Ingle, JN

Abstract Background Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We have recently demonstrated that endoxifen is the most active tamoxifen metabolite with regard to inhibiting the growth of ERα+ breast cancer cells and these studies have led to the development of endoxifen as a novel anti-breast cancer drug for which first-in-human studies are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast (OB) and osteoclast (OC) maturation and gene expression were monitored by cell differentiation assays and real-time PCR. Dual-energy X-ray absorptiometry (DXA), peripheral Quantitative Computed Tomography (pQCT) and micro-Computed Tomography (μCT) were used to determine changes in bone density, mass and architecture following 45 days of oral endoxifen administration (50mg/kg/day) to 3-month-old ovariectomized (OVX) C57BL/6 mice relative to vehicle control treated animals. Alterations in the numbers and activity of OBs and OCs were determined by histomorphometry and serum levels of P1NP and CTX-1 respectively. Results: Endoxifen treatment of mouse derived bone marrow stromal cells and human OBs led to significant increases in the expression of critical bone marker genes such as Runx2, osterix, osteocalcin, osteoprotegerin and alkaline phosphatase in a dose dependent manner. Daily administration of endoxifen to OVX mice led to significant increases in total body bone mineral density (BMD) (6%) and content (BMC) (9%), which was accompanied by a 50% decrease in fat tissue mass as determined by DXA. pQCT analysis of the tibial metaphysis revealed dramatic increases in BMD (35%) and BMC (20%), as well as trabecular density (52%), cortical content (62%), cortical area (60%) and cortical thickness (78%). μCT analysis of the femoral metaphysis revealed increases in bone volume/total volume (200%), trabecular number (38%) and trabecular thickness (18%), as well as decreased trabecular spacing (29%). Interestingly, there was nearly a 50% increase in the numbers of OCs derived from endoxifen treated mice which was associated with elevated expression of OC marker genes such as NFATcl, RANK, c-fms and cathepsin-K compared to control treated animals. Approximately 4 times as many OBs and OCs were observed on the bone surfaces of endoxifen treated mice which correlated with nearly 2-fold increases in serum levels of the bone formation (P1NP) and resorption (CTX-1) markers. Conclusions: These data are the first to demonstrate that endoxifen has anabolic effects on the mouse skeleton which are similar to that of estrogen. Additionally, these data reveal that endoxifen's mechanism of action in bone is different than that reported for tamoxifen and other selective estrogen receptor modulators in mice as it increases, rather than decreases, bone formation and remodeling. Therefore, the use of endoxifen for the treatment of endocrine responsive breast cancer may avoid the detrimental skeletal effects of many conventional endocrine therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-09.

Abstract Background Aromatase inhibitors (AIs) are standard adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer. We previously reported that racial/ethnic minority women receiving an AI in NCIC CTG MA.17 experienced fewer hot flashes, fatigue, and arthralgias than Caucasians (Moy B et al. Ann Oncol 2006;17:1637). Here we examined whether race affected clinical outcomes in the MA.27 AI trial comparing exemestane (E) with anastrozole (A). Methods: Fisher's exact test was used to compare observed side effects (0 vs grades 1–5) between minority and Caucasian women. Adverse events (AE), including menopausal symptoms, were assessed according to the Common Terminology Criteria of the National Cancer Institute (version 3.0). ITT univariate test of race effects on overall survival (OS) was determined with a stratified log-rank test, while multivariate testing was with stratified Cox regression. Results: Among 7312 for whom race was known, distribution was: Caucasian (n=6939; 95%); black (n=235; 3%); Asian/Native Hawaiian/Pacific Islander (n=93); 1%; American Indian (n=39; 1%); and mixed race (n=6). Among women treated with E, minorities reported a significantly lower incidence of hot flashes (45% vs. 56%; p=0.003) and fatigue (34% vs. 46%; p=0.001) compared to Caucasians. Similarly with A, minority women reported significantly fewer hot flashes (47% vs. 58%; p=0.002) and lower cholesterol (12% vs. 18%; p=0.01); however, they reported more headaches than Caucasians (16% vs. 10%; p=0.01). Caucasian women were more likely to discontinue therapy due to AE or side effects (32% vs. 24%). There was a significant OS interaction (p=0.02) between race and treatment with minority women on E having fewer deaths than those on A. Caucasian women had HR of E to A of 0.98 (95% CI 0.81−1.20), p=0.85, while minorities had HR of E to A of 0.72 (95% CI 0.33−1.58), p=0.41. Conclusions: Minority women tolerated AIs better and were also more compliant than Caucasians, supporting our previous findings. A significant interaction between race and type of AI therapy for OS was seen, favoring exemestane in minority women. Replication of these findings in larger cohorts is necessary. Since race/ethnicity may serve as a surrogate for genetic differences, our findings suggest pharmacogenomic differences which require further research. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-14-02.

Abstract Background: The non-steroidal reversible aromatase inhibitor (AI), anastrozole (A) is approved as adjuvant monotherapy in hormone receptor (HR) positive early breast cancer (EBC). The steroidal irreversible AI exemestane (E) is approved in sequence after an initial 2-3 years of tamoxifen. MA27 tested the hypothesis that exemestane would have greater efficacy and better end-organ safety than anastrozole for several reasons: E is an irreversible and more potent AI than A; unlike A, E does not induce intra-tumoral aromatase and; through its mild androgenic activity E may exert a second anti-tumor effect and a more favorable bone and lipid metabolism profile than A. Initially the study also tested the role of a cyclooxygenase-2 inhibitor, celecoxib, used in combination with an AI. However, celecoxib was discontinued early in MA27 following identification of cardiovascular toxicity with COX-2 inhibitors. Methods: Postmenopausal women with hormone receptor positive primary breast cancer were randomized after adequate local treatment to 5 years of adjuvant A (1mg/day) or E (25mg/day) with or without celecoxib/placebo (400mg/plac twice daily for 3 years). The primary objective is the comparison of event free survival (EFS) between women treated with A or E. Secondary objectives include overall survival (OS), distant recurrence (DDFS), incidence of contralateral breast cancer (CBC) and safety. Quality of Life in a subset of women has been evaluated, and a very extensive tissue bank created. Stratification included: prior chemotherapy; lymph node status, celecoxib (during use), aspirin use (during randomization to celecoxib), and trastuzumab use (since November 2005). The primary endpoint (EFS) will look for an improvement from 87.5% to 89.9%; HR 0.80, 2-sided 5% level and 80% power, with 644 events required. Results: 7576 women were randomized between June 2003 and July 2008. Celecoxib/placebo, was discontinued 2 years into the trial in December 2004 after 1635 patients had received celecoxib. The number of events required for final analysis was reached April 2010. Patient characteristics were balanced for age (median 64.1yrs); race; ECOG PS; mastectomy; TNM staging and; prior adjuvant chemotherapy. EFS, OS, DDFS, and CBC as well as clinical fractures, cardiovascular events and adverse events will be presented by arm in the final analysis. Conclusions: NCIC CTG MA.27 is the first definitive EBC trial comparing a non-steroidal and steroidal AI as initial adjuvant therapy. Exemestane is not currently approved as initial adjuvant treatment and superiority or equivalence will lead to its inclusion as a first-line option for EBC in postmenopausal HR positive breast cancer. Efficacy, clinical fractures, cardiovascular events and AI related symptoms will help to define the cost-benefit ratios of these two AIs. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S1-1.

Abstract Background: A major issue surrounding the use of therapeutic drugs for the treatment of breast cancer is the eventual development of resistance. Endoxifen, the most potent tamoxifen metabolite, is being developed as a novel endocrine therapy for the treatment of endocrine responsive breast cancer patients. While numerous studies have investigated the process of tamoxifen resistance, no such data exist regarding the mechanism by which cells develop resistance to endoxifen. Here, we describe the development and characterization of a novel endoxifen resistant MCF7 breast cancer cell line and the identification of a specific treatment to effectively target these resistant cells. Methods: MCF7 cells were chronically exposed to concentrations of endoxifen previously demonstrated to be associated with the greatest reductions in estrogen stimulated proliferation and transcription (1000 nM). Changes in the physiological and molecular properties of these cells were monitored during the course of resistance using a wide range of techniques. Results: Following 15 months of endoxifen exposure, an epithelial to mesenchymal transition (EMT) was induced in MCF7 cells, characterized by loss of E-cadherin expression, up-regulation of fibronectin and vimentin expression and increased responsiveness to TGFβ. Resistant cells exhibit a 7-fold increase in their proliferation rates relative to parental cells and display basal like properties (triple negative) due to silencing of ERα , progesterone receptor and CD24 expression. Resistant cells were confirmed to be estrogen insensitive through the use of cell proliferation and gene expression studies. Wound healing and cell migration assays revealed that resistant cells are highly aggressive with a significant level of metastatic potential. Microarray analysis revealed over 7500 genes to be differentially expressed in the resistant cell line relative to parental cells using a 2-fold cutoff, of which only 52 genes (0.7%) were determined to be up-regulated. Interestingly, a number of the most highly altered genes have previously been implicated in the development of EMT and/or resistance including Twist2, IGF binding protein 5, GATA and β-tubulin. Based on the identification of P-tubulin as the most up-regulated gene in resistant cells, 2-methoxyestradiol (2ME2) was recognized as a candidate drug to specifically target this resistant cell line due to it known roles in blocking tubulin polymerization. Indeed, 2ME2 exposure resulted in the induction of apoptosis and significant cell death in vitro. Conclusions: Chronic exposure of MCF7 cells to high concentrations of endoxifen led to induction of EMT with cells that are basal like, estrogen and SERM insensitive, highly TGFP responsive with significantly increased proliferation rates and metastatic potential relative to parental cells. These findings suggest that the mechanisms of resistance to endoxifen may differ from those observed with long term exposure to tamoxifen and have identified 2ME2 as a potentially successful alternative therapy for endoxifen resistant breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-11.

Abstract Background: NSABP P1 and P2 are the largest prevention trials of tamoxifen and raloxifene in women at high risk of developing breast cancer. Both agents have been shown to decrease, but not eliminate breast cancer risk in these women. The goal of our study was to identify single nucleotide polymorphisms (SNPs) associated with development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Methods: We performed a nested matched case-control genome-wide association study (GWAS) in subjects entered on the P2, which included only postmenopausal women, and subjects at least 50 years old entered on the P1 phase III prevention trials who were treated with tamoxifen or raloxifene. Cases were defined as subjects who developed invasive breast cancer or ductal carcinoma in situ and each was matched to two controls based on trial (P1, P2), age at trial entry, Gail risk score, history of lobular carcinoma in situ, and time on study. The study was restricted to the 94% of subjects self-identified as Caucasian. Genotyping was performed with the Illumina Human610-Quad chip. Eigenstrat analyses were performed to control for population stratification. SNPs were imputed within 200 Kb on either side of regions containing SNPs with p ≤ 3E-05. CYP2D6 genotyping was performed on all subjects. Results: The GWAS included 592 cases and 1171 controls. 547,356 SNPs were used in the analyses after removing 14,019 for call rates less than 98%, 30,560 for minor allele frequencies <1%, and 301 with Hardy-Weinberg equilibrium p-value <1E-06. Eigenvectors did not alter the results when used as covariates. Eleven genotyped SNPs with p-values ranging from 2.12E-06 to 9.98E-06 (conditional logistic regression with eigenvector adjustment) were identified; and 22 SNPs with p < 4E-05 were identified by imputation around these SNPs on chromosomes (Chr) 4, 8, 9, 13, 16 and 22, which were then genotyped with the Invader platform. Following imputation and genotyping, initial functional genomic studies have focused on 8 SNPs on Chr 16 (p-values 1.81E-06 to 9.55E-06, with the lowest p-value for an imputed and fine mapped SNP), all of which were in ZNF423, a gene known to encode a protein that associates with SMAD family members. In lymphoblastoid cell lines genotyped for the 2 top SNPs in ZNF423 (rs8060157 and rs11076499) and stably transfected with estrogen receptor-α, the variant and wild type SNPs displayed striking differences in estradiol-induced expression of ZNF423, SMAD3, BRCA1, and BRCA2. Conclusions: This GWAS revealed SNPs associated with the development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Genome-wide significance was not achieved, but SNPs in ZNF423 identified on Chr 16 were associated with differential expression of ZNF423, SMAD3, BRCA1, and BRCA2 in response to estradiol in vitro, providing direction for future research. (Supported in part by NIH grants U01GM61388, U01GM63173, P50CA116201, U10CA77202, U10CA37377, U10CA69974, U24CA114732, and the Biobank Japan Project funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-02.

Abstract Background: The MA.17 study showed that extended adjuvant endocrine therapy with letrozole (LET) after completing 5 years of tamoxifen (TAM) markedly reduced the risk of recurrence in women with ER+ early stage breast cancer and improved overall survival in women presenting with node +ve disease. The HOXB 13:IL17BR gene expression ratio (signature) has been shown to predict outcome in breast cancer patients treated with adjuvant tamoxifen monotherapy and provides additional information beyond that from known positive (ER and PR) and negative (Her-1 and Her-2) predictors of responsiveness to tamoxifen in node-ve women. We report a case control evaluation of the Breast Cancer Index (BCI; bioTheranostics, Inc.), which combines the HOXB13 and IL17BR twogene and the molecular grade index (MGI) gene expression signatures, with respect to distinguishing which patients are at risk of late recurrences and who would respond to extended endocrine therapy with LET. The prognostic and predictive utility of quantitative immunofluorescence of ER, PR, Her-2, tumor aromatase, COX-2, GATA3 and Nat1 in the TAM-PLACEBO and the TAM-LET cohorts will also be evaluated and compared to results derived by standard immunohistochemistry. Methods: FFPE tumor blocks were collected from patients who experienced a breast cancer recurrence up to unblinding of MA.17. Controls were matched 2:1 for age, tumor size, lymph node status, and prior chemotherapy, and were all disease free for longer than cases. All cases were reviewed for standard histopathology by two independent pathologists. RNA was extracted, amplified, converted to cDNA and subjected to RT-PCR with primers and probes to HOXB13, IL17BR, BUB1A, CENPA, NEK2, RACGAP1 and RRM2. ER, PR HER1, HER2, COX2, Aromatase, GATA3 and NAT1 will be analyzed by routine IHC techniques and by immunoflourescent Automated Quantitative Analysis (AQuA). Results: 105 cases and 210 matched controls are available for evaluation. All sections are under review and tissue microarrays have been performed on all cases and controls. Detailed results on the BCI and ER, PR, Her-2 will be available at the SABCS. Discussion: MA.17 has shown that extended adjuvant endocrine therapy after tamoxifen is effective at preventing disease recurrence given for an additional 5 years. Numerous clinical trials are exploring whether extending AIs will show this benefit, and there is an increasing need to improve the therapeutic index by distinguishing those at risk from those who are not. It is also important to determine which patients will benefit from the therapy and which will recur without benefit. The latter patients could be triaged to clinical trials of novel therapies to overcome endocrine resistance. This study will help to define these issues and pave the way for more effective selection of specific patients for adjuvant endocrine strategies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-26.

Abstract Background: The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity; however, much less is known about the role of ERß. In vitro studies demonstrate a tumor suppressive function for ERß, and we have recently implicated a role for ERα in sensitizing ERα expressing breast cancer cells to the anti-estrogenic effects of endoxifen. However, the in vivo relevance of ERα remains unclear due to conflicting reports. Here, we provide evidence that some of this controversy may be explained by variability in antibody specificity. In addition, we describe the development and characterization of a novel, highly specific monoclonal antibody and provide data regarding ERα expression in human breast cancers. Methods: Five commercially available ERα antibodies were screened for their sensitivity and specificity using western blotting, immunoprecipitation, immunofluorescence and immunohistochemistry in known ERα negative and positive cell lines as well as in normal human tissue samples. A novel monoclonal ERα antibody (C10) was developed and characterized in the same manner. Following identification of two specific antibodies, ERα expression was assessed in 66 breast tumors collected prior to adjuvant therapy. Samples were scored separately for nuclear and cytoplasmic staining. Results: In depth analysis of commercially available ERα antibodies reveled that the majority were non-specific with substantial cross-reactivity to ERα . Only one commercial antibody (PPG5/10), which solely recognizes full-length ERß, and our newly developed monoclonal antibody, which recognizes full-length and all 4 ERα variants, were determined to be sensitive and specific for ERα expression. These same two antibodies resulted in strong staining for endogenous levels of ERα protein in normal prostate tissue by immunohistochemistry. We further assessed these two antibodies in a set of breast tumors. Preliminary analysis revealed significant differences for ERα positivity between these two antibodies. Based on nuclear staining, 92% of tumors were ERα positive using the PPG5/10 antibody while only 34% were positive with C10. Approximately 50% of all tumors exhibited cytoplasmic staining with both antibodies. Conclusions: Our studies demonstrate that the majority of commercially available ERα antibodies are either non-specific or insensitive for the detection of ERα via immunohistochemistry. The present data call into question the relevance of prior studies which tested the association between clinical outcome and ERα expression and demonstrate the need to further analyze the role of ERα in breast cancer using highly specific and validated antibodies. While both the PPG5/10 and C10 antibodies are highly specific for ERß, the significant discrepancy in nuclear staining between them in breast tumors may be due to changes in epitope availability as a result of post-translational processing. Our newly developed C10 antibody could provide additional discriminatory features which may be useful in predicting response to therapy and/or associations with other clinicopathological factors and such studies are currently underway. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-25.

Abstract Background: Mammographic density, the variation in fat, epithelial and stromal tissues seen on screening mammography, is a strong risk factor for breast cancer and can be modified by hormonal agents. Changes in density from tamoxifen or postmenopausal hormone (PMH) use are associated with risk, suggesting that density may be a surrogate marker of therapeutic efficacy. Aromatase inhibitors (AIs) are given as adjuvant therapy in hormone receptor positive postmenopausal breast cancer and are known to decrease levels of estrone and estradiol in both serum and breast tissue. Our goal here was to examine the influence of AIs on mammographic density in women with early breast cancer. Methods: We conducted a case-control study of postmenopausal breast cancer patients initiating adjuvant AI therapy (anastrozole or exemestane) on protocols NCIC CTG MA27, NCCTG N063I and MC (Mayo Clinic) 0532. Eligibility included; an intact contralateral breast with no prior surgery; a screening mammogram within twelve months before AI initiation and at 9-15 months on therapy; no prior endocrine therapy and informed consent. Controls were sampled from the Mayo Mammography Health Study, a cohort of 19,924 receiving screening mammography at the Mayo Clinic, and matched to cases on age, prior PMH use, baseline body mass index (BMI) and interval between mammograms. Pre-treatment and on-study mammograms for cases (corresponding mammograms for controls) were digitized. Change in percent density was estimated on the craniocaudal view of the non-cancerous breast using two methods: a subjective assessment of change by an expert radiologist (within 5%; 5-10% increase, 10-25% increase, 25%+ increase, 5-10% decrease, 10-25% decrease and 25%+ decrease) and a quantitative assessment of absolute change using a computer-assisted thresholding program (Cumulus). Analyses compared magnitude of change in density by both the subjective and quantitative methods between cases and matched controls. Results: 574 pairs were eligible for analyses (MA27-505 cases; N063I-12 cases; MC0532-57 cases). Characteristics of the two groups are shown in the table below. Using either density estimation method, there was a greater decrease in density among women on AI therapy vs. matched controls. In 33% (95% CI: 29-37%) of pairs, there was at least a one greater category decrease for the case relative to her control by subjective estimation. In 14% (95% CI: 11-18%) of the pairs, there was at least a 5% greater decrease for the case relative to her control by quantitative estimation. Data will be available according to AI class (non-steroidal versus steroidal) in November. Conclusions: In the largest report to date to examine the influence of AI therapy on mammographic density, we provide evidence that AI is associated with decreases in density in a small subgroup of women. We are currently examining factors that influence these AI-associated decreases in density and whether these differences are unique to one class of AI. (Supported in part by NIH grants P50CA116201, U01GM61388, U10CA77202, U10CA25224) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-03.

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: 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.