dose of END in comparison to a similar dose of TAM. Supported by NCI contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2607.

Abstract In collaboration with NCI, we are developing endoxifen (END), the active metabolite of tamoxifen (TAM), as a drug for the treatment of estrogen receptor positive breast cancer. Tamoxifen treated women with reduced or absent CYP2D6 enzyme activity have significantly lower END plasma concentrations. In a retrospective analysis of two different prospective tamoxifen trials (NCCTG and ABCSG), we have demonstrated that genetic or drug-induced reductions in CYP2D6 metabolism are associated with a higher risk of breast cancer recurrence in Tam treated ER positive breast cancer. To test the hypothesis that clinically relevant concentrations and exposures of END are achieved following oral administration of END, we characterized the pharmacokinetics of i.v. and oral END and oral TAM in female mice. Endoxifen was synthesized and provided by NCI/DTP. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. Following i.v. administration of a 1 mg/kg dose, peak plasma concentration, terminal elimination half-life and plasma clearance values were 0.26 µM, 6.5 h and 11.8 L/h/kg, respectively. Oral pharmacokinetics and bioavailability were determined following doses of 50 and 200 mg/kg. Peak plasma concentrations of 0.76 µM were achieved with a 50 mg/kg dose and plasma concentrations above 0.1 µM were maintained for longer than 8 h. Peak plasma concentrations of 8 µM were achieved with a 200 mg/kg dose and plasma concentrations above 2 µM were maintained for 24 h. Bioavailability was 50% and greater than 100% following the 50 mg/kg and 200 mg/kg doses, respectively. Based on pharmacokinetic data for oral END and TAM normalized to a 50 mg/kg oral dose, END plasma concentrations and total exposure were 20-fold greater following oral END as compared to oral TAM. In conclusion, END has high oral bioavailability in mice and substantial plasma concentrations are achieved and maintained after a single oral END dose. Compared to an equivalent oral dose of Tam, substantially higher END concentrations are achieved following oral END. These data suggest that the primary administration of End may overcome the limitations related to human CYP2D6 metabolism, and support the ongoing development of endoxifen as a primary therapy for ER breast cancer. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3603.

Abstract Background: We have previously demonstrated that endoxifen is the most potent tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing ERα (Wu, Hawse et al. Cancer Res 2009;69:1722). These studies support the clinical findings which demonstrate that women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. More recently, data has emerged from several laboratories suggesting that expression of ERβ in breast tumors leads to increased benefits from tamoxifen therapy. However, the role of ERβ in mediating endoxifen action has yet to be explored.Methods: Using U2OS osteosarcoma cells, as well as Hs578T and MCF7 breast cancer cells, stably expressing ERβ, we examined the effects of endoxifen on ERβ protein levels by western blotting, ERβ transcriptional activity by real-time RT-PCR, and on ERβ inhibition of breast cancer cell growth through the use of proliferation assays.Results: We have discovered that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. We have also found that endoxifen is a more potent inhibitor of estrogen induced gene expression in MCF7 cells stably expressing ERβ. Concentrations of endoxifen that mimic patients with impaired CYP2D6 activity (20-40 nM) do not significantly alter E2-induced cell proliferation in MCF7 cells expressing only ERα. However, these concentrations of endoxifen significantly reduce the estrogen induced proliferation rate of MCF7 cells expressing ERβ.Conclusions: Our data demonstrate that endoxifen stabilizes ERβ protein, induces ER heterodimerization and more potently inhibits the actions of estrogen in breast cancer cells expressing ERβ. The presence of ERβ sensitizes cells to the anti-estrogenic effects of endoxifen and growth inhibition occurs at substantially lower endoxifen concentrations (20-40 nM) than in cells that express only ERα (100 nm). Our data suggest that tamoxifen therapy may be beneficial even in patients with reduced CYP2D6 metabolism when ERβ is expressed. These studies also suggest that the increased effectiveness of tamoxifen therapy in patients whose tumors express ERβ may be due to the effects of endoxifen. Finally, these findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 405.

Abstract Background: An estimated 1.3 million women worldwide will be diagnosed this year with early stage breast cancer. The majority of women have estrogen receptor (ER) positive breast cancer and are candidates for endocrine therapy. Tamoxifen remains the most commonly employed agent worldwide and substantially reduces recurrence and mortality. Tamoxifen is a pro-drug. CYP2D6 converts tamoxifen to endoxifen, which is primarily responsible for inhibition of estrogen-stimulated breast cancer growth in vitro. There have been conflicting reports regarding the relationship between CYP2D6 genotype/phenotype and tamoxifen clinical outcome. The ITPC was established to collect the worldwide experience relating to genetic variation in CYP2D6 and the outcomes of women with ER positive breast cancer treated with adjuvant tamoxifen. Establishment of the predictive value of CYP2D6 would enable the a priori identification of patients that could be spared ineffective therapy.Methods: Individual patient data including tumor characteristics, genotype, medication data, and recurrence information were requested from all published and non-published reports of clinical outcomes related to CYP2D6 and deposited at PharmGKB. CYP2D6 genotype was available for the following null (*3, 4, 5 and 6) and IM (*10, 17 and 41) alleles. CYP2D6 inhibiting medication data was requested for weak and potent CYP2D6 inhibitors. The primary analysis was to determine the association of CYP2D6 phenotype (combined genotype and medication) with breast cancer recurrence in tamoxifen treated early stage, ER positive invasive breast cancer.Results: At this time, the ITPC has received patient data on 3432 patients from the U.S., Sweden, UK, Germany, Belgium, and Japan. Median age at diagnosis was 62 years. CYP2D6 genotype was available in 3,392 (99%). The allele frequency for CYP2D6*4, the most common allele associated with the CYP2D6 poor metabolizer phenotype, was q=0.23. A total of 1152 recurrences representing first events (either local, regional, distant, or contralateral breast cancer) were recorded. At the time of abstract submission, patient and genotype data from an additional 1300 tamoxifen treated patients were pending submission.Conclusions: The ITPC represents the largest collection of individual patient data in regard to the potential importance of CYP2D6 phenotype in adjuvant tamoxifen therapy. The findings from this consortium are expected to substantially improve the global understanding of CYP2D6 and tamoxifen clinical outcome. Final results will be presented at the meeting Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 33.

Abstract Background. Third generation aromatase inhibitors (AIs) have been shown to be superior to tamoxifen in multiple randomized clinical trials and become the new standard of treatment for postmenopausal women with early or advanced endocrine-sensitive breast cancer. It is thus important to identify biomarkers predicting resistance to AIs so that alternative therapeutic options can be employed. A two gene expression index, HOXB13:IL17BR, and HOXB13 in particular, has been shown to be a negative predictor of clinical benefit from tamoxifen. It is unclear whether HOXB13:IL17BR or HOXB13 is also predictive of AI therapy.Methods. We performed a retrospective analysis of a phase II randomized neoadjuvant study of everolimus+letrozole vs. placebo+letrozole in patients with estrogen receptor-positive breast cancer. Total RNA was extracted from frozen tissue biopsies taken either prior to treatment or after 15 days of treatment. HOXB13:IL13BR and a molecular grade index (MGI) were measured in 107 pre-treatment samples and 111 samples after 15 days of treatment. Antiproliferative response as defined by a ln[Ki767] <1 at day 15 in the samples analyzed was correlated with these gene expression indices.Results. In the placebo+letrozole treatment arm, the antiproliferative response rate was 30% (14/47). Using a pre-specified cutpoint of 0.06, the response rate was 40% for low HOXB13:IL17BR patients, compared to 12% for high HOXB13:IL17BR patients (p=0.05). In the everolimus+letrozole arm, the antiproliferative response rate in the samples analyzed was 55% (33/60), and the response rates for low and high HOXB13:Il17BR patients were 70% and 37% (p=0.02), respectively. When examined separately, high HOXB13 expression was significantly associated with nonresponse to therapy (everolimus+letrozole arm, p=0.02; placebo+letrozle arm, p=0.03), but IL17BR was not (everolimus+letrozole arm, p=0.6; placebo+letrozle arm, p=0.88). As expected, we observed a significant correlation between MGI and Ki67 staining (Spearman r=0.67, p<0.0001).Conclusions. High HOXB13:IL17BR index and high HOXB13 expression may identify a possible subset of patients with estrogen receptor-positive tumor that could be less responsive to aromatase inhibitors such as letrozole. Furthermore, use of MGI may enable rapid assessment of antiproliferative response by fine needle aspiration. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2120.

Abstract Background: Endoxifen, the active metabolite of tamoxifen (Tam), is currently being developed as a drug for the treatment of estrogen receptor (ER) + breast cancer (BC). HER2 expression in ER+ BC is associated with Tam resistance, and in vivo Tam administration to mice bearing ER+/HER2+ xenografts stimulates BC growth (Shou, JNCI 2004). In humans, endoxifen is the most important Tam metabolite responsible for inhibiting estrogen induced BC growth (Wu, Cancer Research 2009). CYP2D6 metabolism affects the concentrations (conc) of endoxifen (Stearns, JNCI, 2003) and associated with worse disease free survival (DFS) in Tam treated BC (Goetz, JCO, 2005). We sought to determine the activity of Tam and its metabolites in ER+/HER2+ BC cell lines and to evaluate the role of CYP2D6 metabolism in Tam-treated patients (pts) with ER+/HER2+ BC. Additionally, we sought to determine endoxifen conc in mice administered oral Tam.Methods: MCF7 (parental and HER2-expressing) and BT474 (ER+/HER2+) cells were used to compare the activity of Tam, 4HT, and endoxifen on estrogen- stimulated growth. Oral tam PK were characterized in mice treated with standard dose of Tam (4 mg/kg; 100 μg). Clinical data were obtained via a retrospective analysis of Tam-treated pts with ER+/ HER2+ BC randomized to receive 5 years of Tam (NCCTG 89-30-52). CYP2D6 metabolism (extensive or decreased) was based on CYP2D6 genotype (*3, 4, 6, 10, 17, 41) and co-administration of a CYP2D6 inhibitor (yes/no). HER2 was determined by immunohistochemistry (IHC) or FISH (tumors 0, 1, or 2+ by IHC). The association between CYP2D6 and DFS was assessed using the log-rank test and proportional hazards modeling.Results: Compared to Tam, endoxifen potently inhibited the growth of estrogen- stimulated BT474 cells. In MCF7 cells, expression of HER2 shifted the conc of endoxifen required for 50% inhibition of growth (IC50) from 54 nM (parental) to 131 nM (HER2 expressing). Using the range of conc of Tam and its metabolites observed in humans (Tam, 300-500 nM; 4HT, 5-10 nM; and endoxifen, 10-200 nM), only endoxifen potently inhibited estrogen- stimulated growth of MCF7HER2+ cells and only at conc achievable in CYP2D6 extensive metabolizers (>50nM). In mice, conc of 4HT and endoxifen were below 15 nM following an oral dose of 4 mg/kg. In NCCTG 89-30-52, both CYP2D6 phenotype and HER2 status was determined in 201/256 randomized pts. HER2 was expressed in 23/215 (11%) but not associated with DFS overall (p=0.62). In the HER2+ subset, pts with decreased CYP2D6 metabolism (n=10) had significantly shorter DFS compared to extensive metabolizers (n=9) (HR 9.5, p=0.03; 95% CI 1.16-76.9).Conclusions: Our in vitro and clinical data provide a simple pharmacological model for understanding HER2 resistance in Tam-treated breast cancer. Mice, which lack the CYP2D6 enzyme, may not be an appropriate model for understanding tam resistance given low conc of both 4HT and endoxifen. Given that the in vitro conc of endoxifen needed to inhibit the growth of ER+/HER-2+ BC are achievable in only a subset of humans (CYP2D6 extensive metabolizers), the primary administration of endoxifen could overcome de novo Tam resistance in ER+/HER2+ BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2006.

Abstract Abstract #57 Background: ABCSG 8 is the only prospective trial examining the value of switching patients (pts) to an aromatase inhibitor after tamoxifen (Tam) in which randomization occurred prior to therapy. This trial demonstrated that 2 years (yrs) of Tam followed by 3 yrs of anastrozole (TAM to A) was superior to 5 yrs of Tam (Jakesz SABC 2005). Tam is metabolically activated by CYP2D6 to endoxifen and pts with impaired CYP2D6 metabolism have a higher risk of recurrence (Goetz JCO 2005). The HOXB13/IL17BR (H/I) index (Ma Cancer Cell 2004) is associated with Tam resistance while a 5-gene molecular grade index (MGI) distinguishes outcome in grade II tumors. H/I and MGI are complementary prognostic factors that define low (low MGI), intermediate (low H/I, high MGI) and high risk groups (high H/I and MGI) (Ma Clin Cancer Res 2008). We sought to validate these biomarkers in ABCSG Trial 8, and hypothesized that they may predict the benefit of sequencing TAM to A versus Tam alone.
 Methods: From 1996-2004, ABCSG 8 randomized 3,901 pts with ER positive breast cancer. The primary endpoint was event-free survival. Assuming 40% of pts had reduced CYP2D6 metabolism, we designed a nested case control study in which 110 Tam treated cases matched to 2 controls would yield 80% power to detect a relative risk of 2.3 for pts with reduced CYP2D6 metabolism. Additionally, we sought to perform an exploratory analysis of the benefit of switching from Tam to A in pts with impaired vs non-impaired CYP2D6 metabolism. DNA and RNA derived from paraffin embedded sections were used to genotype for CYP2D6 and to obtain H/I and MGI RT-PCR profiles.
 Findings: Thus far, CYP2D6 genotyping is complete in 345 pts corresponding to 53 cases/106 controls (Tam) and 62 cases/124 controls (Tam to A). DNA and RNA yields were excellent with a CYP2D6 genotype call rate of >95%. CYP2D6 allele frequencies were as expected: *3 (.01), *4, (.23), *6 (.01), *10 (.02), *17 (.00), and *41 (.10). 44% of pts had reduced CYP2D6 metabolism (at least one CYP2D6 null allele or intermediate metabolizer) and 8% were CYP2D6 poor metabolizers. For H/I and MGI, 340 pts were profiled with 64%, 20% and 16% in the low, intermediate, and high risk groups, respectively. Tissue sectioning/genotyping for additional cases/controls is ongoing.
 Discussion: This is the first retrospective analysis of a large Tam versus Tam to A trial to evaluate the association between 1) CYP2D6, HOXB13/IL17BR, and MGI with TAM treatment outcome and 2) the predictive benefit of these biomarkers in pts randomized to switch from Tam to AI versus Tam alone. Final results will be presented at the meeting. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 57.

Abstract Abstract #404 Background: The oral HDACi vorinostat (V) sensitizes breast cancer cells to tubulin polymerizing agents and anti-VEGF directed therapies in vitro. We sought to determine the safety and efficacy of V plus paclitaxel (P) and bevacizumab (B) as first-line therapy for MBC.
 Methods: Eligible patients (pts) with measurable disease, ECOG PS 0 or 1, and no prior chemotherapy for MBC, received P (90mg/m2) on days 1, 8, 15 and B (10mg/kg) on days 1,15 every 28 days, plus V 200 mg (N=3) or 300 mg (N=28) BID orally for 3 days given the day before, day of, and day after each P dose. Response was assessed after every 3 cycles (RECIST). The trial was designed to distinguish between a response rate of < 40% vs. > 60% (alpha=0.10, beta=0.10) using Simon's optimal 2-stage design. At least 12 responses were required in the first 28 evaluable pts in the 1st stage and 21 of 41 in the 2nd stage.
 Results: There were no dose-limiting toxicities among the first 6 pts treated at the 1st (200 mg BID; N=3) and 2nd (300 mg BID; N=3) dose levels. Among the 26 evaluable pts treated at the recommended phase II dose of 300mg bid in the 1st stage, 14 patients had a partial response (PR-54%; 90% confidence interval [C.I.] 36%-71%), 6 had stable disease (SD) at 3 months and remain on treatment, 2 had SD as the best response with progressive disease (PD) at 11.8 and 15 months, and 4 had PD as their best response. Additionally, 2 of 3 pts treated at the first V dose level had a PR, indicating PR in 16 of 29 overall (55%, 90% C.I 38%-71%). Only 11 of 29 evaluable pts (38%) have progressed after a median followup of 10.5 months (R-2.3-14.9). Two pts had tumor biopsies and peripheral blood mononuclear cells (PBMC) collected before and 4 hours after the 3rd V dose (300 mg BID) which showed increased acetylation of K69 lysine residue of the chaperone protein Hsp 90, upregulation of Hsp70, and downregulation of AKT by Western Blot, providing evidence of Hsp90 inhibition in tumor and PBMC.
 
 Gr 3-4 toxicities included neutropenia (26%), neuropathy (23%), fatigue (16%), thrombosis (10%), vomiting (6%), hypertension (3%), and diarrhea (3%).
 Conclusions: Our preliminary findings indicate that the HDACi V inhibits Hsp90 and downregulates AKT in breast cancer in vivo, and enhances the effectiveness of P plus B in MBC. Accrual is nearly complete and the final efficacy analysis will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 404.

Purpose We performed a case-control genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with musculoskeletal adverse events (MS-AEs) in women treated with aromatase inhibitors (AIs) for early breast cancer. Patients and Methods A nested case-control design was used to select patients enrolled onto the MA.27 phase III trial comparing anastrozole with exemestane. Cases were matched to two controls and were defined as patients with grade 3 or 4 MS-AEs (according to the National Cancer Institute's Common Terminology Criteria for Adverse Events v3.0) or those who discontinued treatment for any grade of MS-AE within the first 2 years. Genotyping was performed with the Illumina Human610-Quad BeadChip. Results The GWAS included 293 cases and 585 controls. A total of 551,358 SNPs were analyzed, followed by imputation and fine mapping of a region of interest on chromosome 14. Four SNPs on chromosome 14 had the lowest P values (2.23E-06 to 6.67E-07). T-cell leukemia 1A (TCL1A) was the gene closest (926-7000 bp) to the four SNPs. Functional genomic studies revealed that one of these SNPs (rs11849538) created an estrogen response element and that TCL1A expression was estrogen dependent, was associated with the variant SNP genotypes in estradiol-treated lymphoblastoid cells transfected with estrogen receptor alpha and was directly related to interleukin 17 receptor A (IL17RA) expression. Conclusion This GWAS identified SNPs associated with MS-AEs in women treated with AIs and with a gene (TCL1A) which, in turn, was related to a cytokine (IL17). These findings provide a focus for further research to identify patients at risk for MS-AEs and to explore the mechanisms for these adverse events.