Ingle, JN

Abstract Abstract #6037 Background: Tamoxifen (Tam) is biotransformed to the potent antiestrogen, endoxifen, by the CYP2D6 enzyme. We previously demonstrated that patients (pts) receiving adjuvant TAM with impaired CYP2D6 metabolism due to CYP2D6 (*4) and/or concurrent administration of a CYP2D6 inhibitor had a higher risk of recurrence. Other studies suggest CYP2C19*17 (Schroth JCO 2007) and SULT1A1*2 (Nowell JNCI 2002) may be associated with treatment outcome. With extended follow-up, we sought to evaluate the importance of comprehensive CYP2D6 genotyping as well as the potential association between CYP2C19*17 and SULT1A1 copy number with clinical outcome in pts randomized to TAM in NCCTG 89-30-52.
 Methods: Using DNA derived from paraffin embedded sections, CYP2D6 and SULT1A1 genotype were determined using quantitative multiplex PCR and CYP2C19 by sequencing. Pts administered the following CYP2D6 inhibitors (fluoxetine, paroxetine, sertraline, cimetidine, amiodarone, doxepin, ticlopidine and haloperidol) were considered as intermediate (IM) or poor metabolizers (PM) based on the potency of CYP2D6 inhibition. CYP2D6 phenotype was defined as follows: extensive metabolizers (EM) were pts not administered an inhibitor who did not carry a null allele (*3, *4, *6) and who were not homozygous for an IM allele (*10, *17, *41). CYP2D6 IM were either heterozygous for a null allele or homozygous for an IM allele but not administered an inhibitor or CYP2D6 EM administered a weak inhibitor. PM were pts homozygous for a null allele, or any patient administered a potent inhibitor. The association between genotype or CYP2D6 phenotype and clinical outcome was determined using the log-rank test. Multivariate Cox modeling was performed using traditional prognostic factors.
 Results: The median follow-up of living pts is now 14.5 years. Of 256 pts. randomized to TAM, genotype was determined for CYP2D6 (n=210), CYP2C19*17 (n=170), and SULT1A1 (n=169) with the following allele frequencies: CYP2D6 *3 (.02), *4(.20), *6 (.01), *10 (.03), *17 (.00), *41(.08), and CYP2C19*17 (.22). The frequency of SULT1A1 copy number alleles (CNA) was 1 (4%), 2 (67%), 3 (20%), and 4+ (9%). 14/227 patients (6%) were administered an inhibitor. A multivariate analysis accounting for nodal status and tumor size demonstrated that compared to CYP2D6 EM, CYP2D6 PM had significantly shorter time to recurrence (TTR) (HR 4.0, p=0.001) and DFS (HR 2.0, p=0.02) and CYP2D6 IM tended to have shorter TTR (HR 1.8, p=0.08) and DFS (HR 1.4, p=0.10). DFS did not differ by SULT1A1 copy number (p=0.62) or CYP2C19 *17 (p=0.47)
 Conclusion: Long term follow-up of pts in NCCTG 89-30-52 confirms the importance of CYP2D6 metabolism and further demonstrates the importance of comprehensive genotyping and phenotyping with CYP2D6 inhibitors. We could not identify an association between CYP2C19*17 or SULT1A1 copy number with recurrence but further evaluation is needed in larger cohorts. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6037.

Abstract Abstract #4033 Background: Aromatase activity within the breast is a source of estrogen that may cause breast cancer. Mammographic density (MD) is a risk factor for breast cancer whose biologic basis is unknown. Our study compared aromatase expression in tissue from dense and non-dense areas of the breasts of healthy volunteers.
 Methods: Participants were 40+ yrs, had a screening mammogram with visible MD, no history of cancer and were not on endocrine therapy. Ultrasound-guided core biopsies were done within 6 months of mammography to obtain three paired cores from mammographically dense and non-dense regions of the breast. Immunostaining for aromatase expression employed the streptavidin-biotin amplification method using the recently developed 677 mouse monoclonal antibody. Immunoreactivity (IR) was scored in terms of proportion of cells staining positive for aromatase (PPC) (0=<1%, 1=1-25%, 2=26-50%, 3=51+% (and 4=75%+ for adipocytes)) and relative immunointensity (0=none, 1=weak, 2=moderate, 3=intense) for each cell type (stroma, normal ductal epithelium, adipocytes). A composite score weighting the PPC by their relative intensity (range 0-9; 0-12 for adipocytes) was also calculated. The sum of the composite score across the three cell types provided a global assessment of aromatase IR. Repeated measures analyses evaluated differences in aromatase IR for dense compared to non-dense tissue within and across cell types. Parameter estimates (β) indicated the average difference between dense and non-dense IR.
 Results: 18 (37%) of the 49 participants were premenopausal (median age 46 yrs). Summing across cell types, the global composite score showed increased aromatase IR on sections sampled from dense vs. non-dense regions (β=5.3,p<0.001). This global difference reflected increased aromatase IR in the stroma and normal ductal epithelium from dense sections (vs. non-dense). For 42 women with >1% stromal cells on each section, there was evidence for increased IR on dense sections as indicated by the greater PPCs (β=0.9), relative intensity (β=0.7) and composite score (β=2.7)(p's<0.001). Similarly, for 20 women with >1% normal ductal epithelium in both dense and non-dense sections, there was a greater composite score (β=1.4,p=0.004) for cells on dense sections although differences for PPC (β=0.3,p=0.11) and relative intensity (β=0.3,p=0.09) did not reach statistical significance. No differences were seen in IR for adipocytes from the two density regions (46 women). Findings were unchanged with adjustment for covariates, including proportions of each cell type per section.
 Conclusions: There is strong evidence for increased aromatase expression in the stromal and normal ductal epithelium of dense vs. non-dense tissue. These results support ongoing research into mammographic density as a biomarker of effect of aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4033.

Abstract Abstract #2131 Introduction: TAS-108 is an oral steroidal anti-estrogen agent that selectively inhibits ERα and is mainly metabolized by CYP3A4. The purpose of this study is to investigate the efficacy and safety of TAS-108 administrated orally in three dose levels in patients with locally advanced, locally recurrent inoperable or metastatic breast carcinoma (BC) in four countries (USA, Russia, Mexico and Chile).
 Methods: Postmenopausal women with confirmed ER and/or PgR positive BC who had previously responded to one or two standard endocrine therapies, with or without one prior chemotherapy were randomly assigned to three doses of TAS-108, 40 mg, 80 and 120 mg daily. Using a modified Panageas' optimal two-stage trinomial design, the enrollment of 60 evaluable patients (first-stage: 19) was required to each individual dose group. Tumor response was assessed every 8 weeks according to RECIST criteria. Adverse events (AEs) were graded by CTC-AE v3.0.
 Results: A total of 146 patients with mean age of 63 years old were enrolled with 61 patients in the 40 mg group and 66 in the 80 mg group. The 120 mg group was terminated at the end of stage 1 with 19 patients enrolled due to lack of efficacy. The mean duration of study treatment was 172 days for the 40 mg group and 160 days for the 80 mg group. Partial response (PR) was documented in 6 (10%) patients in the 40 mg group and 4 (6.7%) patients in the 80 mg group. The rate of disease stabilization (CR+PR+SD) reported by the investigators was 43% (95%CI, 31%,56%) in the 40 mg group and 45% (95%CI, 32%,58%) in the 80 mg group. Adjudicated clinical benefit (CR+PR+SD more than 24 wks) was observed in 22% of patients in the 40 mg group and 20% of patients in the 80 mg group. Clinical benefit was achieved in 25% of patients with 1 prior hormonal therapy and 15% of patients with more than one line of prior hormonal therapy. Median time to progression (TTP): 15 weeks for the 40 mg group and 15.9 weeks for the 80 mg group. Median duration of clinical benefit was 32 weeks in the 40 mg group and 64 weeks in the 80 mg group. In the 40/80/120 mg groups, the commonly reported treatment-related AEs included nausea (15%/11%/16%), fatigue (10%/11%/16%), headache (10%/6%/16%), hot flushes (10%/5%/32%), diarrhea (2%/6%/5%), constipation (3%/3%/5%), and arthralgia (2%/5%/11%). No endometrial cancer and treatment-related deaths occurred during the study.
 Conclusions: TAS-108 has demonstrated anti-tumor activity in this population and was generally well tolerated. The 40 mg dose was chosen as the recommended dose for future clinical evaluation in patients with advanced breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2131.

Abstract Abstract #19 Background: Tamoxifen (TAM) is a standard endocrine therapy for the treatment of women with estrogen receptor (ER) positive breast cancer. TAM is activated by the cytochrome P450 2D6 enzyme system into two potent and active metabolites, 4-hydroxytamoxifen (4HT) and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). While human concentrations of 4HT are negligible and vary little in plasma (5-10 nM), endoxifen concentrations vary widely (10-180 nm), and women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. Despite these observations, endoxifen's contribution to tamoxifen's overall drug effectiveness is uncertain.
 Methods: Using cells endogenously expressing ERa (MCF7, T47D) and cells stably transfected with ERa (Hs578T and U2OS), we examined the relative effects of TAM and its primary metabolites on ERa protein levels by western blotting, ERa transcriptional activity by luciferase reporter assays and real-time RT-PCR, and on ER positive breast cancer cell growth through the use of proliferation assays.
 Results: We have discovered that endoxifen induces ERa protein turnover through proteasomal degradation similar to that of ICI in a concentration and time-dependent manner. These findings are in stark contrast to TAM, N-desmethyl-tamoxifen (NDT) and 4HT, which stabilize the ER. Optimal degradation occurs only at endoxifen concentrations observed in human CYP2D6 extensive metabolizers (> 40 nM) and persists even in the presence of TAM (300 nM), 4HT (7 nM), and NDT (700 nM) at concentrations observed in patients receiving tamoxifen therapy. In contrast, reducing endoxifen concentrations to those observed in a CYP2D6 poor metabolizer (20 nM), without altering TAM, 4HT, and NDT, results in ER stabilization. High endoxifen concentrations (100-1000 nM) completely block estrogen (E2)-induced ER transcriptional activity even in the presence of TAM, 4HT, and NDT, while low endoxifen concentrations (20-40 nM) do not. Further, low concentrations of endoxifen (20 nM) do not significantly alter E2-induced cell proliferation; however, high concentrations of endoxifen (100-1000 nM) completely block this process. Discussion: Our data demonstrate that endoxifen is a potent anti-estrogen that targets ERa for proteasomal degradation, blocks ERa transcriptional activity and inhibits E2-induced breast cancer cell proliferation. Importantly, these effects of endoxifen are observed at concentrations found in CYP2D6 extensive metabolizers and are maintained even in the presence of TAM, 4HT and NDT. These studies suggest that endoxifen may be the primary metabolite responsible for tamoxifen's effectiveness in the treatment of ER positive breast cancer and provide the impetus to evaluate whether TAM-related side-effects, such as thrombo-embolism and endometrial hyperplasia/carcinoma, are inversely associated with a patient's ability to metabolically activate tamoxifen. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 19.

Abstract Abstract #12 Background: Meta-analyses (EBCTCG, Lancet 2005; 365:2687) have established in estrogen receptor (ER) positive breast cancer (BC) that 5 yrs of adjuvant tamoxifen (Tam) substantially reduces recurrence and mortality. The aromatase inhibitors (AIs) anastrozole, exemestane and letrozole have been studied in prospective trials in postmenopausal patients (pts) in comparison with Tam either as initial monotherapy (Cohort 1) or after 2 to 3 years of Tam in a switching strategy (Cohort 2); both to a total of 5 years (yrs) of endocrine therapy.
 Methods: Outcomes data submitted to EBCTCG were used in separate meta-analyses of Cohort 1 (trials: ATAC, BIG 1-98/IBCSG 18-98) and of Cohort 2 (trials: ABCSG 8, ARNO 95, IES/BIG 2-97, ITA). The primary analysis involved pts with tumors reported to be ER positive with endpoints of BC recurrence (all, local only, contralateral BC only, distant), death with and without recurrence, any death. Subgroup analyses included progesterone receptor (PgR) status, age, tumor grade and nodal status. All p-values were 2-sided.
 Results: Highlights include: Cohort 1: 9,856 pts with 50,000 woman-years of follow-up. At 5 yrs, AI therapy was associated with an absolute 2.7% (standard error [SE] 0.7) decrease in BC recurrence (10.7% vs 13.4%, relative decrease 20% [SE 5], p=0.00004). There appeared to be greater proportional decreases in isolated local recurrence (30% [SE 10], p=0.003) and in contralateral disease (38% [SE 12], p=0.003) than in distant recurrence (12% [SE 6], p= 0.04). AIs yielded an absolute 1.0% (SE 0.5) decrease in BC mortality (5.5% vs 6.5%, relative decrease 7% [SE 7], p=0.28). Cohort 2: 9,015 pts with 33,000 woman-years of follow-up. At 6 yrs from treatment divergence (i.e., 8-9 yrs from diagnosis), AI therapy was associated with an absolute 3.5% (SE 1.1) decrease in BC recurrence (12.6% vs 16.1%, relative decrease 29% [SE 6], p<0.00001). There appeared to be greater proportional reductions in isolated local recurrence (40% [SE 13], p=0.002) and in contralateral disease (35% [SE 16], p=0.03) than in distant recurrence (24% [SE 7], p= 0.001). AIs yielded an absolute 1.6% (SE 0.8) decrease in BC mortality (6.3% vs 8.0%, relative decrease 22% [SE 9], p=0.02). Subset analyses with respect to PgR status, age, tumor grade and nodal status revealed no apparent heterogeneity between the proportional reductions in recurrence and no indication of an increase or decrease in non-breast deaths with AIs in either cohort 1 or 2.
 Conclusions: In 2 separate meta-analyses considering both the initial monotherapy setting and the switching setting after 2 to 3 yrs of Tam, AIs produced significantly lower BC recurrence rates compared with Tam. Additional follow-up data will provide important information on long-term survival. Meta-analyses can be helpful even in the era of relatively large clinical trials. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 12.

Abstract Abstract #1134 Background: The selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene reduce the risk of ER+ (but not ER-) invasive breast cancers in healthy women at high risk for developing breast cancer. Aromatase inhibitors (AIs) given as adjuvant therapy to treatment-naïve or post-tamoxifen patients significantly reduce the risk of in-breast recurrences (IBRs) and contralateral breast cancers (CBCs) and are currently in clinical trials for breast cancer prevention (NCIC CTG MAP.3 and IBIS-II). It is hypothesized that SERMS inhibit promotion of ER+ breast cancer whereas AIs may reduce both ER+ and ER- breast cancer by inhibiting both tumor initiation and promotion. Little is known about the characteristics of IBRs and CBCs that arise on AI therapy. We present the ER/PR expression and clinicopathologic features of IBRs and CBCs that occurred on MA.17.
 Methods: We examined ER/PR status of IBRs and CBCs that arose on letrozole vs. placebo among women enrolled in MA.17, a placebo-controlled (PLAC) trial of letrozole (LET) following 5 years of tamoxifen in postmenopausal women with early stage breast cancer.
 Results: Seventy-one patients (pts) developed an IBR and 87 developed a CBC on trial. Consistent with results previously reported, fewer IBRs (LET 20 vs PLAC 51) and CBCs (LET 35 vs PLAC 52) were observed in the LET group. ER and PR status is currently available on 35 women with an IBR and 39 with a CBC. The majority of IBRs were ER+ in both the LET and PLAC groups (10/11 [91%] vs 18/24 [75%], respectively; p=NS) but numbers of both ER+ and – IBRs were less in LET group, suggesting that letrozole may decrease both ER+ and ER- IBRs. CBCs that arose on PLAC were more likely to be ER+ than on LET (16/22 [73%] vs 6/19 pts [32%], respectively; p=0.01), suggesting that letrozole predominantly prevents ER+ CBCs. Discordance in ER expression between primary breast cancer and IBRs among women randomized to LET vs. PLAC was observed in 1/11 [9%] and 6/24 [26%] women respectively (p=NS) and between primary breast cancer and CBCs in 12/18 pts [67%] vs. 6/21 [29%] women respectively (p=0.01). Other clinicopathologic characteristics such as grade, tumor size, PR, HER-2/neu, and nodal status of IBRs and CBCs will be presented at the meeting.
 Conclusion: Extended adjuvant endocrine therapy with letrozole results in fewer IBRs and CBCs compared with placebo as previously reported. Our data suggests that letrozole may decrease both ER+ and ER- IBRs. Letrozole appears to prevent ER+ CBCs but has little or no apparent effect on the development of ER- CBCs. These results need confirmation in the primary prevention trials of AIs.
 
 Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1134.