Goetz, Matthew P.

Abstract Background. Half of all breast cancers are early stage, lymph node negative, and hormone receptor positive. A 21-gene (Oncotype DX®; Genomic Health, Inc., Redwood City, CA) recurrence score (RS) is prognostic for recurrence and predictive of chemotherapy benefit. We explored the ability of oncologists to predict the RS using standard prognostic criteria. Methods. Standard demographic and tumor prognostic criteria were obtained from patients with an available RS. Two academic pathologists provided tumor grade, histologic type, and hormone receptor status. Six academic oncologists predicted the RS category (low, intermediate, or high) and provided a recommendation for therapy. The oncologists were then given the actual RS and provided recommendations for therapy. Analysis for agreement was performed. Results. Thirty-one cases, including nine additional cases with variant pathology reads, were presented. There was substantial agreement in oncologists' ability to discriminate between true low or true intermediate and true high (κ = 0.75; p < .0001). Predictions between low and intermediate were not consistent. The most common discrepancies were predictions of a low RS risk when cases were true intermediate and predictions of an intermediate RS risk when cases were true low. The actual RS resulted in a change in the treatment recommendations in 19% of cases. Of the 186 scenarios and six oncologists in aggregate, five fewer chemotherapy recommendations resulted with the actual RS. Conclusions. Oncologists are able to differentiate between a low or intermediate RS and a high RS using standard prognostic criteria. However, provision of the actual RS changed the treatment recommendations in nearly 20% of cases, suggesting that the RS may reduce chemotherapy use. This effect was observed in particular in intermediate-risk cases. Prospective clinical trials are necessary to determine whether decisions based on the RS change outcomes.

Abstract Background: Controversy exists regarding the association between CYP2D6 enzyme activity and tamoxifen effectiveness in the adjuvant treatment of invasive breast cancer; however, this association in the primary prevention of breast cancer is unknown. Methods: We conducted a nested case–control study in the context of the NSABP P1 and P2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, and metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both noninvasive and invasive) while on 5 years of selective estrogen receptor modulators therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was conducted for alleles associated with absent (*3, *4, *5, and *6), reduced (*10, *17, and *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded. Results: A total of 591 cases were matched to 1,126 controls and DNA was genotyped in more than 97%. In patients treated with tamoxifen, there was no association of CYP2D6 genotype [OR (extensive/poor metabolizer): 0.90; 95% CI: 0.46–1.74, P = 0.74), use of a potent CYP2D6 inhibitor (OR 0.92; 95% CI: 0.575–1.486), or CYP2D6 metabolizer status (OR 1.03; 95% CI: 0.669–1.607) with breast cancer occurrence. Likewise, there was no association between any CYP2D6 metabolism parameter with breast cancer events in raloxifene-treated patients. Conclusion: In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy. Clin Cancer Res; 17(21); 6944–51. ©2011 AACR.

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.