Search

Background: We recently showed that low dose (5mg/d) tamoxifen (babytam) for 3 years can halve the incidence of new breast neoplastic events in hormone sensitive or unknown breast pre-invasive neoplasia after surgery with limited toxicity (DeCensi et al JCO 2019; 37:1629-37). Here we report the results of circulating surrogate endpoint biomarkers of breast cancer risk with special attention to the risk of ovarian stimulation observed with the full dose in premenopausal women. Methods: Five hundred women 75 years old or younger were randomized to babytam or placebo (PLA). A subgroup of 406 women consented for collection of morning fasting serum at baseline (0), 1 (1Y) and 3 years (3Y) of treatment. There was a loss of about 25% of blood sampling at 3Y. Serum IGF-I, IGFBP-3, SHBG and C-reactive Protein (CRP) were performed on all available samples. Estradiol and testosterone were determined in a subsample (n=285) to determine the extent of ovarian stimulation by babytam in premenopausal women. We used Mann-Whitney test for univariate comparisons and linear regression modeling for multivariate analyses setting changes from baseline (1Y or 3Y minus baseline values) as dependent variable and treatment arm, age, BMI and baseline biomarkers values as explanatory factors. We tested treatment*menopausal status as interaction term. COX P-H model was used to calculate hazard ratio for CRP increase. Results: At baseline, all biomarkers were evenly balanced between arms (data not shown). IGF-I decreased significantly on babytam as compared to PLA. The difference of the changes between arms was -20 ng/mL after 1Y (p Citation Format: Harriet Johansson, Matteo Puntoni, Debora Macis, Valentina Aristarco, Aliana Guerrieri-Gonzaga, Davide Serrano, Silvia Caviglia, Laura Cortesi, Cristiana Taverniti, Antonio Ponti, Maria Grazia Pacquola, Marcella Gulisano, Fabio Falcini, Maria Digennaro, Anna Carriello, Katia Cagossi, Graziella Pinotti, Tania Buttiron Webber, Matteo Lazzeroni, Bernardo Bonanni, Luca Boni, Andrea DeCensi. Effects of low dose tamoxifen on circulating risk biomarkers in a phase III trial in breast pre-invasive disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD3-08.

Background: Neoadjuvant endocrine therapy (NET) with gonadotropin-releasing hormone (GnRH) agonist and aromatase inhibitors is effective in selected premenopausal patients (pts). Degarelix, an antagonist of GnRH, has immediate onset of action through binding to GnRH receptors in the pituitary gland and thereby suppressing the production of LH and FSH. Its suppressing activity in premenopausal women might be faster and free of estrodial breakthrough on continued treatment compared with a GnRH angonist, and thereby provide significant clinical value for pts who are candidates for short-term NET. Methods: Eligible pts were premenopausal women with cT2-4b, any nodal stage, ER and PgR >50%, HER2-negative (by IHC and/or ISH) breast cancer who were not candidates for breast conserving surgery. Premenopausal status was determined locally with estradiol (E2) levels >54 pg/mL (or >198 pmol/L), measured within 14 days prior to randomization. Pts were randomized 1:1 to Triptorelin (T) 3.75 mg i.m. on day 1 of every cycle or Degarelix (D) 240 mg s.c. given as two injections of 120 mg on day 1 of cycle 1, then 80 mg s.c. on day 1 of cycles 2-6 with letrozole (L) 2.5 mg/day for 6 cycles. Each cycle was 28 days. Definitive surgery was performed within 2-3 weeks after the last administration of T or D. Serum was collected prior to the first injection (baseline), 24 and 72 hours, 7 and 14 days, then prior to injection on day 1 of cycles 2-6. The primary endpoint was time to optimal ovarian function suppression (OFS) calculated as time from the first injection of D or T to the first assessment of centrally assessed 17-β-estradiol (E2) level in the range of optimal OFS (≤2.72 pg/mL or ≤10 pmol/L) during the 6 cycles of NET. The trial had 90% power to detect a difference using a logrank test, 2-sided α=0.05. Secondary endpoints included tolerability, Ki67changes, PEPI score, best overall response. NCT02005887 Results: TREND completed accrual of 51 pts in January 2017. A preliminary analysis based on the first 45 pts is reported here. 89% of patients were ≥40 yrs, 76% had T1-2 and 22% T3 tumors, and 51% were node-positive. Dominant histology type was ductal (93%). The table summarizes centrally-assessed E2 according to treatment at baseline and for the first 5 assessment time points indicating immediate suppression for the D+L arm. E2 levels on day 1 of cycles 2-6 were all below the limit of quantification (0.625 pg/mL) for the D+L arm. For the T+L arm continued OFS was not maintained in 4 pts. BaselineCycle 1Cycle 2Day:01371429No. Pts D+L222221212221T+L232321232222Median (IQR) D+L96.2 (64.2,206.8)10.1 (4.0,21.8)0.6 (0.6,1.0)0.6 (0.6,0.6)0.6 (0.6,0.6)0.6 (0.6, 0.6)T+L85.1 (49.7,118.0)37.4 (17.9,59.2)12.8 (7.7,23.8)9.0 (1.2,29.7)0.6 (0.6,1.4)0.6 (0.6, 0.6) Conclusion: Evidence from this first analysis demonstrates rapid and maintained OFS with the combination of D+L as a NET in premenopausal breast cancer patients. The final analysis of the total population, including secondary endpoints, will be presented at the symposium. Citation Format: Colleoni M, Gray K, Munzone E, Dellapasqua S, Zamagni C, Gianni L, Johansson H, Viale G, Kammler R, Maibach R, Rabaglio-Poretti M, Di Leo A, Coates AS, Gelber RD, Regan MM, Goldhirsch A. A randomized phase II trial evaluating the endocrine activity and efficacy of neoadjuvant degarelix versus triptorelin in premenopausal patients receiving letrozole for primary endocrine responsive breast cancer (TREND; IBCSG 41-13) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-06.

Background Polymorphisms of genes involved in estrogen production have been linked to breast cancer risk, prognosis and treatment response. Polymorphisms of the aromatase gene CYP19A1 influence its activity. The UGT2B17 catalyzes glucuronic acid transfer to a variety of substrates, including steroids and drugs like the aromatase inhibitor exemestane. We investigated the impact of two variants of CYP19A1 (rs10046, rs4646) and the UGT2B17 gene deletion on disease outcome in 125 postmenopausal women operated for ER-positive primary breast cancer enrolled in a randomized pre-surgical trial. Patients Briefly, upon informed consent, postmenopausal patients with ER-positive breast cancer (stage T1–2, N0–1, M0) eligible for surgery were randomized to receive either exemestane (25 mg/day), or celecoxib (800 mg/day), or placebo for 6 weeks prior to surgery at the European Institute of Oncology (2004-2008). Exemestane showed a significant 10% absolute reduction in Ki67 labeling index compared to the other two arms. Serum and whole blood was taken at baseline and the day before surgery and stored at -80°C until assayed. Methods DNA was extracted from blood by QIAamp DNA Blood Kits. The CYP19A1 rs1004/rs46466 were analyzed by Taqman genotyping assays in real-time PCR. The UGT2B17 deletion was estimated by copy number assay (Lifetechnologies). Serum estradiol (E2) and estrone (E1) levels were measured by gas chromatography tandem mass spectrometry detection (GS-MS/MS) after liquid-liquid extraction. The lower limit of quantitation were 0.625 pg/mL for estradiol and 1.56 pg/mL for estrone. The association of genetic polymorphisms with “any event” was assessed by the Cox proportional hazards models adjusted for confounders. Results The genetic polymorphisms did not deviate from Hardy-Weinberg equilibrium (P-value >0.41) and minor allele frequency of rs10046 (A/G), rs4646 (C/A), and UGT2B17Del were 0.45, 0.22, and 0.31, respectively. The rs10046 A and rs4646 C alleles were associated with higher estrogen levels. Carriers of rs10046 AA had median levels of 7.57 pg/ml E2 and 35.9 pg/mL E1 versus 3,9 pg/mL E2 and 27.4 E1 pg/mL in CA/AA genotypes (P Conclusions Our analysis confirms previous findings of an association of CYP19A1 rs10046/rs4646 with estrogen levels in postmenopausal women. Interestingly, the carriers of the variants associated with lower estrogen levels at diagnosis had better prognosis. Further genomic profiling in larger trials aimed to enhance tailored treatment efficacy in endocrine-responsive postmenopausal breast cancer are warranted. Citation Format: Johansson H, Gandini S, Aristarco V, Macis D, Guerrieri-Gonzaga A, Serrano D, Pruneri G, Lazzeroni M, Viale G, Toesca A, Rajasekaran A, Bonanni B, DeCensi A. Impact of common polymorphisms of CYP19A1 and UGT2B17 gene deletion on early endocrine-responsive postmenopausal breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-08-05.

Introduction. DCIS is a potential precursor of invasive breast cancer (IBC). Immune escape might play a critical role in the transition from DCIS to IBC. We aim to identify an immune-related gene expression signature of DCIS to distinguish hazardous lesions, and to avoid the burden of overtreatment in women with less harmful DCIS. Since tumor-infiltrating lymphocytes (TILs) occur more frequently in hormone receptor-negative, Her-2 positive DCIS, we focused the analysis on this subtype. Methods. We performed a prospective nested case-control study involving women with pure DCIS (hormone receptor-negative, Her-2 positive, TILs ≥ 20%), treated with breast conservative surgery with or without radiotherapy. Cases were defined as women with DCIS developing subsequent ipsilateral breast event (IBE), controls as women with DCIS without IBE at 8 years of follow-up. Next-generation sequencing gene expression assay (Oncomine™ Immune Response Research Assay) targeting 395 genes associated with tumor-immune systems interactions was performed on RNA extracted from DCIS samples of 25 cases and 25 age- and radiotherapy-matched controls. In order to obtain reliable results, we considered adequate samples with mapped reads > 1 million and valid reads > 800.000. Normalized gene-level count data generated from the run were further processed with Affymetrix™. A Volcano plot was also employed in order to distinguish genes differently expressed between cases and controls according to the log-gene-level fold change and the ANOVA test (p values < 0.05). Univariate (Log-rank) and multivariate Cox proportional hazard models identified genes associated with the disease free survival. Results. Thirty-one samples (15 cases; 16 controls) reached the quality parameters. The analysis identified 11 genes with a significant different expression between cases and controls. The upregulation of three immune-related genes (VTCN1, NT5E and CMKLR1) remained significantly associated with disease free survival. Notably, VTCN1 and NT5E expressions were significantly associated with the risk of invasive local recurrence. Conclusions. This exploratory analysis of hormone receptor-negative, Her-2 positive DCIS with TILs ≥ 20% showed significant differences in immune-related gene expression profiles between women with subsequent IBE and controls. VTCN1 and NT5E may play an important role in immune surveillance mechanisms of DCIS and may be targeted by immune-based therapy to slow or prevent DCIS progression. These results are well worth further validation on future studies. Citation Format: Matteo Lazzeroni, Caternia Fumagalli, Federica Corso, Alberto Ranghiero, Debora Macis, Elena Guerini Rocco, Valentina Aristarco, Harriet Johansson, Aliana Guerrieri-Gonzaga, Davide Serrano, Andrea DeCensi, Sara Gandini, Massimo Barberis, Bernardo Bonanni. Identification of an immune gene expression signature for progression of breast ductal carcinoma in situ (dcis) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1705.

Background:The assessment of breast cancer (BC) risk is a key step for an effective preventive treatment. Besides the established risk assessment models, validation of independent predictive factors such as circulating biomarkers would improve patient selection and treatment efficacy. Obesity and metabolic imbalance play an important role in BC risk in menopausal women. The role of adipocytes in energy homeostasis is currently under investigation for their emerging relationship with BC. Adipokines (such as leptin and adiponectin) are linked to insulin sensitivity and have been related to BC risk and prognosis. Adiponectin, a peptide hormone secreted by the adipose tissue, has been inversely related to BC risk both in observational studies and in a phase II chemoprevention trial in premenopausal women at increased risk. Aim:We measured baseline serum adiponectin and leptin levels as well as HOMA index, in 534 postmenopausal women enrolled in 16 Italian centers and randomized in one of the two international phase III trials for BC prevention -the IBIS-II(Prevention) and IBIS-II(DCIS) trials- to assess whether these biomarkers were different in the healthy women at increased risk for BC cohort compared to the DCIS cohort. Methods:Healthy postmenopausal women (aged 40-70) at increased risk for BC (on an age-dependent risk model; n=186) or DCIS patients who underwent radical surgery in the previous 6 months (n=348) were eligible according to the two separate protocol entry criteria. At baseline, fasting blood was collected, processed and stored at -80°C till biomarkers measurement. Insulin and glucose levels were measured with the Architect system (Abbott Laboratories, Abbott Park, IL 60064 USA). Serum adiponectin and leptin levels were determined with Immunoassays by R&D (Minneapolis, USA). Results:Participant characteristics and biomarker levels (median, IQ range) by disease status are reported below. Table 1Healthy (n=186)DCIS (n=348)Age at entry59 (55, 63)60 (56, 65)BMI (kg/m2)25.2 (22.9, 28.4)25.0 (22.4, 28.1)Adiponectin (ng/mL)13063 (10279, 18157)11498 (7722, 16909)Leptin (pg/mL)16181 (9594, 26391)17284 (9675, 26173)L/A ratio1.33 (0.60, 2.09)1.46 (0.69, 3.19)Glucose (mg/dL)89 (81, 97)93 (86, 103)Insulin (μU/mL)6.6 (5.0, 9.6)7.5 (5.4, 10.6)HOMA index1.39 (1.04, 2.02)1.79 (1.18, 2.61)L/A, Leptin/Adiponectin ratio Adiponectin was significantly negatively correlated with leptin, L/A ratio, HOMA index and BMI, while leptin was positively correlated with L-A ratio, BMI, HOMA. Logistic regression has been used and Odds Ratios (ORs) have been calculated to assess the association of DCIS with biomarkers.DCIS patients were significantly more frequent in the lowest quartile of adiponectin compared to the highest quartile (60% vs 75%; OR=2.49; 95% CI, 1.39-4.44, p= 0.0003) adjusting for center, BMI, HOMA index and age. Conclusions:Low serum adiponectin levels in postmenopausal women are more frequent in DCIS patients compared to healthy at risk subjects independently of BMI, HOMA index and age and results are similar to premenopausal women. Future investigations in both trials will assess whether adiponectin is also associated with BC events. Acknowledgments: J.Forbes and T.Howell, Co-Chairmen,IBIS-II Steering Committee. Citation Format: Aliana Guerrieri-Gonzaga, Debora Macis, Sara Gandini, Valentina Aristarco, Harriet Johansson, Giorgia Bollani, Teresa Roth, Maria-Teresa Sandri, Jill Knox, Jack Cuzick, Bernardo Bonanni. Low serum adiponectin level is an independent risk factor of DCIS in postmenopausal women at increased risk of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-16.

Background: Single nucleotide polymorphisms (SNPs) of the aromatase enzyme (CYP19A1) and estrogen receptor alpha (ESR1) may be associated with breast cancer susceptibility and endocrine-mediated side effects. The IBCSG Tamoxifen (Tam) and Exemestane (Exe) Trial (TEXT) includes a translational research project to assess whether selected SNPs may influence treatment efficacy and toxicity. We report on early-onset hot flashes and sweating (HF/S) and musculoskeletal symptoms (MS; myalgia, arthralgia, stiffness) with respect to CYP19A1 and ESR1 SNPs under combined endocrine therapy. Patients and Methods: 2,672 premenopausal women with HR+ early breast cancer were randomized to treatment with the GnRH-agonist triptorelin (Trip)+Tam or Trip+Exe for 5 years. Randomization was stratified according to intended use of chemotherapy (yes/no) and lymph node status (N- vs N+). Estrogen-depletion side effects (HF/S and MS) were recorded at baseline, 3-monthly during the first year and 6-monthly thereafter using the NCI CTCAE v3.0. DNA was centrally extracted from whole blood with Qiagen kits. SNPs of CYP19A1 (rs4646 and rs10046) and ESR1 (rs207764, rs2234693 and rs9340799) were analyzed by a pyrosequencing method (Diatech Pharmacogenetics S.r.l., Jesi, Italy). Control genotypes (wt/wt; wt/v; v/v) for all SNPs were processed in each run. Logistic regression was used to analyze two endpoints: presence or absence of grade (gr) 2-3 HF/S during first 6 months and gr 2-4 MS during first 12 months. Four genetic models were used to explore associations with side effects: genetic (wt/wt; wt/v; v/v), additive, dominant and recessive. Results: DNA was isolated and genotyped for 1970 (74%) consenting women. Clinical characteristics and outcomes of this cohort were consistent with the overall trial. At baseline median age was 44, median BMI 24 kg/m2 and 86% had regular menses. During follow-up, 43% reported gr 2-3 HF/S and 27% reported gr 2-4 MS. The 5 SNPs did not deviate from Hardy-Weinberg equilibrium (p>0.30) and minor allele frequency ranged from 29%-48%. The CYP19A1 SNP rs10046 (C>T) was associated with gr 2-3 HF/S. Specifically, women with variant homozygote genotype (T/T) had a reduced risk of HF/S (39% T/T vs 45%). The univariate odds ratio (OR) was 0.77 (95%CI: 0.62-0.96; p=0.02) compared with other variant groups. The multivariate model showed consistent results after adjusting for age, BMI, menstrual status, chemo use, treatment allocation (Tam vs Exe) and baseline HF/S. The other 4 SNPs were not associated with the selected side effects. Conclusions: Our analysis indicates association of HF/S with CYP19A1 rs10046 genetic variants. The lack of association between early-onset of selected estrogen-depletion side effects and the other 4 SNPs may be masked by the concurrent Trip. Ovarian reserve before treatment may also influence the impact of SNP genotypes on early-onset side effects. Further analysis with details of concurrent medications and treatment adherence will contribute to the interpretation of these results. Funded by Susan G. Komen for the Cure. Citation Format: Harriet Johansson, Kathryn P Gray, Olivia Pagani, Meredith M Regan, Giuseppe Viale, Valentina Aristarco, Debora Macis, Antonella Puccio, Susanne Roux, Rudolf Maibach, Marco Colleoni, Manuela Rabaglio-Poretti, Alan S Coates, Richard D Gelber, Aron Goldhirsch, Roswitha Kammler, Bernardo Bonanni, Barbara A Walley. CYP19A1 and ESR1 polymorphisms and selected early-onset side effects during combined endocrine therapy in the IBCSG TEXT trial for premenopausal women with hormone receptor-positive (HR+) early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-01.

Background Exemestane (EXE) is an aromatase inactivator used in the prevention and treatment of breast cancer (BC). The majority of EXE and its active metabolite 17-dihydroEXE are excreted as glucuronide conjugates by uridine diphosphate glucuronosyltransferase (UGT). The UGT2B17 enzyme is the most expressed in human liver. A deletion spanning the entire UGT2B17 gene (*2) decreased the EXE glucuronidation process by 14-fold in human liver microsomes (HLM) from UGT2B17(*2/*2) genotype subjects compared to wild-type UGT2B17(*1/*1) HLMs. Aim The aim of this study was to investigate whether the UGT2B17 deletion is associated with increased serum levels of EXE and 17-dihydroEXE and to assess if this deletion predicts the anti-proliferative effect of EXE in BC tissue as measured by Ki67 changes 6 weeks apart. Methods In a phase II pre-surgical trial, 50 postmenopausal women with histologically-confirmed ER positive BC; stage T1-2, N0-1, M0 were assigned to EXE 25 mg/d for 6 weeks before surgery. Morning fasting blood was collected at baseline (B) and after 6 weeks and stored at -80° C until assayed. Time of last drug intake was collected at blood draw. DNA was extracted from whole blood (Qiagen, Italy). We used Taqman copy number variation assay (Life Technologies, Monza, Italy) for the UGT2B17 genotyping. EXE and 17-dihydroEXE concentrations were determined by mass spectrometry (MS) using Waters® Xevo™ TQ MS in electrospray positive ionization mode (Waters, Manchester, UK), optimized by multiple reaction monitoring mode. We used Zorbax Eclipse Plus C18 columns and mobile phase MeOH/H2O with 0.1% formic acid. EXE pure substance was provided by Pfizer Inc.,17β-hydroxy EXE and EXE-19-d3 purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Wilcoxon Signed Rank Test was used to assess differences in serum concentrations of EXE, its metabolites, and post-pretreatment tissue Ki67 changes according to UGT2B17 genotypes. Results Median age and BMI were 62 years and 26 kg/m2, respectively. The UGT2B17 genotype (n=50) was 24 homozygote wt (*1/*1), 20 heterozygote (*1/*2) and 6 homozygote variant (*2/*2). Minor allele frequency = 0.32. Hardy-Weinberg Equilibrium (P=0.57) was respected. Median and interquartile ranges (IQRs) of EXE and 17-dihydroEXE at 6 weeks, by time elapsed since last drug intakeTime since last drugEXE (nM)17-dihydroEXE (nM)≤24 hours (n=32)10.25 (5.55, 17.25)2.75 (1.85, 3.6)>24 hours (n=15)2.10 (1.20, 2.20)*1.50 (1.20, 2.00)*** P=.0001 vs ≤24h; **P=.0003 vs ≤24h ; Median and IQRs of EXE, 17-dihydroEXE and Ki67 change from B by UGT2B17 genotypesGenotypeEXE (nM)17-dihydroEXE (nM)Ki67 changeBMIUGT2B17 *1/*1 (n=22)5.3 (2.2, 11.4)1.85 (1.4, 3.06)-9 (-16, -5)28 (24, 32)UGT2B17 *1/*2, *2/*2 (n=25)9.3 (2.6, 17.6)*2.5 (2.0, 3.6)**-11 (-19, -3)♦26 (23, 30)♦♦P-values between genotype groups:*P=.28; **P=.04; ♦P=.82; ♦♦P=.41 Conclusions Our study demonstrates a significant association of the UGT2B17 deletion with increased serum concentrations of 17-dihydroEXE, irrespective of time since last drug intake. Neither 17-dihydroEXE, nor genotype explained the significant anti-proliferative effect of EXE on BC tissue. Larger studies should determine the clinical implications of this gene on EXE efficacy. Citation Format: Harriet Johansson, Valentina Aristarco, Jennifer Gjerde, Sara Gandini, Aliana Guerrieri-Gonzaga, Matteo Lazzeroni, Serena Mora, Debora Macis, Davide Serrano, Antonio Toesca, Luca Bottiglieri, Gunnar Mellgren, Giuseppe Viale, Andrea DeCensi, Bernardo Bonanni. A genetic deletion in the uridine diphosphate glucuronosyltransferase 2B17 affects the pharmacokinetics of exemestane [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-43.

Background: In a presurgical trial in 200 non-diabetic women with breast cancer, we previously showed a heterogeneous effect of metformin on the Ki67, with a decreased proliferation in women with insulin resistance (HOMA>2.8) and an opposite effect in women with HOMA≤ 2.8 (Bonanni et al. JCO 30:2593, 2012). Here we determined the effect of metformin on noninvasive proliferative disorders. Methods: Patients with operable breast cancer were randomly assigned to metformin, 850 mg or placebo once daily on days 1-3 followed by two 850 mg tablets after dinner on days 4 to 28. A total of 3-5 specimens of adjacent (≤1 cm from tumor) and distant (>1 cm from tumor) tissue were obtained from the surgical specimens to assess systematically the prevalence of LCIS and DCIS adjacent to invasive cancer and of distant ductal hyperplasia (DH) in normal tissue. We also determined the effect of metformin on Ki67 in these lesions overall and by molecular subtype. All HER2 2+ DCIS by IHC were assessed by FISH. Results: Overall, the prevalence of LCIS, DCIS and DH was 4.5% (9/200), 66% (132/200) and 35% (69/200), respectively. The Ki67 was positively associated with DCIS grade (p-trend Median (IQR) Ki67 level (%) in premalignant disorders by treatment armPremalignant groupPremalignant subgroupMetformin armPlacebo armp-value*p-interaction‡LCIS (n=9) 15 (5-15)5 (4-6)0.1 DH (n=69) 3 (1-4)3 (1-4)0.5 All DCIS (n=132) 12 (8-20)10 (7-24)0.9 DCIS grade 1/2 (n=108)10 (7-16)10 (6-17)0.90.2 DCIS grade 3 (n=24)33 (25-55)40 (32-40)0.2 HER2+ve (n=22)22 (11-32)35 (30-40)0.060.04 HER2-ve (n=58)16 (10-20)17 (8-26)0.7 ER+ve/HER2+ve (n=15)12 (7-18)32 (27-42)0.0040.001 ER+ve/HER2-ve (n=53)16 (10-20)15 (8-22)0.8 PR+ve/HER2+ve (n=12)18 (12-18)32 (24-44)0.020.05 PR+ve/HER2-ve (n=48)16 (10-20)12 (8-20)0.6 *Wilcoxon rank-sum test; ‡ p-interaction between treatment and DCIS grade or HER2 status from a linear regression model, adjusted for age and BMI. The effect of metformin on DCIS was different by HER2 status (p-interaction=.04) and, among this molecular subtype, by ER and PR status (p-interaction=.001 and .02, respectively). In HER2+ve DCIS, metformin decreased Ki67 by 40% overall (p=.06), by over 60% in ER+ve/HER2+ve DCIS (p=.004). and by 45% in PR+ve/HER2+ve DCIS (p=.02) There was no effect of metformin in HER2-ve DCIS, regardless of ER or PR status. Metformin did not affect Ki67 in DH overall, but showed a trend towards a decrease in women with abdominal adiposity (p-interaction=.05). Conclusions: Window of opportunity pre-surgical models provide insight into a drug’s preventive potential by targeting intraepithelial proliferations adjacent to invasive cancer. The model shows a high prevalence of preinvasive lesions (field cancerization) in apparently normal breast tissue adjacent to breast cancer. Metformin selectively decreased Ki67 in HER2+ve DCIS, particularly in the ER+ve subgroup, in line with a selective inhibitory effect on the HER2 pathway observed both in in vitro and in vivo preclinical models. Our results provide the background for a phase III trial of metformin in HER2+ve DCIS (ISRCTN16493703, Supported by AIRC, LILT and Health Ministry 2009-RF-153222). Citation Format: Bernardo Bonanni, Andrea DeCensi, Aliana Guerrieri-Gonzaga, Giancarlo Pruneri, Matteo Puntoni, Massimiliano Cazzaniga, Andrea Vingiani, Davide Serrano, Oreste Gentilini, Harriet Johansson, Valentina Aristarco, Matteo Lazzeroni, Clara Varicchio, Marilena Petrera, Giuseppe Viale. Metformin decreases Ki67 in HER2+ve ductal carcinoma in situ in a window of opportunity trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-12-02.

Introduction: Obesity is a risk factor for postmenopausal breast cancer (BC) and evidence supports the pivotal role of adipokines in the association between obesity and BC. In the present study, we aim to investigate the role of adiponectin and leptin as independent risk markers for postmenopausal BC in a prospective nested case-control study within the cohort of women from the International Breast cancer Intervention prevention Study II (IBIS-II). Methods and Results: In the IBIS-II Prevention trial, 3864 healthy postmenopausal women at increased risk for BC were randomized to receive 1 mg/day oral anastrozole or matching placebo for 5 years. We measured serum adiponectin and leptin at baseline and 12-months in 119 cases and 336 controls in the placebo arm, and 57 cases and 165 controls in the anastrozole arm. We performed the adipokines measurements on the automated immunoassay platform ELLA (ProteinSimple, Biotechne). Baseline characteristics (age, body mass index (BMI), smoking status, and oophorectomy) were not different between cases and controls in both trial arms, except for hormone replacement therapy (HRT) use (38.7% of cases vs 51.5% of controls) and hysterectomy (25.2% of cases vs 37.5% of controls) in the placebo arm. We did not observe any statistically significant difference in adiponectin and leptin at baseline between cases and controls in both trial arms. When we considered the change in adiponectin levels between baseline and 12-months of follow-up, we observed a borderline significant higher proportion of BC cases in subjects with a decrease in adiponectin (36.4%) compared to subjects with an increase in adiponectin (25.7%). The multivariate Cox model indicated that women on placebo with a decrease in adiponectin between baseline and 12-months had a 36% significant increased risk in BC (HR=0.64; CI 0.42-0.98; adjusted for age, BMI, previous HRT use, smoking, oophorectomy, hysterectomy). Changes in leptin were not significantly associated with BC incidence overall. However, the multivariate Cox model for the anastrozole arm, indicated a significant higher BC risk for women with the highest increase (>10 ng/ml) vs the lowest decrease ( Citation Format: Debora Macis, Ivana Sestak, Valentina Aristarco, Harriet Johansson, Aliana Guerrieri-Gonzaga, Andrea Decensi, Bernardo Bonanni, Jack Cuzick, Sara Gandini. Adiponectin, leptin and breast cancer in high risk postmenopausal women: Results from a nested case-control study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4216.

Background: A high proportion of Breast Cancer (BC) patients with a positive family history is not explained by mutations in BRCA1 and BRCA2 genes and the biology and BC outcome are different compared to patients with BRCA mutations. Low serum 25-hydroxyvitamin D (25(OH)D) has been found to be associated with an increased cancer incidence and poorer prognosis. Single nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR) and vitamin D binding protein (VDBP) genes (also called GC (Globulin Complex)), may contribute to final vitamin D activity. The aim of this study is to assess the role of 25(OH)D and VDBP serum levels, VDR and GC SNPs on BC recurrence and survival in a cohort of patients with positive family history, wild type (WT) for BRCA1 and BRCA2. Methods: VDR (ApaI, FokI, TaqI, BsmI) and GC (rs2282679, rs4588, rs7041) SNPs were analyzed, by TaqMan SNP Genotyping Assays (Thermo Fisher Scientific), for 431 WT patients. Serum levels of 25(OH)D and VDBP were measured using an electro-chemiluminescence immunoassay (Abbott) and Human Vitamin D BP Quantikine ELISA kit (R&D) respectively. Results: A cohort of 431 BC patients was followed from 2001 to 2015 (median follow-up 9 years). Women with the GC rs228679 variant had significantly lower levels of circulating VDBP and 25(OH)D compared to carriers of the major allele, whereas the GC rs7041 variant was associated with a greater level of VDBP and 25(OH)D. Multivariate Cox models showed that BsmI is significantly associated with BC outcome: patients with the major allele of BsmI (C) have half the risk of recurrence (CC vs. TT HR=0.43; 95%CI: 0.27-0.68; and CC vs. CT HR=0.66; 95%CI: 0.45-0.99). Finally, low serum VDBP concentrations are significantly associated with a higher risk of BC recurrence (VDBP321: HR=1.79; 95%CI: 1.03-3.13) and 25(OH)D>30 ng/ml in late summer/autumn vs Conclusions: 25(OH)D and VDBP serum levels were related with survival and breast cancer recurrence and influenced by genetic variants of GC SNPs. An allele VDR dose-related decrease of breast cancer recurrence was achieved with the increasing number of allele C of BsmI rs1544410(C>T). Our study provides evidence for the pivotal role of vitamin D metabolism in breast cancer outcome of high risk patients with a positive family history, wild type for BRCA1 or BRCA2 genes. Citation Format: Valentina Aristarco, Davide Serrano, Monica Barile, Davide Bondavalli, Mariarosaria Calvello, Debora Macis, Harriet Johansson, Aliana Guerrieri Gonzaga, Irene Feroce, Valeria Pensotti, Sara Gandini, Bernardo Bonanni. Association of vitamin D related polymorphisms with hereditary breast cancer in 431 patients wild type for BRCA1 and BRCA2 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 248.

Background: Epidemiologic data support an inverse association between green tea intake and breast cancer (BC) risk. Greenselect® Phytosome® (GSP) is a lecithin formulation of a caffeine-free green tea catechin extract. The purpose of the study was to determine the tissue distribution of Epigallocatechin 3-O-gallate (EGCG) and its effect on cell proliferation and other biomarkers in BC patients undergoing surgery. Methods: Twelve early BC patients received GSP, 300 mg daily for 4 weeks prior to surgery. The EGCG levels were measured before (free-) and after (total-) enzymatic hydrolysis by HPLC-MS/MS in plasma, urine, BC tissue and surrounding normal tissue. Fasting blood samples were taken at baseline, before the last administration and 2 hours later. Women were asked to avoid any tea for at least 14 days prior the beginning of the study and for the entire duration of the study. Results: All patients were fully compliant and completed the treatment program. No toxicity was observed. Free-EGCG and Total-EGCG were undetectable in baseline samples except for 1 patient with total-EGCG of 1 ng/mL in plasma. Despite a high between-subject variability, total EGCG was detectable in all tumor tissue samples collected (n = 8). Repeated administration of GSP achieved levels of total-EGCG ranging from 17 to 121 ng/mL in plasma, and up to 8 ng/g in BC tissue. Median total-EGCG concentration was higher in the tumor as compared to the adjacent normal tissue (P = 0.01).Urine EGCG levels correlated inversely with concentration in normal tissue (P = 0.01). Free-EGCG concentrations ranged from 8 to 65 ng/mL in plasma, but were under the limit of quantitation in tumor tissue and present in traces in adjacent normal breast tissue. Conclusions: Compared to other green tea extracts, GSP taken orally, appears to increase bioavailability of EGCG which selectively accumulates in breast tumor tissue. Biomarkers of activity will be presented at the conference. Median (Q1-Q3) concentrations after supplementation:Urine-final ng/mLPlasma-end ng/mLPlasma-end-2h ng/mLBreast-K ng/gBreast-N ng/gTotal-EGCG4.7 (3.3, 5.4)20.3 (14.7, 30.7)89.0 (50.9, 111.4)3.2 (2.7, 4.6)0.50 (0.5,2.3)end: before the last administration; end-2h: 2 hours after the last administration; value below the detectable level were imputed as 0.5. K: cancer tissue; N: normal tissue; P-value from Wilcoxon-rank test. Citation Format: Matteo Lazzeroni, Aliana Guerrieri-Gonzaga, Sara Gandini, Harriet Johansson, Davide Serrano, Pietro Caldarella, Gianmatteo Pagani, Paolo Arnone, Giancarlo Pruneri, Valentina Aristarco, Antonella Puccio, Serena Mora, Antonella Riva, Giovanna Petrangolini, Paolo Morazzoni, Bernardo Bonanni. A presurgical study of lecithin formulation of green tea extract in women with early breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT122.

The prevalence of metabolic syndrome is increasing along with breast cancer incidence worldwide. Because fenretinide improves insulin action and glucose tolerance in insulin-resistant obese mice and because tamoxifen has shown to regulate several markers involved in metabolic syndrome, we sought to investigate the effect of fenretinide or tamoxifen at low dose on features linked to insulin resistance in premenopausal women at risk for breast cancer. We randomized 235 women to low-dose tamoxifen (5 mg/daily), fenretinide (200 mg/daily), or their combination or placebo for 2 years. We used the homeostasis model assessment (HOMA; fasting insulin × glucose/22.5) to estimate insulin sensitivity. Women were considered to improve insulin sensitivity when they shifted from a HOMA ≥2.8 to

Background: Vitamin D plays an important role in many normal body functions, including regulation of cell growth, bone formation, inflammation, neuromuscular and immune function. Lower serum levels of 25-hydroxyvitamin D, an index of vitamin D status, are associated with greater risk of cancer. Genetic variants in vitamin D related genes have been linked to breast cancer (BC) risk. These include the vitamin D receptor (VDR) and the GC gene that encodes for the vitamin D binding protein. Aims: To determinate the frequency of 5 single nucleotide polymorphisms (SNPs) of VDR and GC in a large cohort of subjects candidate to BRCA 1/2 genetic testing and to assess any interaction between vitamin D and BRCA pathways on disease outcome. Methods: From 2002 to 2012 blood samples from 1025 subjects with family history of ovarian cancer (OC) or BC were collected after informed consent was signed during the genetic counseling. DNA was extracted from whole EDTA treated blood and Allelic Discrimination (Applied Biosystems’ Taqman assays) of VDR (BsmI, FokI, TaqI, ApaI) and GC SNPs is ongoing. Results: At last follow-up in February 2014 our cohort included 272 unaffected subjects (117 BRCA carriers, 37 WT and 118 true negative) and 753 affected subjects (276 BRCA1/2 carriers, 469 WT and 1 true negative subjects). Amongst BC patients (N = 675), 221 were BRCA carriers, 446 WT, and 8 true negative. Those affected ofBC and OC (N = 27), included 23 BRCA carriers and 4 WT, while OC patients (N = 51) included 32 BRCA carriers and 19 WT. Here we present the preliminary data obtained for VDR and GC SNPs in 833 and 710 subjects, respectively. None of the genotypes deviated from the Hardy-Weinberg equilibrium. At the AACR meeting the genotyping of all subjects will be completed. Conclusions: Our results of vitamin D genotyping will be correlated with BC tumor molecular subtype in affected subjects, and any association with BRCA mutation and outcome will be assessed. The study was funded by IEO Foundation and AVON. Description of vitamin D genetic polymorphisms and their frequenciesSNPsType of polymorphismMinor Allele Frequency (MAF)MAF HapMapHWE Pvalue(1df chi-square test)rs 1544410; VDR BsmITransition C/TT: 0.420.440.50rs2228570; VDR FokITransition A/GA: 0.360.410.72rs731236; VDR TaqITransition A/GC: 0.410.440.52rs7975232; VDR ApaITransversion A/CG: 0.430.430.91rs22282679; GCTransversion G/TG: 0.280.260.52 Citation Format: Valentina Aristarco, Harriet Johansson, Debora Macis, Aliana Guerrieri Gonzaga, Sara Gandini, Davide Serrano, Irene Feroce, Monica Barile, Antonella Puccio, Lorenzo Brocca, Bernardo Bonanni. Associations of Vitamin D related polymorphisms with hereditary breast cancer in 1025 subjects undergoing BRCA 1/2 testing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2015-1909

Background: Window-of-opportunity (WOP), presurgical models often use Ki-67 labeling index (LI) as the main surrogate biomarker for screening therapeutic activity of candidate agents and characterize their mechanism of action. Ki-67 LI in the placebo arm has been frequently reported to be higher at surgery than at baseline biopsy in WOP studies, a finding that has been ascribed to the higher proliferative activity in the tumor edge of surgical specimens that cannot be detected in the baseline core biopsy. Since this observation has important implications in sample size calculation and effect interpretation, we investigated which factors affected the changes of Ki-67 LI in a series of patients allocated to the placebo arms of a number of WOP trials carried out at IEO. Methods: Data from 181 patients with pT1-2 invasive breast cancer from the placebo arms of three WOP randomized trials were pooled with those of 98 untreated patients, who had a diagnostic core biopsy preceding surgery but declined participation to the study or were ineligible for histological characteristics. Ki-67 LI was measured by evaluating the prevalence of neoplastic cells showing any definite nuclear immunostaining with the Mib-1 monoclonal antibody, in the whole invasive component of baseline core biopsies and in at least 2000 invasive neoplastic cells of surgical samples, in accordance to the recently licensed international recommendations. Results: We collected data of 273 breast cancer patients with information on the changes in Ki-67 LI: 46 (17%) were Luminal A, 85 (31%) Luminal B/HER2−, 24 (9%) Luminal B/HER2+, 38 (14%) HER2+ and 81 (30%) Triple Negative (TN). Median (IQR) Ki-67LI at baseline in each molecular subtype was 10% (7–11), 20% (16–29), 28% (22–35), 30% (25–45) and 50% (32–75) in the Luminal A, Luminal B/HER2−, Luminal B/HER2+, HER2+ and TN, respectively. Median (IQR) age was 50 years (44–60), median BMI was 24 (22–27), 50% were post-menopausal. The median (range) time elapsed from biopsy to surgery was 41 days (33–48). Overall, the median change in Ki-67 LI between baseline biopsies and surgical samples was 0 (IQR, −2, 5). Results from multivariate analysis showed that none of the factors investigated, including patient and tumor characteristics, time elapsed from biopsy to surgery and circulating biomarkers (IGF-I, SHBG and ultrasensitive CRP) were associated with the Ki-67 LI change except for the immunohistochemically defined molecular subtype, which explained most of the variability of the changes (p = 0.004). As a matter of fact, we observed a 5%, significant increase of Ki-67 LI both in HER2+ and TN tumors, after adjustment for baseline values. Conclusions: We reported a significant increase in Ki-67 LI between baseline biopsy and endpoint surgery in the placebo arms of HER2+ and TN tumors. This association suggests a real biological increase in proliferation rather than an analytical artifact, and should be taken into account in designing future WOP studies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD06-06.

Background: To further investigate tamoxifen (T) minimal active dose, we conducted a window of opportunity study in estrogen receptor (ER) positive breast cancer (BC) patients. The study was a three arms randomized phase II double blind clinical trial. The participants were randomized to tamoxifen 10 mg a week vs. raloxifene 60 mg a day vs. placebo for 6 weeks, which was the average waiting list in our Institute at the time of this study was conducted. The primary endpoint was tissue change of Ki67LI. This result was previously reported and overall we did not observe any KI67LI modulation by treatment. The clinical relevance of CYP2D6 polymorphisms is controversial, and recently negative data have been published. Here we present a significant biomarkers modulation by low dose tamoxifen based on CYP2D6 subjects phenotype. Methods: DNA was extracted from whole blood by the use of QIAamp DNA blood kit. We employed the INFINITI analyzer for CYP2D6 genotyping, according to the protocol of the manufacturer. A total of 14 SNPs were analyzed. To determine the concentrations of tamoxifen and its metabolites, we used a high-pressure liquid chromatography-tandem mass spectrometry system. Expression of Ki-67, ER and PgR were determined by immunohistochemistry. Serum concentration of SHBG and estradiol were measured by chemiluminescent immunometric assay; IGF1 and IGFBP3 were determined by enzymatic colorimetric methods. Results: CYP2D6 was analyzed in the 50 women who took tamoxifen. We found 40% of EM (extensive metabolizer), 48% of IM (intermidiet metabolizer), 6% of PM (poor metabolizer) and 6% UM (ultra-rapid metabolizer). The phenotypes were grouped in two classes: active (EM + UM) and with a reduced activity (IM + PM). Among T metabolites a significant accumulation of N-desmethyl-Tamoxifen was present in the reduced activity group (mean plasma level 19.9 vs. 14.4 ng/ml reduced vs. active group p=.05). Endoxifen and 4-OH-Tamoxifen were not significantly different in the two phenotype groups. Then we correlated the phenotype with biomarkers modulations. The active phenotype showed a significant different modulation in Ki67 with a median chage of −2.5 in the active group and +3 in the reduced one. No differences on ER or PgR expression were determined by the phenotype. Among the circulating biomarkers, SHBG in the active group showed a significant difference compared to the reduced one (median change 8.35 and 2 p=0.05) whereas the estradiol and IGF1/BP3 did not show any difference between groups. Conclusions: CYP2D6 phenotypes interact with the biological activity of low dose T. A reduced activity of the enzyme induces an accumulation of N-desmethyl-Tamoxifen, whereas no significant differences for Endoxifen. In spite of a similar Endoxifen concentration, a reduced activity phenotype may influence T activity as demonstrated by the different modulation of Ki67 and SHBG biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2664. doi:1538-7445.AM2012-2664

Use of metformin has been associated with reduced breast cancer risk in observational studies in diabetic patients, but evidence for antitumor activity in randomized clinical studies is lacking. In a randomized trial in non-diabetic women with operable breast cancer who were allocated after tumor biopsy to either metformin, 850 mg bid (n=100) or placebo (n=100) for 4 weeks prior to surgery, we recently showed that the effect of the drug on breast cancer proliferation (Ki-67 labeling index) varies according to insulin resistance as assessed by HOMA index. Here we report effects of metformin on circulating markers of insulin resistance, including C-peptide, IGFBP-1, testosterone, SHBG and adiponectin. We observed a non-homogenous effect of metformin on some of these biomarkers, in a manner that is related to the dual effect of metformin on tumor Ki-67. Overall, metformin did not affect C-peptide (p=0.74) or IGFBP-1 levels (p=0.48), but there was a significant interaction with BMI (p=0.02), with a 19% decrease in C-peptide (p=0.105) in women with BMI >27 (75th percentile), and a 4% increase (p=0.367) in women with BMI β27. In women with baseline IGFBP-1 levels Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3561. doi:1538-7445.AM2012-3561

Background Tamoxifen is the most widely used drug for the prevention and treatment of hormone-receptor positive breast cancer (BC). Nevertheless, its clinical effectiveness varies among individuals. Tamoxifen is extensively metabolized by cytochrome P450 (CYP) enzymes, and recent in vivo studies have shown that women with genetically impaired CYP2D6 have reduced production of endoxifen, the main active metabolite. This impairment has been linked to a higher risk of breast cancer recurrence. Despite these observations, the contribution of endoxifen to the overall drug effectiveness of tamoxifen remains uncertain. Methods and results This study presents a pooled analysis of four chemoprevention trials assessing three different low dose tamoxifen schedules: 1mg/day (n=52), 10 mg/week (n=152) and 5 mg/day (n=158). Each participant had signed an informed consent. Genomic DNA was extracted from whole blood by the QIAamp DNA Blood Kit (Qiagen, Valencia, CA, USA), and the CYP2D6 polymorphisms analyzed by the use of the INFINITI CYP450 2D6T Assay (AutoGenomics, Carlsbad, CA, USA). The following alleles were identified: Full enzyme activity: *1, *2; reduced activity: *9, *41 *29; no activity: *3, *4, *5, *6. A total of 336 out of the 362 study participants were genotyped. Circulating concentrations of tamoxifen and its metabolites were determined on cryo-conserved (−80°C) serum samples collected at different treatment intervals according to study design. The women were defined as extensive metabolizers (EM) if they had 2 fully active alleles; intermediate (IM) if they carried reduced alleles or one non-functional allele, while poor metabolizers (PM) carried two non-functional alleles. We found the following phenotype frequencies: 146/336 (43%) EM, 171/336 (51%) IM, 19/336 (6%) PM. When analyzing the effects of phenotypes on serum concentrations of tamoxifen and its metabolites we found a statistically significant effect of phenotype on tamoxifen and metabolites. EM had lower levels of tamoxifen (8,6 ng/mL; P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3675. doi:10.1158/1538-7445.AM2011-3675