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Women with early-stage oestrogen receptor (ER)-positive (ER+) breast cancer who receive standard endocrine therapy for 5 years remain at risk of distant recurrence for at least 15 years after treatment discontinuation. The extension of the duration of adjuvant endocrine therapy to 10 years has been shown to reduce the risk of recurrence only in a subset of women and, to date, predictive biomarkers of benefit from therapy do not exist. In this Review, we briefly explore the epidemiology of late recurrence (>5 years after diagnosis) in patients with ER+ breast cancer. The mechanisms underlying this phenomenon remain poorly understood; we discuss the evidence currently available on processes such as alterations of gene expression or specific genomic aberrations and examine several models used for risk prognostication and for estimating the presence of minimal residual disease, as well as the relevance of these prediction tools for clinicians and patients. Our aim is to enable clinicians to make well-informed decisions on whether to extend endocrine therapy for each individual patient..

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Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection..

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors..

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Importance: Multiple molecular signatures are available for managing estrogen receptor (ER)-positive breast cancer but with little direct comparative information to guide the patient's choice. Objective: To conduct a within-patient comparison of the prognostic value of 6 multigene signatures in women with early ER-positive breast cancer who received endocrine therapy for 5 years. Design, Setting, and Participants: This retrospective biomarker analysis included 774 postmenopausal women with ER-positive ERBB2 (formerly HER2)-negative breast cancer. This analysis was performed as a preplanned secondary study of data from the Anastrozole or Tamoxifen Alone or Combined randomized clinical trial comparing 5-year treatment with anastrozole vs tamoxifen with 10-year follow-up data. The signatures included the Oncotype Dx recurrence score, PAM50-based Prosigna risk of recurrence (ROR), Breast Cancer Index (BCI), EndoPredict (EPclin), Clinical Treatment Score, and 4-marker immunohistochemical score. Data were collected from January 2009, through April 2015. Main Outcomes and Measures: The primary objective was to compare the prognostic value of these signatures in addition to the Clinical Treatment Score (nodal status, tumor size, grade, age, and endocrine treatment) for distant recurrence for 0 to 10 years and 5 to 10 years after diagnosis. Likelihood ratio (LR) statistics were used with the χ2 test and C indexes to assess the prognostic value of each signature. Results: In this study of 774 postmenopausal women with ER-positive, ERBB2-negative disease (mean [SD] age, 64.1 [8.1] years), 591 (mean [SD] age, 63.4 [7.9] years) had node-negative disease. The signatures providing the most prognostic information were the ROR (hazard ratio [HR], 2.56; 95% CI, 1.96-3.35), followed by the BCI (HR, 2.46; 95% CI, 1.88-3.23) and EPclin (HR, 2.14; 95% CI, 1.71-2.68). Each provided significantly more information than the Clinical Treatment Score (HR, 1.99; 95% CI, 1.58-2.50), the recurrence score (HR, 1.69; 95% CI, 1.40-2.03), and the 4-marker immunohistochemical score (HR, 1.95; 95% CI, 1.55-2.45). Substantially less information was provided by all 6 molecular tests for the 183 patients with 1 to 3 positive nodes, but the BCI (ΔLR χ2 = 9.2) and EPclin (ΔLR χ2 = 7.4) provided more additional prognostic information than the other signatures. Conclusions and Relevance: For women with node-negative disease, the ROR, BCI, and EPclin were significantly more prognostic for overall and late distant recurrence. For women with 1 to 3 positive nodes, limited independent information was available from any test. These data might help oncologists and patients to choose the most appropriate test when considering chemotherapy use and/or extended endocrine therapy. Trial Registration: isrctn.com Identifier: ISRCTN18233230..

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Purpose Estimating risk of late distant recurrence (DR) is an important goal for managing women with hormone receptor-positive breast cancer after 5 years of endocrine treatment without recurrence. We developed and validated a simple clinicopathologic tool (Clinical Treatment Score post-5 years [CTS5]) to estimate residual risk of DR after 5 years of endocrine treatment. Patients and Methods The ATAC (Arimidex, Tamoxifen, Alone or in Combination) data set (N = 4,735) was used to create a prognostic score for post-5-year risk of DR. Validity of CTS5 (ATAC) was tested in the BIG 1-98 data set (N = 6,711). Time to late DR, 5 years after finishing scheduled endocrine therapy, was the primary end point. Cox regression models estimated the prognostic performance of CTS5 (ATAC). Results CTS5 (ATAC) was significantly prognostic for late DR in the ATAC cohort (hazard ratio, 2.47; 95% CI, 2.24 to 2.73; P < .001) and BIG 1-98 validation cohort (hazard ratio, 2.07; 95% CI, 1.88 to 2.28; P < .001). CTS5 (ATAC) risk stratification defined in the training cohort as low (< 5% DR risk, years 5 to 10), intermediate (5% to 10%), or high (> 10%) identified 43% of the validation cohort as low risk, with an observed DR rate of 3.6% (95% CI, 2.7% to 4.9%) during years 5 to 10. From years 5 to 10, 63% of node-negative patients were low risk, with a DR rate of 3.9% (95% CI, 2.9% to 5.3%), and 24% with one to three positive nodes were low risk, with a DR rate of 1.5% (95% CI, 0.5% to 3.8%). A final CTS5 for future use was derived from pooled data from ATAC and BIG 1-98. Conclusion CTS5 is a simple tool based on information that is readily available to all clinicians. CTS5 was validated as highly prognostic for late DR in the independent BIG 1-98 study. The final CTS5 algorithm identified 42% of women with < 1% per-year risk of DR who could be advised of the limited potential value of extended endocrine therapy..

To investigate the impact of sampling methodology on gene expression data from primary estrogen receptor–positive (ER+) breast cancer biopsies, global gene expression was measured in core-cut biopsies at baseline and surgery from patients randomly assigned to receive either two weeks of presurgical aromatase inhibitor (AI; n = 157) or no presurgical treatment (n = 56). Those genes most markedly altered in the AI group (eg, FOS, DUSP1, RGS1, FOSB) were similarly altered in the no treatment group; some widely investigated genes that were apparently unaffected in the AI group (eg, MYC) were counter-altered in the control group, masking actual AI-dependent changes. In the absence of a control group, these artefactual changes would likely lead to the most affected genes being the erroneous focus of research. The findings are likely relevant to all archival collections of ER+ breast cancer..

Purpose To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in > 10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended work-up for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. Find additional information at www.asco.org/breast-cancer-guidelines ..

PURPOSE: - To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. METHODS: - Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. RECOMMENDATIONS: - Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in >10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended workup for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥2.0; average HER2 copy number <4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥4.0 and <6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results..

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588)..

Purpose The Letrozole (Femara) Versus Anastrozole Clinical Evaluation (FACE) study compared the efficacy and safety of adjuvant letrozole versus anastrozole in postmenopausal patients with hormone receptor (HR) -positive and node-positive early breast cancer (eBC). Methods Postmenopausal women with HR-positive and node-positive eBC were randomly assigned to receive adjuvant therapy with either letrozole (2.5 mg) or anastrozole (1 mg) once per day for 5 years or until recurrence of disease. Patients were stratified on the basis of the number of lymph nodes and human epidermal growth factor receptor 2 status. The primary end point was 5-year disease-free survival (DFS), and the key secondary end points were overall survival and safety. Results A total of 4,136 patients were randomly assigned to receive either letrozole (n = 2,061) or anastrozole (n = 2,075). The final analysis was done at 709 DFS events (letrozole, 341 [16.5%]; anastrozole, 368 [17.7%]). The 5-year estimated DFS rate was 84.9% for letrozole versus 82.9% for anastrozole arm (hazard ratio, 0.93; 95% CI, 0.80 to 1.07; P = .3150). Exploratory analysis showed similar DFS with letrozole and anastrozole in all evaluated subgroups. The 5-year estimated overall survival rate was 89.9% for letrozole versus 89.2% for anastrozole arm (hazard ratio, 0.98; 95% CI, 0.82 to 1.17; P = .7916). Most common grade 3 to 4 adverse events (> 5% of patients) reported for letrozole versus anastrozole were arthralgia (3.9% v 3.3%, and 48.2% v 47.9% for all adverse events), hypertension (1.2% v 1.0%), hot flushes (0.8% v 0.4%), myalgia (0.8% v 0.7%), dyspnea (0.8% v 0.5%), and depression (0.8% v 0.6%). Conclusion Letrozole did not demonstrate significantly superior efficacy or safety compared with anastrozole in postmenopausal patients with HR-positive, node-positive eBC..

Purpose At least 94 common single nucleotide polymorphisms (SNPs) are associated with breast cancer. The extent to which an SNP panel can refine risk in women who receive preventive therapy has not been directly assessed previously. Materials and Methods A risk score on the basis of 88 SNPs (SNP88) was investigated in a nested case-control study of women enrolled in the International Breast Intervention Study (IBIS-I) or the Royal Marsden study. A total of 359 women who developed cancer were matched to 636 controls by age, trial, follow-up time, and treatment arm. Genotyping was done using the OncoArray. Conditional logistic regression and matched concordance indices (mC) were used to measure the performance of SNP88 alone and with other breast cancer risk factors assessed using the Tyrer-Cuzick (TC) model. Results SNP88 was predictive of breast cancer risk overall (interquartile range odds ratio [IQ-OR], 1.37; 95% CI, 1.14 to 1.66; mC, 0.55), but mainly for estrogen receptor-positive disease (IQ-OR, 1.44; 95% CI, 1.16 to 1.79; P for heterogeneity = .10) versus estrogen receptor-negative disease. However, the observed risk of SNP88 was only 46% (95% CI, 19% to 74%) of expected. No significant interaction was observed with treatment arm (placebo IQ-OR, 1.46; 95% CI, 1.13 to 1.87; tamoxifen IQ-OR, 1.25; 95% CI, 0.96 to 1.64; P for heterogeneity = .5). The predictive power was similar to the TC model (IQ-OR, 1.45; 95% CI, 1.21 to 1.73; mC, 0.55), but SNP88 was independent of TC (Spearman rank-order correlation, 0.012; P = .7), and when combined multiplicatively, a substantial improvement was seen (IQ-OR, 1.64; 95% CI, 1.36 to 1.97; mC, 0.60). Conclusion A polygenic risk score may be used to refine risk from the TC or similar models in women who are at an elevated risk of breast cancer and considering preventive therapy. Recalibration may be necessary for accurate risk assessment..

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BACKGROUND: An earlier age at onset of breast development and longer time between pubertal stages has been implicated in breast cancer risk. It is not clear whether associations of breast cancer risk with puberty or predictors of onset of puberty, such as weight and height, are mediated via mammographic density, an important risk factor for breast cancer. METHODS: We investigated whether childhood body size and pubertal timing and tempo, collected by questionnaire, are associated with percentage and absolute area mammographic density at ages 47-73 years in 1105 women recruited to a prospective study. RESULTS: After controlling for adult adiposity, weight at ages 7 and 11 years was strongly significantly inversely associated with percentage and absolute dense area (p trend <0.001), and positively associated with absolute non-dense area. Greater height at age 7, but not age 11, was associated with lower percentage density (p trend = 0.016). Later age at menarche and age at when regular periods were established was associated with increased density, but additional adjustment for childhood weight attenuated the association. A longer interval between thelarche and menarche, and between thelarche and regular periods, was associated with increased dense area, even after adjusting for childhood weight (p trend = 0.013 and 0.028, respectively), and was independent of age at pubertal onset. CONCLUSIONS: Greater prepubertal weight and earlier pubertal onset are associated with lower adult breast density, but age at pubertal onset does not appear to have an independent effect on adult density after controlling for childhood adiposity. A possible effect of pubertal tempo on density needs further investigation..

IMPORTANCE: A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown. OBJECTIVE: To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial. DESIGN, SETTING, AND PARTICIPANTS: The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2). INTERVENTIONS: Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines. MAIN OUTCOMES AND MEASURES: Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models. RESULTS: Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P < .001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (≥2 to <5) group (DFS: 3-way ITT Pvalue for interaction = .07; censored = .02; IPW = .03; OS ITT Pvalue for interaction = .007; censored = .04; IPW = .03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction = .06; OS = .02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab. CONCLUSIONS AND RELEVANCE: Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (≥2 to <5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00045032..

PURPOSE: ESR1 mutations are selected by prior aromatase inhibitor (AI) therapy in advanced breast cancer. We assessed the impact of ESR1 mutations on sensitivity to standard therapies in two phase III randomized trials that represent the development of the current standard therapy for estrogen receptor-positive advanced breast cancer. MATERIALS AND METHODS: In a prospective-retrospective analysis, we assessed ESR1 mutations in available archived baseline plasma from the SoFEA (Study of Faslodex Versus Exemestane With or Without Arimidex) trial, which compared exemestane with fulvestrant-containing regimens in patients with prior sensitivity to nonsteroidal AI and in baseline plasma from the PALOMA3 (Palbociclib Combined With Fulvestrant in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer After Endocrine Failure) trial, which compared fulvestrant plus placebo with fulvestrant plus palbociclib in patients with progression after receiving prior endocrine therapy. ESR1 mutations were analyzed by multiplex digital polymerase chain reaction. RESULTS: In SoFEA, ESR1 mutations were found in 39.1% of patients (63 of 161), of whom 49.1% (27 of 55) were polyclonal, with rates of mutation detection unaffected by delays in processing of archival plasma. Patients with ESR1 mutations had improved progression-free survival (PFS) after taking fulvestrant (n = 45) compared with exemestane (n = 18; hazard ratio [HR], 0.52; 95% CI, 0.30 to 0.92; P = .02), whereas patients with wild-type ESR1 had similar PFS after receiving either treatment (HR, 1.07; 95% CI, 0.68 to 1.67; P = .77). In PALOMA3, ESR1 mutations were found in the plasma of 25.3% of patients (91 of 360), of whom 28.6% (26 of 91) were polyclonal, with mutations associated with acquired resistance to prior AI. Fulvestrant plus palbociclib improved PFS compared with fulvestrant plus placebo in both ESR1 mutant (HR, 0.43; 95% CI, 0.25 to 0.74; P = .002) and ESR1 wild-type patients (HR, 0.49; 95% CI, 0.35 to 0.70; P < .001). CONCLUSION: ESR1 mutation analysis in plasma after progression after prior AI therapy may help direct choice of further endocrine-based therapy. Additional confirmatory studies are required..

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Candidate gene studies have reported CYP19A1 variants to be associated with endometrial cancer and with estradiol (E2) concentrations. We analyzed 2937 single nucleotide polymorphisms (SNPs) in 6608 endometrial cancer cases and 37 925 controls and report the first genome wide-significant association between endometrial cancer and a CYP19A1 SNP (rs727479 in intron 2, P=4.8×10−11). SNP rs727479 was also among those most strongly associated with circulating E2 concentrations in 2767 post-menopausal controls (P=7.4×10−8). The observed endometrial cancer odds ratio per rs727479 A-allele (1.15, CI=1.11–1.21) is compatible with that predicted by the observed effect on E2 concentrations (1.09, CI=1.03–1.21), consistent with the hypothesis that endometrial cancer risk is driven by E2. From 28 candidate-causal SNPs, 12 co-located with three putative gene-regulatory elements and their risk alleles associated with higher CYP19A1 expression in bioinformatical analyses. For both phenotypes, the associations with rs727479 were stronger among women with a higher BMI (Pinteraction=0.034 and 0.066 respectively), suggesting a biologically plausible gene-environment interaction..

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Automated methods are needed to facilitate high-throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large-scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37-0.87) and study (kappa range = 0.39-0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p-value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000-4,500 cells: kappa = 0.78) than those with lower counts (50-500 cells: kappa = 0.41; p-value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre- and post-analytical quality control procedures are necessary in order to ensure satisfactory performance..

BACKGROUND: The value of KI67 in breast cancer prognostication has been questioned due to concerns on the analytical validity of visual KI67 assessment and methodological limitations of published studies. Here, we investigate the prognostic value of automated KI67 scoring in a large, multicentre study, and compare this with pathologists' visual scores available in a subset of patients. METHODS: We utilised 143 tissue microarrays containing 15,313 tumour tissue cores from 8088 breast cancer patients in 10 collaborating studies. A total of 1401 deaths occurred during a median follow-up of 7.5 years. Centralised KI67 assessment was performed using an automated scoring protocol. The relationship of KI67 levels with 10-year breast cancer specific survival (BCSS) was investigated using Kaplan-Meier survival curves and Cox proportional hazard regression models adjusted for known prognostic factors. RESULTS: Patients in the highest quartile of KI67 (>12 % positive KI67 cells) had a worse 10-year BCSS than patients in the lower three quartiles. This association was statistically significant for ER-positive patients (hazard ratio (HR) (95 % CI) at baseline = 1.96 (1.31-2.93); P = 0.001) but not for ER-negative patients (1.23 (0.86-1.77); P = 0.248) (P-heterogeneity = 0.064). In spite of differences in characteristics of the study populations, the estimates of HR were consistent across all studies (P-heterogeneity = 0.941 for ER-positive and P-heterogeneity = 0.866 for ER-negative). Among ER-positive cancers, KI67 was associated with worse prognosis in both node-negative (2.47 (1.16-5.27)) and node-positive (1.74 (1.05-2.86)) tumours (P-heterogeneity = 0.671). Further classification according to ER, PR and HER2 showed statistically significant associations with prognosis among hormone receptor-positive patients regardless of HER2 status (P-heterogeneity = 0.270) and among triple-negative patients (1.70 (1.02-2.84)). Model fit parameters were similar for visual and automated measures of KI67 in a subset of 2440 patients with information from both sources. CONCLUSIONS: Findings from this large-scale multicentre analysis with centrally generated automated KI67 scores show strong evidence in support of a prognostic value for automated KI67 scoring in breast cancer. Given the advantages of automated scoring in terms of its potential for standardisation, reproducibility and throughput, automated methods appear to be promising alternatives to visual scoring for KI67 assessment..

BACKGROUND: The purpose of this study was (i) to test the hypothesis that combining Ki67 with residual cancer burden (RCB) following neoadjuvant chemotherapy, as the residual proliferative cancer burden (RPCB), provides significantly more prognostic information than either alone; (ii) to determine whether also integrating information on ER and grade improves prognostic power. PATIENTS AND METHODS: A total of 220 patients treated with neoadjuvant chemotherapy for primary breast cancer were included in the study. Analyses employed a Cox proportional hazard model. Prognostic indices (PIs) were created adding in Ki67, grade and ER to RCB. Leave-one-out cross-validation was used to reduce bias. The overall change in χ(2) of the best model for each index was used to compare the prognostic ability of the different indices. RESULTS: All PIs provided significant prognostic information for patients with residual disease following neoadjuvant chemotherapy. RPCB (χ(2) = 61.4) was significantly more prognostic than either RCB (χ(2) = 38.1) or Ki67 (χ(2) = 53.8) alone P < 0.001. A PI incorporating RCB, Ki67 grade and ER provided the most prognostic information overall and gave χ(2) = 73.8. CONCLUSIONS: This study provides proof of principle that the addition of post-treatment Ki67 to RCB improves the prediction of long-term outcome. Prediction may be further improved by addition of post-treatment grade and ER and warrants further investigation for estimating post-neoadjuvant risk of recurrence. These indices may have utility in stratifying patients for novel therapeutic interventions after neoadjuvant chemotherapy..

PURPOSE: Not all breast cancers respond to lapatinib. A change in Ki67 after short-term exposure may elucidate a biomarker profile for responsive versus nonresponsive tumors. EXPERIMENTAL DESIGN: Women with primary breast cancer were randomized (3:1) to 10 to 14 days of preoperative lapatinib or placebo in a multicenter phase II trial (ISRCTN68509377). Biopsies pre-/posttreatment were analyzed for Ki67, apoptosis, HER2, EGFR, ER, PgR, pAKT, pERK, and stathmin by IHC. Further markers were measured by RT-PCR. Primary endpoint was change in Ki67. HER2(+) was defined as 2+/3+ by IHC and FISH(+). RESULTS: One hundred twenty-one patients (lapatinib, 94; placebo, 27) were randomized; of these, 21% were HER2(+), 78% were HER2(-) nonamplified, 26% were EGFR(+). Paired samples containing tumor were obtained for 98% (118 of 121). Ki67 fell significantly with lapatinib (-31%; P < 0.001), but not with placebo (-3%). Whereas Ki67 reduction with lapatinib was greatest in HER2(+) breast cancer (-46%; P = 0.003), there was a significant Ki67 decrease in HER2(-) breast cancer (-27%; P = 0.017) with 14% of HER2(-) breast cancer demonstrating ≥50% Ki67 reduction with lapatinib. Among HER2(+) patients, the only biomarker predictive of Ki67 response was the EGFR/HER4 ligand epiregulin (EREG) (rho = -0.7; P = 0.002). Among HER2(-) tumors, only HER3 mRNA levels were significantly associated with Ki67 response on multivariate analysis (P = 0.01). In HER2(-) breast cancer, HER2 and HER3 mRNA levels were highly correlated (rho = 0.67, P < 0.001), with all Ki67 responders having elevated HER3 and HER2 expression. CONCLUSIONS: Lapatinib has antiproliferative effects in a subgroup of HER2(-) nonamplified tumors characterized by high HER3 expression. The possible role of high HER2:HER3 heterodimers in predicting response to lapatinib merits investigation in HER2(-) tumors..

Understanding the complex relationship between obesity and breast cancer is fundamental to our knowledge of the etiology of this malignancy; changes in the composition of the hormonal milieu are implicit in this process. Estrogens are synthesized from androgens by aromatase in the gonads and in peripheral tissues, principally, adipose tissue. Obesity in women, regardless of their age, leads to more aromatase and more extra-glandular estrogen production. In postmenopausal women, in whom ovarian estrogen production is absent, the increased incidence of breast cancer in women with high body mass index has been attributed to the relatively high plasma levels of estradiol from subcutaneous fat. In contrast, obesity in premenopausal women is associated with a previously unexplained reduced incidence of breast cancer. In obese premenopausal women, the cumulative effect of higher levels of estrogens synthesized in the peripheral tissues, together with ovarian estrogen production, results in a negative feedback on the hypothalamic pituitary controlled release of gonadotrophins and a resultant diminution in ovarian steroid production. As a consequence, the normal balance of estrogen and progesterone levels is disrupted: while estrogen levels are normalized, progesterone production is markedly decreased. Progesterone is a promoter of proliferation in the breast. The low levels of progesterone in obese premenopausal women are consistent with, and we propose, are responsible for, the reduction in breast cancer incidence in these women..

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Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes. .

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PURPOSE: We have previously shown that the PAM50-based risk of recurrence (ROR) score is significantly correlated with distant recurrence in both the translational research cohort within the Arimidex, Tamoxifen Alone or in Combination (ATAC) trial (TransATAC) and Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG 8) randomized trials. Here, we focus on the ROR score for predicting distant recurrence after 5 years of follow-up in a combined analysis of these two randomized trials. METHODS: Long-term follow-up data and tissue samples were obtained from 2,137 postmenopausal women with hormone receptor-positive early-stage breast cancer from the ABCSG 8 and TransATAC trials. We used Cox proportional hazard regression models to determine the prognostic value of ROR for distant recurrence beyond 5 years in the combined data set. RESULTS: A total of 2,137 women who did not have a recurrence 5 years after diagnosis were included in the combined analyses. The Clinical Treatment Score (CTS) was the strongest prognostic factor 5 years after diagnosis (univariable: likelihood ratio [LR] χ(2) = 94.12, bivariable: LR χ(2) = 61.43). The ROR score was significantly prognostic by itself in years 5 to 10. In the node-negative/human epidermal growth factor receptor 2-negative subgroup, more prognostic value for late distant recurrence was added by the ROR score compared with the CTS. CONCLUSION: The ROR score added clinically meaningful prognostic information to the CTS in all patients and all subgroups in the late follow-up period. These results suggest that the ROR score may be helpful for separating patients into risk groups who could be spared or potentially benefit from extended hormonal therapy beyond 5 years of treatment..

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Purpose Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. Patients and Methods Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor–alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. Results The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer –specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. Conclusion A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs. .

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Trastuzumab-containing therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17 (CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity. .

Recent studies have provided strong evidence for the emergence of substantial numbers of constitutively active ESR1 mutations in estrogen receptor-positive metastatic breast cancer that are undetected in primary disease. Some of these mutants remain partially sensitive to current anti-estrogen therapies but effective therapeutics targeted at them may require new approaches..

PURPOSE: To investigate potential associations between gene modules representing key biologic processes and response to aromatase inhibitors (AI) in estrogen receptor-positive (ER(+)) breast cancer. PATIENTS AND METHODS: Paired gene expression and Ki67 protein expression were available from 69 postmenopausal women with ER(+) early breast cancer, at baseline and 2 weeks post-anastrozole treatment, in the presurgical setting. Functional gene modules (n = 26) were retrieved from published studies and their module scores were computed before and after elimination of proliferation-associated genes (PAG). Ki67 and module scores were assessed at baseline and 2 weeks post-anastrozole. Unsupervised clustering was used to assess associations between modules and Ki67. RESULTS: Proliferation-based modules were highly correlated with Ki67 expression both pretreatment and on-treatment. At baseline with and without PAGs, Ki67 expression was significantly inversely correlated with ERG, ESR1.2, SET, and PIK3CA modules. Modules measuring estrogen signaling strongly predicted antiproliferative response to therapy with and without PAGs. Baseline expression of insulin-like growth factor-1 (IGF-I) module predicted a poor change in Ki67-implicating genes within the module as involved in de novo resistance to AIs. High expression of Immune.2.STAT1 module pretreatment predicted poor antiproliferative response to therapy. A significant association between estrogen-regulated genes modules (ESR1, ESR1-2, SET, and ERG) was evident post AI. CONCLUSIONS: Multiple processes and pathways are affected by AI treatment in ER(+) breast cancer. Modules closely associated with ESR1 expression were predictive of good antiproliferative response to AIs, but modules representing immune activity and IGF-I/MAPK were predictive of poor Ki67 response, supporting their therapeutic targeting in combination with AIs..

PURPOSE: Endocrine therapies include aromatase inhibitors and the selective estrogen receptor (ER) downregulator fulvestrant. This study aimed to determine whether the reported efficacy of fulvestrant over anastrozole, and high- over low-dose fulvestrant, reflect distinct transcriptional responses. EXPERIMENTAL DESIGN: Global gene expression profiles from ERα-positive breast carcinomas before and during presurgical treatment with fulvestrant (n = 22) or anastrozole (n = 81), and corresponding in vitro models, were compared. Transcripts responding differently to fulvestrant and estrogen deprivation were identified and integrated using Gene Ontology, pathway and network analyses to evaluate their potential significance. RESULTS: The overall transcriptional response to fulvestrant and estrogen deprivation was correlated (r = 0.61 in presurgical studies, r = 0.87 in vitro), involving downregulation of estrogen-regulated and proliferation-associated genes. The transcriptional response to fulvestrant was of greater magnitude than estrogen deprivation (slope = 0.62 in presurgical studies, slope = 0.63 in vitro). Comparative analyses identified 28 genes and 40 Gene Ontology categories affected differentially by fulvestrant. Seventeen fulvestrant-specific genes, including CAV1/2, SNAI2, and NRP1, associated with ERα, androgen receptor (AR), and TP53, in a network regulating cell cycle, death, survival, and tumor morphology. Eighteen genes responding differently to fulvestrant specifically predicted antiproliferative response to fulvestrant, but not anastrozole. Transcriptional effects of low-dose fulvestrant correlated with high-dose treatment, but were of lower magnitude (ratio = 0.29). CONCLUSIONS: The transcriptional response to fulvestrant has much in common with estrogen deprivation, but is stronger with distinctions potentially attributable to arrest of estrogen-independent ERα activity and involvement of AR signaling. Genes responding differently to fulvestrant may have predictive utility. These data are consistent with the clinical efficacy of fulvestrant versus anastrozole and higher dosing regimens..

Prospective cohort studies examining sex hormones in relation to cancer risk have generally collected blood samples at 1 time point, with an assumption that hormone levels measured in these samples will be reliable markers of true levels at other times. In postmenopausal women, body fat is a major source of estradiol; therefore, changes in adiposity may affect the correlation of single measurements to more relevant long-term averages. To estimate the intraclass correlation coefficient (ICC) for estradiol and testosterone, we collected repeat blood samples from 119 postmenopausal women (average age = 59.4 (standard deviation, 4.7) years) from the United Kingdom during 2004-2005 and again during 2010-2011. The ICCs (adjusted for assay variation) were 0.73 (95% confidence interval: 0.63, 0.82) for total estradiol and 0.59 (95% confidence interval: 0.47, 0.72) for total testosterone. The ICCs were 3%-5% larger after adjustment for change in body mass index (weight (kg)/height (m)(2)) or leptin, which are 2 markers of change in adiposity. There was no increase in ICCs after adjustment for change in age, alcohol consumption, smoking, exercise, time between waking and blood collection, or season. The results suggest that other factors account for within-woman variation in these sex hormones. .

PURPOSE: To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. METHODS: ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. RESULTS: The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. RECOMMENDATIONS: The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing..

BACKGROUND: Mammographic density and sex hormone levels are strong risk factors for breast cancer, but it is unclear whether they represent the same aetiological entity or are independent risk factors. METHODS: Within the Breakthrough Generations Study cohort, we conducted a case-control study of 265 postmenopausal breast cancer cases and 343 controls with prediagnostic mammograms and blood samples. Plasma was assayed for oestradiol, testosterone and sex hormone-binding globulin (SHBG) concentrations and mammographic density assessed by Cumulus. RESULTS: Oestradiol and testosterone were negatively and SHBG positively associated with percentage density and absolute dense area, but after adjusting for body mass index the associations remained significant only for SHBG. Breast cancer risk was independently and significantly positively associated with percentage density (P=0.002), oestradiol (P=0.002) and testosterone (P=0.007) levels. Women in the highest tertile of both density and sex hormone level were at greatest risk, with an odds ratio of 7.81 (95% confidence interval (CI): 2.89-21.1) for oestradiol and 4.57 (95% CI: 1.75-11.9) for testosterone and high density compared with those who were in the lowest tertiles. The cumulative risk of breast cancer in the highest oestradiol and density tertiles, representing 8% of controls, was estimated as 12.8% at ages 50-69 years and 19.4% at ages 20-79 years, and in the lowest tertiles was 1.7% and 4.3%, respectively. Associations of breast cancer risk with tertiles of mammographic dense area were less strong than for percentage density. CONCLUSIONS: Endogenous sex hormone levels and mammographic density are independent risk factors for postmenopausal breast cancer, which in combination can identify women who might benefit from increased frequency of screening and chemoprophylaxis..

BACKGROUND: Recent data have raised concern about the clinical efficacy of aromatase inhibitors in overweight and/or obese breast cancer patients. We report in vivo aromatase inhibition and plasma and tissue oestrogen levels in relation to body mass index (BMI) status among breast cancer patients treated with different aromatase inhibitors. METHODS: We compared data on in vivo aromatase inhibition (64 patients) as well as plasma and tissue oestrogen levels from patients participating in our studies to BMI values. RESULTS: We found a weak positive correlation between pretreatment aromatisation level and BMI (n=64; R=0.236; p=0.060) but no correlation between on-treatment aromatisation levels or percentage aromatase inhibition and BMI within patient subgroups treated with any of a panel of aromatase inhibitors. Pre-treatment levels of plasma estradiol (p<0.001), estrone (p=0.001) and estrone sulphate (p=0.002) correlated to BMI. While on-treatment levels of plasma estrane sulphate correlated to BMI in patients on letrozole (R=0.601; p=0.001; n=25 for all) or anastrozole (n=12; R=0.611; p=0.035) therapy, letrozole suppressed plasma estrone sulphate more than anastrozole independent of BMI. No correlation between on-treatment tumour oestrogen levels and BMI was recorded. CONCLUSIONS: Our unique data do not support a lack of effective aromatase inhibition in overweight patients or therefore a need for alternative therapy. The higher levels of estrogens in overweight postmenopausal breast cancer patients before and during aromatase inhibition may be due to effects of BMI on oestrogen metabolism rather than aromatisation..

The TACT trial is the largest study assessing the benefit of taxanes as part of adjuvant therapy for early breast cancer. The goal of this translational study was to clarify the predictive and prognostic value of Tau within the TACT trial. Tissue microarrays (TMA) were available from 3,610 patients. ER, PR, HER2 from the TACT trial and Tau protein expression was determined by immunohistochemistry on duplicate TMAs. Two parallel scoring systems were generated for Tau expression ('dichotomised' vs. 'combined' score). The positivity rate of Tau expression was 50 % in the trial population (n = 2,483). Tau expression correlated positively with ER (p < 0.001) and PR status (p < 0.001); but negatively with histological grade (p < 0.001) and HER2 status (p < 0.001). Analyses with either scoring systems for Tau expression demonstrated no significant interaction between Tau expression and efficacy of docetaxel. Contrary to the hypothesis that taxane benefit would be enriched in Tau negative/low patients, the only groups with a suggestion of a reduced event rate in the taxane group were the HER2-positive, Tau positive subgroups. Tau expression was seen to be a prognostic factor on univariate analysis associated with an improved DFS, independent of the treatment group (p < 0.001). It had no prognostic value in ER-negative tumours and the weak prognostic effect of Tau in ER-positive tumours (p = 0.02) diminished, when considering ER as an ordinal variable. On multivariable analyses, Tau had no prognostic value in either group. In addition, no significant interaction between Tau expression and benefit from docetaxel in patients within the PR-positive and negative subsets was seen. This is now the second large adjuvant study, and the first with quantitative analysis of ER and Tau expression, failing to show an association between Tau and taxane benefit with limited utility as a prognostic marker for Tau in ER-positive early breast cancer patients..

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BACKGROUND: [(18)F]fluorothymidine (FLT) has been proposed as a positron emission tomography (PET)-imaging biomarker of proliferation for breast cancer. The aim of this prospective study was to assess the feasibility of FLT-PET-CT as a technique for predicting the response to neoadjuvant chemotherapy (NAC) in primary breast cancer and to compare baseline FLT with Ki-67. METHODS: Twenty women with primary breast cancer had a baseline FLT-PET-CT scan that was repeated before the second cycle of chemotherapy. Expression of Ki-67 in the diagnostic biopsy was quantified. From the FLT-PET-CT scans lesion maximum and mean standardised uptake values (SUVmax, SUVmean) were calculated. RESULTS: Mean baseline SUVmax was 7.3, and 4.62 post one cycle of NAC, representing a drop of 2.68 (36.3%). There was no significant association between baseline, post chemotherapy, or change in SUVmax and pathological response to NAC. There was a significant correlation between pre-chemotherapy Ki-67 and SUVmax of 0.604 (P=0.006). CONCLUSIONS: Baseline SUVmax measurements of FLT-PET-CT were significantly related to Ki-67 suggesting that it is a proliferation biomarker. However, in this series neither the baseline value nor the change in SUVmax after one cycle of NAC were able to predict response as most patients had a sizeable SUVmax reduction..

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PURPOSE: To document the rate and outcome of trastuzumab-associated cardiac dysfunction in patients following 1 or 2 years of adjuvant therapy. PATIENTS AND METHODS: The Herceptin Adjuvant (HERA) trial is a three-arm, randomized trial comparing 2 years or 1 year of trastuzumab with observation in 5,102 patients with human epidermal growth factor receptor 2 (HER2) -positive early-stage breast cancer. Cardiac function was closely monitored. Eligible patients had left ventricular ejection fraction (LVEF) ≥ 55% at study entry following neoadjuvant chemotherapy with or without radiotherapy. This 8-year median follow-up analysis considered patients randomly assigned to 2 years or 1 year of trastuzumab or observation. RESULTS: The as-treated safety population for 2 years of trastuzumab (n = 1,673), 1 year of trastuzumab (n = 1,682), and observation (n = 1,744) is reported. Cardiac adverse events leading to discontinuation of trastuzumab occurred in 9.4% of patients in the 2-year arm and 5.2% of patients in the 1-year arm. Cardiac death, severe congestive heart failure (CHF), and confirmed significant LVEF decrease remained low in all three arms. The incidence of severe CHF (0.8%, 0.8%, and 0.0%, respectively) and confirmed significant LVEF decrease (7.2%, 4.1%, and 0.9%, respectively) was significantly higher in the 2-year and 1-year trastuzumab arms compared with the observation arm. Severe CHF was the same for 2-year and 1-year trastuzumab. Of patients with confirmed LVEF decrease receiving 2-year trastuzumab, 87.5% reached acute recovery. Of patients with confirmed LVEF decrease receiving 1-year trastuzumab, 81.2% reached acute recovery. CONCLUSION: Long-term assessment at 8-year median follow-up confirms the low incidence of cardiac events for trastuzumab given sequentially after chemotherapy and radiotherapy, and cardiac events were reversible in the vast majority of patients..

The purpose of this study is to determine if there are differences in the expression of estrogen-regulated genes (ERGs), proliferation-associated genes and the progesterone effector RANKL, in premenopausal ER+ breast cancer as a result of the major changes in hormone levels that occur through the menstrual cycle. Primary ER+ tumours from 174 patients were assigned to one of three menstrual cycle windows: W1 (days 27-35 + 1-6), W2 (days 7-16) and W3 (days 17-26). RNA expression of 42 genes, including 24 putative genes associated with plasma E2 levels, seven proliferation genes and RANKL was measured. Expression of PGR, TFF1, GREB1 and PDZK1 followed the previously reported pattern: a higher level in W2 compared to W1 while W3 had an intermediate value, mirroring changes in plasma estradiol. Of the other 20 ERGs, four (RUNX1, AGR2, SERPINA3 and SERPINA5) showed significant differences (p = 0.009-0.049) in expression across the menstrual cycle. The expression of six of seven proliferation-associated genes varied across the cycle but differently from the ERGs, being 20-35 % lower in W3 compared to W1 and W2 (p = 0.004-0.031). Expression of RANKL was 2.5 to 3-fold highest in W3 (p = 0.0001) and negatively correlated to the expression of the proliferation-associated genes (r = -0.37; p < 0.0001). Expression of proliferation-associated genes and RANKL in ER+ breast tumours varies across the menstrual cycle showing a different rhythm to that of ERGs. This may affect the interpretation of gene expression profiles but may be exploitable as an endogenous test of endocrine responsiveness. .

BACKGROUND: Aromatase inhibitors prevent breast cancer in postmenopausal women at high risk of the disease but are associated with accelerated bone loss. We assessed effectiveness of oral risedronate for prevention of reduction in bone mineral density (BMD) after 3 years of follow-up in a subset of patients in the IBIS-II trial. METHODS: The double-blind IBIS-II trial recruited 3864 healthy, postmenopausal women at increased risk of breast cancer and randomly allocated them oral anastrozole (1 mg/day) or matched placebo. 1410 (36%) postmenopausal women were then enrolled in a bone substudy and stratified at baseline according to their lowest baseline T score at spine or femoral neck (stratum I: T score at least -1·0; stratum II: T score at least -2·5 but less than -1·0; stratum III: T score less than -2·5 but greater than -4·0). Women in stratum I were monitored only; women in stratum III were all given risedronate (35 mg/week). Women in stratum II were randomly assigned (1:1) to risedronate (35 mg/week) or matched placebo by use of a block randomisation schedule via a web-based programme. The primary outcome of this per-protocol analysis (done with all women with a baseline and 3 year DXA assessment) was the effect of risedronate versus placebo for osteopenic women in stratum II randomly allocated to anastrozole (1 mg/day). Secondary outcomes included effect of anastrozole (1 mg/day) on BMD in women not receiving risedronate (strata I and II) and in osteoporotic women who were all treated with risedronate (stratum III). The trial is ongoing, but no longer recruiting. This trial is registered, number ISRCTN31488319. FINDINGS: Between Feb 2, 2003, and Sept 30, 2010, 150 (58%) of 260 women in stratum II who had been randomly allocated to anastrozole and either risedronate or placebo had baseline and 3 year assessments. At the lumbar spine, 3 year mean BMD change for the 77 women receiving anastrozole/risedronate was 1·1% (95% CI 0·2 to 2·1) versus -2·6% (-4·0 to -1·3) for the 73 women receiving anastrozole/placebo (p<0·0001). For the total hip, 3 year mean BMD change for women receiving anastrozole/risedronate was -0·7% (-1·6 to 0·2) versus -3·5% (-4·6 to -2·3) for women receiving anastrozole/placebo (p=0·0001). 652 (65%) of 1008 women in strata I and II who were not randomly allocated to risedronate had both baseline and 3 year assessments. Women not receiving risedronate in stratum I and II who received anastrozole (310 women) had a significant BMD decrease after 3 years of follow-up compared with women who received placebo (342 women) at the lumbar spine (-4·0% [-4·5 to -3·4] vs -1·2% [-1·7 to -0·7], p<0·0001) and total hip (-4·0% [-4·4 to -3·6] vs -1·8% [-2·1 to -1·4], p<0·0001). 106 (79%) of 149 women in stratum III had a baseline and a 3 year assessment. The 46 women allocated to anastrozole had a modest BMD increase of 1·2% (-0·1 to 2·6) at the spine compared with a 3·9% (2·6 to 5·2) increase for the 60 women allocated to placebo (p=0·006). For the total hip, a small 0·3% (-0·9 to 1·5) increase was noted for women allocated anastrozole compared with a 1·5% (0·5 to 2·5) increase for women allocated placebo, but the difference was not significant (p=0·12). The most common adverse event reported was arthralgia (stratum I: 94 placebo and 114 anastrozole; stratum II: 39 placebo/placebo, 25 placebo/risedronate, 34 anastrozole/placebo, and 34 anastrozole/risedronate; stratum III: 21 placebo/risedronate, 17 anastrozole/risedronate). Other adverse events included hot flushes, alopecia, abdominal pain, and back pain. INTERPRETATION: Risedronate counterbalances the effect of anastrozole-induced bone loss in osteopenic and osteoporotic women and might be offered in combination with anastrozole treatment to provide an improved risk-benefit profile. FUNDING: Cancer Research UK (C569/A5032), National Health and Medical Research Council Australia (GNT300755, GNT569213), Sanofi-Aventis, and AstraZeneca..

Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here, we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor-subtype specific, and it did not change during treatment in tumors with partial or no response. However, lower pretreatment genetic diversity was significantly associated with pathologic complete response. In contrast, phenotypic diversity was different between pre- and posttreatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution. .

BACKGROUND: Aromatase inhibitors effectively prevent breast cancer recurrence and development of new contralateral tumours in postmenopausal women. We assessed the efficacy and safety of the aromatase inhibitor anastrozole for prevention of breast cancer in postmenopausal women who are at high risk of the disease. METHODS: Between Feb 2, 2003, and Jan 31, 2012, we recruited postmenopausal women aged 40-70 years from 18 countries into an international, double-blind, randomised placebo-controlled trial. To be eligible, women had to be at increased risk of breast cancer (judged on the basis of specific criteria). Eligible women were randomly assigned (1:1) by central computer allocation to receive 1 mg oral anastrozole or matching placebo every day for 5 years. Randomisation was stratified by country and was done with blocks (size six, eight, or ten). All trial personnel, participants, and clinicians were masked to treatment allocation; only the trial statistician was unmasked. The primary endpoint was histologically confirmed breast cancer (invasive cancers or non-invasive ductal carcinoma in situ). Analyses were done by intention to treat. This trial is registered, number ISRCTN31488319. FINDINGS: 1920 women were randomly assigned to receive anastrozole and 1944 to placebo. After a median follow-up of 5·0 years (IQR 3·0-7·1), 40 women in the anastrozole group (2%) and 85 in the placebo group (4%) had developed breast cancer (hazard ratio 0·47, 95% CI 0·32-0·68, p<0·0001). The predicted cumulative incidence of all breast cancers after 7 years was 5·6% in the placebo group and 2·8% in the anastrozole group. 18 deaths were reported in the anastrozole group and 17 in the placebo group, and no specific causes were more common in one group than the other (p=0·836). INTERPRETATION: Anastrozole effectively reduces incidence of breast cancer in high-risk postmenopausal women. This finding, along with the fact that most of the side-effects associated with oestrogen deprivation were not attributable to treatment, provides support for the use of anastrozole in postmenopausal women at high risk of breast cancer. FUNDING: Cancer Research UK, the National Health and Medical Research Council Australia, Sanofi-Aventis, and AstraZeneca..

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INTRODUCTION: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age /=15 years (OR(het) = 0.84, 95% CI 0.75, 0.94; OR(hom) = 0.81, 95% CI 0.51, 1.30; P(trend) = 0.002) but not for those who had their menarche age

BACKGROUND: We have found that the platelet-derived growth factor receptor (PDGFR)/Abl signaling pathway is up-regulated as a determinant of the acquisition of resistance to estrogen deprivation in vitro. We aimed to determine its clinical relevance in aromatase inhibitor (AI)-resistant breast cancer. PATIENTS AND METHODS: We identified a cohort of 45 patients with estrogen receptor-positive breast cancer who had been treated with an AI, subsequently relapsed and had biopsy material available from both the presentation and post-AI recurrent lesion. PDGFRα, PDGFRβ and Abl expression was assessed in formalin-fixed paraffin-embedded sections. RESULTS: Tumor protein expression of PDGFRα (1.39-fold, P=0.0065), PDGFRβ (4.32-fold, P=0.006) and Abl (1.8-fold, P=0.001) was increased at the point of relapse. Tumor and stromal expression of PDGFRα as well as PDGFRβ was significantly correlated in pre-treatment and relapse samples. High post-treatment tumor and stromal PDGFRβ levels were associated with a short time to treatment failure (TTF). Expression of PDGFRα in relapsing tumor specimens was correlated with Abl expression and Ki67 levels. Furthermore, changes in Abl correlated significantly with changes in ER expression. CONCLUSIONS: These clinical data support a role for enhanced PDGF/Abl signaling in AI-resistant disease and provide a rationale for targeting the pathway in endocrine-resistant breast cancer..

PURPOSE: To evaluate the benefit of adjuvant trastuzumab in patients diagnosed with human epidermal growth factor receptor 2 (HER2) -positive invasive lobular carcinoma (ILC) enrolled onto the Herceptin Adjuvant (HERA) trial. PATIENTS AND METHODS: Patients randomly assigned to receive one year of trastuzumab and one year of observation in the HERA trial were included (n = 3,401). Centrally reviewed estrogen receptor (ER), progesterone receptor (PgR), and HER2 copy numbers were used. First site-specific relapse pattern was evaluated for ILC and invasive ductal carcinoma (IDC). The magnitude of trastuzumab benefit was assessed using the Cox proportional hazards model for disease-free survival (DFS) and overall survival (OS). RESULTS: Median follow-up time was 4 years. A total of 187 ILC and 3,213 IDC patients were included. High Allred scores (6 to 8) were more common in patients with ILC than IDC for both ER (36.9% v 22.7%) and PgR (44.1% v 28.5%). A trend toward decreased HER2 copy number was observed in the ILC group. The ILC and IDC subgroups had similar patterns of first site of disease relapse. DFS hazard ratios (HRs) comparing 1 year of trastuzumab versus observation were 0.63 for ILC (95% CI, 0.34 to 1.15) and 0.77 for IDC (95% CI, 0.67 to 0.89; P for interaction = .49). The OS HRs comparing 1 year of trastuzumab versus observation were 0.60 for ILC (95% CI, 0.27 to 1.31) and 0.86 for IDC (95% CI, 0.71 to 1.06; P for interaction = .29). CONCLUSION: In this retrospective analysis, there was no suggestion that patients in the ILC cohort experienced a different magnitude of benefit from adjuvant trastuzumab than those in the IDC cohort..

PURPOSE: Risk of distant recurrence (DR) among women with estrogen receptor (ER) -positive early breast cancer is the major determinant of recommendations for or against chemotherapy. It is frequently estimated using the Oncotype DX recurrence score (RS). The PAM50 risk of recurrence (ROR) score provides an alternative approach, which also identifies intrinsic subtypes. PATIENTS AND METHODS: mRNA from 1,017 patients with ER-positive primary breast cancer treated with anastrozole or tamoxifen in the ATAC trial was assessed for ROR using the NanoString nCounter. Likelihood ratio (LR) tests and concordance indices (c indices) were used to assess the prognostic information provided beyond that of a clinical treatment score (CTS) by RS, ROR, or IHC4, an index of DR risk derived from immunohistochemical assessment of ER, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and Ki67. RESULTS: ROR added significant prognostic information beyond CTS in all patients (Δ LR-χ(2) = 33.9; P < .001) and in all four subgroups: node negative, node positive, HER2 negative, and HER2 negative/node negative; more information was added by ROR than by RS. C indices in the HER2-negative/node-negative subgroup were 0.73, 0.76, and 0.78 for CTS, CTS plus RS, and CTS plus ROR, respectively. More patients were scored as high risk and fewer as intermediate risk by ROR than by RS. Relatively similar prognostic information was added by ROR and IHC4 in all patients but more by ROR in the HER2-negative/node-negative group. CONCLUSION: ROR provides more prognostic information in endocrine-treated patients with ER-positive, node-negative disease than RS, with better differentiation of intermediate- and higher-risk groups..

PURPOSE: To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. METHODS: ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. RESULTS: The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. RECOMMENDATIONS: The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to > 10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. This guideline was developed through a collaboration between the American Society of Clinical Oncology and the College of American Pathologists and has been published jointly by invitation and consent in both Journal of Clinical Oncology and the Archives of Pathology & Laboratory Medicine..

Plasma estradiol (E2) and progesterone vary markedly through the menstrual cycle. Data on whether these differences in hormone levels affect gene expression in oestrogen receptor-positive (ER+) tumours are inconsistent. We wished to determine whether there are substantial changes in the expression of oestrogen-regulated genes (ERGs) in ER+ breast cancer through the menstrual cycle. One hundred and seventy five paraffin-embedded ER+ breast carcinomas from premenopausal patients were analysed. Timing of the ovarian cycle was confirmed using serum progesterone levels. Patients were ascribed to one of three pre-defined menstrual cycle windows: 1 (days 27-35 + 1-6), 2 (days 7-16) and 3 (days 17-26). The RNA expression of ESR1, four ERGs (PGR, GREB1, TFF1 and PDZK1), and three proliferation genes (MKI67, TOP2A and CDC20) were compared between the windows. Gene expression of the four ERGs was 53-129 % higher in window 2 than window 1 (p = 0.0013, 0.0006, 0.022 and 0.066 for PGR, GREB1, TFF1 and PDZK1, respectively) and lower (9-41 %) in window 3 compared to window 2 (p = 0.079, 0.31, 0.031 and 0.065 for PGR, GREB1, TFF1 and PDZK1, respectively). Their average expression (AvERG) was 64 % higher in window 2 than window 1 (p < 0.0001) and 21 % lower in window 3 than window 2 (p = 0.0043). There were no significant differences between the windows for ESR1 and proliferation genes. In agreement with the gene expression data, progesterone receptor protein levels measured by immunohistochemistry (IHC) were 164 and 227 % higher in windows 2 and 3, respectively, compared to window 1 (30.7 and 37.9 % cells positive vs. 11.6 %; p = 0.0003 and 0.0004, respectively), while no difference in ER IHC score was observed. In conclusion, we observed significant differences in the expression of ERGs in ER+ breast tumours across the menstrual cycle. This variability may affect the interpretation of gene expression profiles incorporating ERGs and may be exploitable as an endogenous test of endocrine responsiveness..

Up to 50 % of women receiving aromatase inhibitor (AI) complain of AI-associated musculoskeletal symptoms (AIMSS) and 15 % discontinue treatment. We conducted a randomized, sham-controlled trial to evaluate whether acupuncture improves AIMSS and to explore potential mechanisms. Postmenopausal women with early stage breast cancer, experiencing AIMSS were randomized to eight weekly real or sham acupuncture sessions. We evaluated changes in the Health Assessment Questionnaire Disability Index (HAQ-DI) and pain visual analog scale (VAS) following the intervention compared to baseline. Serum estradiol, β-endorphin, and proinflammatory cytokine concentrations were measured pre and post-intervention. We enrolled 51 women of whom 47 were evaluable, including 23 randomized to real and 24 to sham acupuncture. Baseline characteristics were balanced between groups with the exception of a higher HAQ-DI score in the real acupuncture group (p = 0.047). We did not observe a statistically significant difference in reduction of HAQ-DI (p = 0.30) or VAS (p = 0.31) between the two groups. Following eight weekly treatments, we observed a statistically significant reduction of IL-17 (p ≤ 0.009) in both groups. No significant modulation was seen in estradiol, β-endorphin, or other proinflammatory cytokine concentrations in either group. We did not observe a significant difference in AIMSS changes between real and sham acupuncture. As sham acupuncture used in this study may not be equivalent to placebo, further studies with a non-acupuncture arm may be required to establish whether acupuncture is beneficial for the treatment of AIMSS..

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BACKGROUND: Adjuvant endocrine therapy beyond 5 years reduces recurrence in patients with estrogen receptor-positive breast cancer. We have previously shown that immunohistochemical markers (IHC4) and two gene expression profile tests (recurrence score [RS] and PAM50 risk of recurrence [ROR]) are associated with time to distant recurrence, and we have now assessed the value of each of these scores and routine clinical variables for predicting outcome, specifically in years 5 to 10. METHODS: We used univariate and multivariable proportional hazards models to determine the prognostic value of all variables and scores (IHC4, RS, ROR) for distant recurrence, separately in years 0 to 5 and specifically for years 5 to 10 for all patients. All statistical tests were two-sided. RESULTS: Nodal status and tumor size were at least as strong in years 5 to 10 as in years 0 to 5 (nodal status, years 5-10: χ² = 21.72 vs years 0-5: χ² = 11.08, both P < .001; tumor size, years 5-10: χ² = 10.52 vs years 0-5: χ² = 10.82, both P = .001). Ki67 and the overall IHC4 score were the only statistically significant biomarkers related to distant recurrence univariablely in the 5 to 10 year period (χ² = 8.67, χ² = 13.22, respectively). The ROR score was the strongest molecular prognostic factor in the late follow-up period (χ² = 16.29; P < .001), whereas IHC4 (χ² = 7.41) and RS (χ² = 5.55) were only weakly prognostic in this period. Similar results were seen for all subgroups and for all recurrences. CONCLUSIONS: None of the IHC4 markers provided statistically significant prognostic information in years 5 to 10, except for nodal status and tumor size. ROR gave the strongest prognostic information in years 5 to 10. These results may help select patients who could benefit most from hormonal therapy beyond 5 years of treatment..

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Results from the NSABP B-28 trial suggest AKT activation may predict reduced benefit from taxanes following standard anthracycline therapy. Pre-clinical data support a link between PI3 K/AKT signalling and taxane resistance. Using the UK taxotere as adjuvant chemotherapy trial (TACT), we tested the hypothesis that activation of AKT or downstream markers, p70S6K or p90RSK, identifies patients with reduced benefit from taxane chemotherapy. TACT is a multi-centre open-label phase III trial comparing four cycles of standard FEC (fluorouracil, epirubicin, cyclophosphamide) followed by four cycles of docetaxel versus eight cycles of anthracycline-based chemotherapy. Samples from 3,596 patients were available for the current study. We performed immunohistochemical analysis of activation of AKT, p70S6 K and p90RSK. Using a training set with multiple cut-offs for predictive values (10 % increments in expression), we found no evidence for a treatment by marker interaction for pAKT473, pS6 or p90RSK. pAKT473, pS6 and p90RSK expression levels were weakly correlated. A robust, preplanned statistical analysis in the TACT trial found no evidence that pAKT473, pS6 or p90RSK identifies patients deriving reduced benefit from adjuvant docetaxel. This result is consistent with the recent NASBP B28 study where the pAKT473 effect is not statistically significant for the treatment interaction test. Therefore, neither TACT nor NASBP-B28 provides statistically robust evidence of a treatment by marker interaction between pAKT473 and taxane treatment. Alternative methods for selecting patients benefitting from taxanes should be explored..

The higher incidence of breast cancer in developed countries has been tempered by reductions in mortality, largely attributable to mammographic screening programmes and advances in adjuvant therapy. Optimal systemic management requires consideration of clinical, pathological and biological parameters. Oestrogen receptor alpha (ERα), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) are established biomarkers evaluated at diagnosis, which identify cardinal subtypes of breast cancer. Their prognostic and predictive utility effectively guides systemic treatment with endocrine, anti-HER2 and chemotherapy. Hence, accurate and reliable determination remains of paramount importance. However, the goals of personalized medicine and targeted therapies demand further information regarding residual risk and potential benefit of additional treatments in specific circumstances. The need for biomarkers which are fit for purpose, and the demands placed upon them, is therefore expected to increase. Technological advances, in particular high-throughput global gene expression profiling, have generated multi-gene signatures providing further prognostic and predictive information. The rational integration of routinely evaluated clinico-pathological parameters with key indicators of biological activity, such as proliferation markers, also provides a ready opportunity to improve the information available to guide systemic therapy decisions. The additional value of such information and its proper place in patient management is currently under evaluation in prospective clinical trials. Expanding the utility of biomarkers to lower resource settings requires an emphasis on cost effectiveness, quality assurance and possible international variations in tumor biology; the potential for improved clinical outcomes should be justified against logistical and economic considerations..

CONTEXT: Endogenous sex hormones are risk factors for postmenopausal breast cancer. A potential route for favorable hormonal modification is weight loss. OBJECTIVE: The objective of the study was to measure change in plasma estradiol and testosterone levels in postmenopausal women in relation to change in body mass index (BMI) and plasma leptin. SETTING: The setting was a cohort study of over 100,000 female volunteers from the general population, United Kingdom. PARTICIPANTS: The participants were a sample of 177 postmenopausal women aged over 45 years who provided blood samples during 2004-2005 and again during 2010-2011. MAIN OUTCOME MEASURE: Outcomes were percentage change in plasma estradiol and testosterone levels per 1 kg/m² change in BMI and per 1 ng/mL change in plasma leptin. RESULTS: Among women with reduction in BMI, estradiol decreased 12.7% (95% confidence interval: [6.4%, 19.5%]; P < .0001) per kg/m² and among women with increased BMI estradiol increased 6.4% [0.2%, 12.9%] (P = .042). The corresponding figures for testosterone were 10.7% [3.0%, 19.0%] (P = .006) and 1.9% [-5.4%, 9.7%] (P = .61) per kg/m². For women with decreases and increases in leptin, estradiol decreased by 3.6% [1.3%, 6.0%] (P = .003) per ng/mL and increased by 1.7% [-0.3%, 3.6%] (P = .094), respectively. The corresponding figures for testosterone were 4.8% [2.0%, 7.8%] (P = .009) and 0.3% [-2.0%, 2.6%] (P = .82) per ng/mL. CONCLUSIONS: In postmenopausal women, changes in BMI and plasma leptin occurring over several years are associated with changes in estradiol and testosterone levels. The results suggest that fat loss by an individual can result in substantial decreases in postmenopausal estradiol and testosterone levels and provides support for weight management to lessen breast cancer risk..

BAG1 is a multifunctional anti-apoptotic protein located on chromosome 9q12, which binds to Bcl-2. BAG1 is present as a separate module in the GHI-RS 21-gene panel. It may provide additional prognostic information as an immunohistochemical marker when added to IHC4. Analysis of BAG1 was performed on archival tumour blocks from patients from the anastrozole and tamoxifen arms of the ATAC trial of 5 years endocrine therapy in postmenopausal women with oestrogen receptor (ER)-positive primary breast cancer. Staining was scored separately as nuclear or cytoplasmic. Statistical analyses were performed on data from median 10-year follow-up with distant recurrence as primary endpoint. Data on both nuclear and cytoplasmic BAG1 as well as the IHC4 markers (ER, PgR, HER2 and Ki67) were available on 963 ER-positive cases of which 860 were HER2-negative. Cytoplasmic and nuclear BAG1 were highly correlated (Spearman r = 0.79, p < 00001). Women with higher BAG1 expression developed 30 % fewer distant recurrences compared to those with low expression. Nuclear BAG1 contributed significantly to the clinical and IHC4 models with added information being greater in node-positive cases. Similar results were seen if all recurrences were the endpoints. BAG1 expression provides significant prognostic information when added to the classical clinicopathological parameters and IHC4, particularly in node-positive patients. .

Prosurvival protein BCL-2 is overexpressed in estrogen receptor positive (ER(+)) breast cancer. In this issue of Cancer Cell, Vaillant and colleagues demonstrate that targeting BCL-2 with BH3 mimetics improves the response of xenografts from primary ER(+) breast tumors to endocrine therapy and reduces tamoxifen-induced endometrial hyperplasia, a strategy with potential clinical applicability. .

The study of large prospective collections of plasma samples from women prior to the development of breast cancer has firmly established certain sex steroids as being significantly associated with risk. The strongest associations have been found in postmenopausal women in whom the within person variability of most hormones is markedly reduced but some positive associations have also been seen in premenopausal women. Plasma estrogens show the strongest correlations with risk and these are strengthened by measurement or calculation of the proportion of estradiol that circulates free of sex hormone binding globulin (SHBG), consistent with this being the most active fraction. The relationships have been reported to potentially explain virtually all of the association of breast cancer with body mass index in postmenopausal women; this is likely to be due to non-ovarian estrogen synthesis being prominent in subcutaneous fat. These strong relationships have led to plasma and urine estrogen levels being used as intermediate end-points in the search for genes that affect breast cancer risk via their role in steroid disposition. Plasma androgen levels also show a relationship with breast cancer risk that is weakened but not eliminated by 'correction' for estrogen levels. This has been argued to be evidence of the local production of estrogens being important in the etiology of breast cancer. Given that plasma steroid levels do not correlate closely with mammographic density, which is strongly associated with risk, the opportunity exists to combine the two factors in assessing breast cancer risk but the low availability of suitable estrogen assays is a major impediment to this. In established breast cancer, plasma estrogens have been found to correlate with gene expression of estrogen dependent genes and the expression of these varies across the menstrual cycle of premenopausal women. There is infrequently a need for routine measurement of plasma estrogen levels but it has been important in the comparative pharmacology and dose-related effectiveness of aromatase inhibitors. Measurement may be needed to identify residual ovarian function in women who have amenorrhea subsequent to cytotoxic chemotherapy indicating their unsuitability for aromatase inhibitor treatment. Use of highly sensitive assays has also revealed that the association between BMI and plasma estrogen levels persists in patients on 3rd generation aromatase inhibitors and that measurable increments in plasma estrogen levels occur with some vaginal estrogen preparations that are of concern in relation to treatment efficacy. .

BACKGROUND: The optimum endocrine treatment for postmenopausal women with advanced hormone-receptor-positive breast cancer that has progressed on non-steroidal aromatase inhibitors (NSAIs) is unclear. The aim of the SoFEA trial was to assess a maximum double endocrine targeting approach with the steroidal anti-oestrogen fulvestrant in combination with continued oestrogen deprivation. METHODS: In a composite, multicentre, phase 3 randomised controlled trial done in the UK and South Korea, postmenopausal women with hormone-receptor-positive breast cancer (oestrogen receptor [ER] positive, progesterone receptor [PR] positive, or both) were eligible if they had relapsed or progressed with locally advanced or metastatic disease on an NSAI (given as adjuvant for at least 12 months or as first-line treatment for at least 6 months). Additionally, patients had to have adequate organ function and a WHO performance status of 0-2. Participants were randomly assigned (1:1:1) to receive fulvestrant (500 mg intramuscular injection on day 1, followed by 250 mg doses on days 15 and 29, and then every 28 days) plus daily oral anastrozole (1 mg); fulvestrant plus anastrozole-matched placebo; or daily oral exemestane (25 mg). Randomisation was done with computer-generated permuted blocks, and stratification was by centre and previous use of an NSAI as adjuvant treatment or for locally advanced or metastatic disease. Participants and investigators were aware of assignment to fulvestrant or exemestane, but not of assignment to anastrozole or placebo. The primary endpoint was progression-free survival (PFS). Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, numbers NCT00253422 (UK) and NCT00944918 (South Korea). FINDINGS: Between March 26, 2004, and Aug 6, 2010, 723 patients underwent randomisation: 243 were assigned to receive fulvestrant plus anastrozole, 231 to fulvestrant plus placebo, and 249 to exemestane. Median PFS was 4·4 months (95% CI 3·4-5·4) in patients assigned to fulvestrant plus anastrozole, 4·8 months (3·6-5·5) in those assigned to fulvestrant plus placebo, and 3·4 months (3·0-4·6) in those assigned to exemestane. No difference was recorded between the patients assigned to fulvestrant plus anastrozole and fulvestrant plus placebo (hazard ratio 1·00, 95% CI 0·83-1·21; log-rank p=0·98), or between those assigned to fulvestrant plus placebo and exemestane (0·95, 0·79-1·14; log-rank p=0·56). 87 serious adverse events were reported: 36 in patients assigned to fulvestrant plus anastrozole, 22 in those assigned to fulvestrant plus placebo, and 29 in those assigned to exemestane. Grade 3-4 adverse events were rare; the most frequent were arthralgia (three in the group assigned to fulvestrant plus anastrozole; seven in that assigned to fulvestrant plus placebo; eight in that assigned to exemestane), lethargy (three; 11; 11), and nausea or vomiting (five; two; eight). INTERPRETATION: After loss of response to NSAIs in postmenopausal women with hormone-receptor-positive advanced breast cancer, maximum double endocrine treatment with 250 mg fulvestrant combined with oestrogen deprivation is no better than either fulvestrant alone or exemestane..

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BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032..

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PURPOSE: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor-positive (ER+) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment. EXPERIMENTAL DESIGN: Baseline and 2-week posttreatment biopsies were obtained from 112 postmenopausal women with ER+ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response. RESULTS: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an independent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort. CONCLUSIONS: The molecular response to aromatase inhibitor treatment varies greatly between patients consistent with the variable clinical benefit from aromatase inhibitor treatment. Higher baseline expression of an inflammatory signature is associated with poor antiproliferative response and should be assessed further as a novel biomarker and potential target for aromatase inhibitor-treated patients..

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BACKGROUND: In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67's value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study. METHODS: Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided. RESULTS: Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation. CONCLUSIONS: Substantial variability in Ki67 scoring was observed among some of the world's most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited..

BACKGROUND: Trastuzumab has established efficacy against breast cancer with overexpression or amplification of the HER2 oncogene. The standard of care is 1 year of adjuvant trastuzumab, but the optimum duration of treatment is unknown. We compared 2 years of treatment with trastuzumab with 1 year of treatment, and updated the comparison of 1 year of trastuzumab versus observation at a median follow-up of 8 years, for patients enrolled in the HERceptin Adjuvant (HERA) trial. METHODS: The HERA trial is an international, multicentre, randomised, open-label, phase 3 trial comparing treatment with trastuzumab for 1 and 2 years with observation after standard neoadjuvant chemotherapy, adjuvant chemotherapy, or both in 5102 patients with HER2-positive early breast cancer. The primary endpoint was disease-free survival. The comparison of 2 years versus 1 year of trastuzumab treatment involved a landmark analysis of 3105 patients who were disease-free 12 months after randomisation to one of the trastuzumab groups, and was planned after observing at least 725 disease-free survival events. The updated intention-to-treat comparison of 1 year trastuzumab treatment versus observation alone in 3399 patients at a median follow-up of 8 years (range 0-10) is also reported. This study is registered with ClinicalTrials.gov, number NCT00045032. FINDINGS: We recorded 367 events of disease-free survival in 1552 patients in the 1 year group and 367 events in 1553 patients in the 2 year group (hazard ratio [HR] 0·99, 95% CI 0·85-1·14, p=0·86). Grade 3-4 adverse events and decreases in left ventricular ejection fraction during treatment were reported more frequently in the 2 year treatment group than in the 1 year group (342 [20·4%] vs 275 [16·3%] grade 3-4 adverse events, and 120 [7·2%] vs 69 [4·1%] decreases in left ventricular ejection fraction, respectively). HRs for a comparison of 1 year of trastuzumab treatment versus observation were 0·76 (95% CI 0·67-0·86, p<0·0001) for disease-free survival and 0·76 (0·65-0·88, p=0·0005) for overall survival, despite crossover of 884 (52%) patients from the observation group to trastuzumab therapy. INTERPRETATION: 2 years of adjuvant trastuzumab is not more effective than is 1 year of treatment for patients with HER2-positive early breast cancer. 1 year of treatment provides a significant disease-free and overall survival benefit compared with observation and remains the standard of care. FUNDING: F Hoffmann-La Roche (Roche)..

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Caveolin-1 is the essential constituent protein of specialised plasma membrane invaginations called caveolae. The unique topology of caveolin-1 facilitates the role of caveolae as molecular hubs, integrating the activity of a multitude of signalling molecules. Despite improvements in our understanding of caveolin-1 interactions and the function of caveolae, the relationship between dysfunctional caveolin-1 and tumourigenesis remains contentious. Perhaps most intriguing has been the demonstration of both oncogenic and tumour suppressor function within particular tumour types, including breast cancer. In this review, the biological and clinical relevance of caveolin-1 in human breast cancer are considered. Evidence is systematically presented for the potential tumour suppressor and oncogenic functions of caveolin-1. Specific reference is made to interactions between caveolin-1 and signalling pathways in the clinical and biological subtypes of breast cancer. Areas of controversy are discussed and technical considerations are highlighted. Translational implications and potential for specific therapeutic manipulation of caveolin-1 are evaluated in the context of evidence from in vitro and in vivo studies..

Caveolin-1 is the principal constituent protein of caveolae, which are specialised plasma membrane invaginations with diverse biological roles. Caveolin-1 is suggested to have tumour suppressive functions and CAV1 gene mutations have been reported in 20% of breast cancers. The aim of the present study was to evaluate the frequency of CAV1 mutations in a large cohort of optimally accrued breast cancers. Two independent series of breast cancer samples were analysed: 82 fresh-frozen grade 3 and 158 formalin-fixed paraffin-embedded invasive ductal carcinomas of no special type were consecutively accrued and subjected to microdissection of neoplastic epithelial cells prior to DNA extraction. Thirty-nine human breast cancer cell lines were also included in this study. The trans-membrane region of CAV1 and adjacent sequences, where mutations are reported to cluster, were amplified by PCR, followed by direct sequencing and mutational analysis. None of the reported CAV1 gene mutations, including CAV1 (P132L), were identified in either clinical samples (95% CI: 0-1.5%) or human breast cancer cell lines analysed. One novel non-synonymous germline polymorphism was detected within a reported region of high mutational frequency. This study does not corroborate the reported frequent occurrence of CAV1 gene mutations, including CAV1 (P132L), in primary human breast carcinomas. Our findings demonstrate that if CAV1 mutations do exist, their overall mutational frequency is substantially lower than positive reports have suggested. Taken together with other studies, which have also failed to identify CAV1 mutations, our data call into question the existence and biological and clinical relevance of CAV1 gene mutations in human breast cancer..

CONTEXT: Abiraterone acetate is a small-molecule cytochrome P450 17A1 (CYP17A1) inhibitor that is active in castration-resistant prostate cancer. OBJECTIVE: Our objective was to determine the impact of abiraterone with and without dexamethasone treatment on in vivo steroidogenesis. DESIGN AND METHODS: We treated 42 castrate, castration-resistant prostate cancer patients with continuous, daily abiraterone acetate and prospectively collected blood and urine before and during abiraterone treatment and after addition of dexamethasone 0.5 mg daily. RESULTS: Treatment with single-agent abiraterone acetate was associated with accumulation of steroids with mineralocorticoid properties upstream of CYP17A1. This resulted in side effects, including hypertension, hypokalemia, and fluid overload, in 38 of 42 patients that were generally treated effectively with eplerenone. Importantly, serum and urinary androgens were suppressed by more than 90% from baseline. Urinary metabolites of 17-hydroxypregnenolone and 17-hydroxyprogesterone downstream of 17α-hydroxylase remained unchanged. However, 3α5α-17-hydroxypregnanolone, which can be converted via the backdoor pathway toward 5α-dihydrotestosterone, increased significantly and correlated with levels of the major 5α-dihydrotestosterone metabolite androsterone. In contrast, urinary metabolites of 11-deoxycortisol and active glucocorticoids declined significantly. Addition of dexamethasone to abiraterone acetate significantly suppressed ACTH and endogenous steroids, including 3α5α-17-hydroxypregnanolone. CONCLUSION: CYP17A1 inhibition with abiraterone acetate is characterized by significant suppression of androgen and cortisol synthesis. The latter is associated with a rise in ACTH that causes raised mineralocorticoids, leading to side effects and incomplete 17α-hydroxylase inhibition. Concomitant inhibition of 17,20-lyase results in diversion of 17-hydroxyprogesterone metabolites toward androgen synthesis via the backdoor pathway. Addition of dexamethasone reverses toxicity and could further suppress androgens by preventing a rise in substrates of backdoor androgen synthesis..

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BACKGROUND: Epidemiological studies have provided strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that are associated with hormone levels and the risk of breast cancer in premenopausal women. METHODS: We measured urinary levels of estrone glucuronide (E1G) using a protocol specifically developed to account for cyclic variation in hormone levels during the menstrual cycle in 729 healthy premenopausal women. We genotyped 642 single-nucleotide polymorphisms (SNPs) in these women; a single SNP, rs10273424, was further tested for association with the risk of breast cancer using data from 10 551 breast cancer case patients and 17 535 control subjects. All statistical tests were two-sided. RESULTS: rs10273424, which maps approximately 50 kb centromeric to the cytochrome P450 3A (CYP3A) gene cluster at chromosome 7q22.1, was associated with a 21.8% reduction in E1G levels (95% confidence interval [CI] = 27.8% to 15.3% reduction; P = 2.7 × 10(-9)) and a modest reduction in the risk of breast cancer in case patients who were diagnosed at or before age 50 years (odds ratio [OR] = 0.91, 95% CI = 0.83 to 0.99; P = .03) but not in those diagnosed after age 50 years (OR = 1.01, 95% CI = 0.93 to 1.10; P = .82). CONCLUSIONS: Genetic variation in noncoding sequences flanking the CYP3A locus contributes to variance in premenopausal E1G levels and is associated with the risk of breast cancer in younger patients. This association may have wider implications given that the most predominantly expressed CYP3A gene, CYP3A4, is responsible for metabolism of endogenous and exogenous hormones and hormonal agents used in the treatment of breast cancer..

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BACKGROUND: Intravaginal estradiols (VE) have been proposed as safe alternatives to systemic estrogen therapy in breast cancer survivors. PATIENTS AND METHODS: Postmenopausal women with estrogen receptor-positive breast cancer or at high risk for breast cancer (n = 24) who were taking an aromatase inhibitor (AI) or a selective estrogen receptor modulator (SERM) and VE for ≥ 90 days for atrophic vaginitis and 24 controls taking AI only participated in the study. Serum samples were drawn from VE ring patients before insertion and 30 and 60 days postinsertion, from VE tablet patients the morning before insertion and approximately 12 hours postinsertion, and once from controls. Samples were assayed for E2 concentrations by using highly sensitive radioimmunoassay after ether extraction. RESULTS: Mean E2 levels in controls were 3.72 pmol/L (range, < 3.0-7.7 pmol/L); mean E2 levels preinsertion and 12 weeks postinsertion in the VE ring patients were significantly greater than controls (P < .001 for each comparison). Mean preinsertion E2 levels in patients using VE tablets were not significantly different than those of controls (P = .48), and postinsertion levels were 76 pmol/L higher than preinsertion (P < .001). CONCLUSION: VE treatment increased E2 levels. Preinsertion levels for patients receiving VE tablets were not elevated compared with those of controls, suggesting that E2 elevations with this preparation may not be continuously sustained. We conclude that VE treatment, regardless of type, results in elevated circulating E2 levels in this population and should be used with caution..

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Estrogen receptor (ER) and HER2 are well established as predictive markers for treatment benefit, although methodological deficiencies can still affect their predictive accuracy. The shift towards earlier diagnosis poses a challenge in identifying those low-risk patients who may safely avoid adjuvant chemotherapy for early breast cancer. Therefore, recent research has focused on developing biomarkers to quantify residual risk on adjuvant endocrine therapy. For widespread adoption into clinical practice, these must be validated in well-designed clinical trials and provide additional information to current standards using reproducible and cost-effective methodologies. Furthermore, evidence from preoperative studies indicates that on- or post-treatment biomarkers can be more predictive than at baseline. In particular, Ki67 has recently emerged as an intermediate marker of long-term outcome. The power of Ki67 to predict treatment benefit from endocrine therapy has facilitated the design of studies where Ki67 is the primary end-point. This has also led to investigations into the predictive power of Ki67 to determine benefit from signal transduction inhibitors and chemotherapy in several recent and ongoing trials..

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BACKGROUND: Mammographic breast density and endogenous sex-hormone levels are both strong risk factors for breast cancer. This study investigated whether there is evidence for a shared genetic basis between these risk factors. METHODS: Using data on 1,286 women from 617 families, we estimated the heritabilities of serum estradiol, testosterone, and sex-hormone binding globulin (SHBG) levels and of three measures of breast density (dense area, nondense area, and percentage density). We tested for associations between hormone levels and density measures and estimated the genetic and environmental correlations between pairs of traits using variance and covariance components models and pedigree-based maximum likelihood methods. RESULTS: We found no significant associations between estradiol, testosterone, or SHBG levels and any of the three density measures, after adjusting for body mass index (BMI). The estimated heritabilities were 63%, 66%, and 65% for square root-transformed adjusted percentage density, dense area, and nondense area, respectively, and 40%, 25%, and 58% for log-transformed-adjusted estradiol, testosterone, and SHBG. We found no evidence of a shared genetic basis between any hormone levels and any measure of density, after adjusting for BMI. The negative genetic correlation between dense and nondense areas remained significant even after adjustment for BMI and other covariates (ρ = -0.34; SE = 0.08; P = 0.0005). CONCLUSIONS: Breast density and sex hormones can be considered as independent sets of traits. IMPACT: Breast density and sex hormones can be used as intermediate phenotypes in the search for breast cancer susceptibility loci..

INTRODUCTION: Estrogen receptor-α (ER) and human epidermal growth factor receptor 2 (HER2) positivity are inversely correlated by standard criteria. However, we investigated the quantitative relation between ER and HER2 expression at both RNA and protein levels in HER2+ve and HER2-ve breast carcinomas. METHODS: ER and HER2 levels were assessed with immunohistochemistry (IHC) and (for HER2) fluorescent in situ hybridization (FISH) and by quantitative reverse transcription-polymerase chain reaction (q-RT-PCR) in formalin-fixed primary breast cancers from 448 patients in the National Cancer Research Institute (NCRI) Adjuvant Breast Cancer Trial (ABC) tamoxifen-only arm. Relations at the RNA level were assessed in 1,139 TransATAC tumors. RESULTS: ER and HER2 RNA levels were negatively correlated as expected in HER2+ve (IHC 3+ and/or FISH-amplified) tumors (r = -0.45; P = 0.0028). However, in HER2-ve tumors (ER+ve and ER-ve combined), a significant positive correlation was found (r = 0.43; P < 0.0001), HER2 RNA levels being 1.74-fold higher in ER+ve versus ER-ve tumors. This correlation was maintained in the ER+veHER2-ve subgroup (r = 0.24; P = 0.0023) and confirmed in this subgroup in 1,139 TransATAC tumours (r = 0.25; P < 0.0001). The positive relation extended to IHC-detected ER in ABC: mean ± 95% confidence interval (CI) H-scores were 90 ± 19 and 134 ± 19 for 0 and 1+ HER2 IHC categories, respectively (P = 0.0013). A trend toward lower relapse-free survival (RFS) was observed in patients with the lowest levels of ER and HER2 RNA levels within the ER+veHER2-ve subgroup both for ABC and TransATAC cohorts. CONCLUSIONS: ER and HER2 expression is positively correlated in HER2-ve tumors. The distinction between HER2+ve and HER2-ve is greater in ER-ve than in ER+ve tumors. These findings are important to consider in clinical trials of anti-HER2 and anti-endocrine therapy in HER2-ve disease. TRIAL REGISTRATION: Clinical trial identifier: ISRCTN31514446..

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INTRODUCTION: Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results. METHODS: BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation. RESULTS: Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies. CONCLUSIONS: The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy..

INTRODUCTION: The majority of breast tumors at primary diagnosis are estrogen receptor positive (ER+). Estrogen (E) mediates its effects by binding to the ER. Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ breast cancer. However, resistance remains a major clinical problem. METHODS: To identify molecular mechanisms associated with resistance to E-deprivation, we assessed the temporal changes in global gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of estradiol (E2), long term estrogen deprived (LTED), that leads to recovery of proliferative status and models resistance to an aromatase inhibitor (AI). The expression levels of proteins were determined by western blotting. Proliferation assays were carried out using the dual platelet derived growth factor receptor (PDGFR)/Abelson tyrosine kinase (Abl) inhibitor nilotinib. Luciferase reporter assays were used to determine effects on ER-mediated transactivation. Changes in recruitment of cofactors to the gene regulated by estrogen in breast cancer 1 (GREB1) promoter were determined by chromatin immunoprecipitation (ChIP). Gene expression data were derived from 81 postmenopausal women with ER+ BC pre-treatment and at two-weeks post-treatment with single agent anastrozole in a neoadjuvant trial. RESULTS: The PDGF/Abl canonical pathway was significantly elevated as early as one week post E-deprivation (P = 1.94 E-04) and this became the top adaptive pathway at the point of proliferative recovery (P = 1.15 E-07). Both PDGFRβ and Abl protein levels were elevated in the LTED cells compared to wild type (wt)-MCF7 cells. The PDGF/Abl tyrosine kinase inhibitor nilotinib, suppressed proliferation in LTED cells in the presence or absence of E. Nilotinib also suppressed ER-mediated transcription by destabilizing the ER and reducing recruitment of amplified in breast cancer-1 (AIB1) and the CREB binding protein (CBP) to the promoter of the E-responsive gene GREB1. High PDGFRβ in primary ER+ breast cancer of 81 patients prior to neoadjuvant treatment with an AI was associated with poorer antiproliferative response. Additionally PDGFRβ expression increased after two weeks of AI therapy (1.25 fold, P = 0.003). CONCLUSIONS: These preclinical and clinical data indicate that the PDGF/Abl signaling pathway merits clinical evaluation as a therapeutic target with endocrine therapy in ER+ breast cancer..

PURPOSE: To investigate whether suppression of plasma estradiol and estrone sulfate levels by the aromatase inhibitors (AIs) anastrozole and letrozole is related to body mass index (BMI) in postmenopausal women with early estrogen receptor (ER) -positive breast cancer. Recent studies have reported that the AI anastrozole has lower effectiveness than tamoxifen in women with high BMI. This effect with high BMI might hypothetically be a result of reduced inhibition of aromatase and suppression of plasma estrogen levels and might be overcome by the use of an increased dose of anastrozole or, alternatively, the use of a more potent AI such as letrozole. PATIENTS AND METHODS: Plasma estradiol and estrone sulfate levels from a highly sensitive radioimmunoassay were available for 44 postmenopausal patients who received anastrozole (1 mg per day) for 3 months followed by letrozole (2.5 mg per day) for 3 months or the opposite sequence. Correlations between the estrogen suppression by each AI and BMI were assessed. RESULTS: Baseline values of estradiol and estrone sulfate were significantly correlated with BMI (r = 0.57; P < .001, and r = 0.38; P = .006, respectively). Levels of estrogen in patients receiving treatment were greater at higher levels of BMI with both AIs, but although this was significant with letrozole (r = 0.35; P = .013, and r = 0.30; P = .035 for estradiol and estrone sulfate, respectively), it was not with anastrozole. Suppression of both estrogen types was greater with letrozole across the full range of BMIs in this study. CONCLUSION: The suppressed levels of plasma estradiol and estrone sulfate in postmenopausal women with early ER-positive breast cancer treated with the AIs anastrozole and letrozole are related to BMI..

BACKGROUND: Adjuvant tamoxifen therapy substantially decreases the risk of recurrence and mortality in women with hormone (estrogen and/or progesterone) receptor-positive breast cancer. Previous studies have suggested that metabolic conversion of tamoxifen to endoxifen by cytochrome P450 2D6 (CYP2D6) is required for patient benefit from tamoxifen therapy. METHODS: Tumor specimens from a subset of postmenopausal patients with hormone receptor-positive early-stage (stages I, II, and IIIA) breast cancer, who were enrolled in the randomized double-blind Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial, were genotyped for variants in CYP2D6 (N = 1203 patients: anastrozole [trade name: Arimidex] group, n = 615 patients; tamoxifen group, n = 588 patients) and UDP-glucuronosyltransferase-2B7 (UGT2B7), whose gene product inactivates endoxifen (N = 1209 patients; anastrozole group, n = 606 patients; tamoxifen group, n = 603 patients). Genotyping was performed using polymerase chain reaction-based TaqMan assays. Based on the genotypes for CYP2D6, patients were classified as poor metabolizer (PM), intermediate metabolizer (IM), or extensive metabolizer (EM) phenotypes. We evaluated the association of CYP2D6 and UGT2B7 genotype with distant recurrence (primary endpoint) and any recurrence (secondary endpoint) by estimating the hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) using Cox proportional hazards models. All statistical tests were two-sided. RESULTS: After a median follow-up of 10 years, no statistically significant associations were observed between CYP2D6 genotype and recurrence in tamoxifen-treated patients (PM vs EM: HR for distant recurrence = 1.25, 95% CI = 0.55 to 3.15, P = .64; HR for any recurrence = 0.99, 95% CI = 0.48 to 2.08, P = .99). A near-null association was observed between UGT2B7 genotype and recurrence in tamoxifen-treated patients. No associations were observed between CYP2D6 and UGT2B7 genotypes and recurrence in anastrozole-treated patients. CONCLUSION: The results do not support the hypothesis that CYP2D6 genotype predicts clinical benefit of adjuvant tamoxifen treatment among postmenopausal breast cancer patients..

BACKGROUND: The immunohistochemical (IHC) 4+C score is a cost-effective prognostic tool that uses clinicopathologic factors and four standard IHC assays: oestrogen receptor (ER), PR, HER2 and Ki67. We assessed its utility in personalising breast cancer treatment in a clinical practice setting, through comparison with Adjuvant! Online (AoL) and the Nottingham Prognostic Index (NPI). METHODS: We prospectively gathered clinicopathologic data for postmenopausal patients with hormone receptor-positive, HER2-negative, N0-3 resected early breast cancer treated consecutively at our institution. We retrospectively calculated and compared prognostic scores. The primary endpoint was the proportion of patients reclassified from AoL-defined intermediate-risk by application of the IHC4+C score. RESULTS: The median age of the 101 patients included in the analysis was 63. In all, 15 of the 26 patients classified as intermediate-risk by AoL were reallocated to a low-risk group by application of the IHC4+C score and no patient was reclassified as high-risk group. Of the 59 patients classified as intermediate-risk group by the NPI, 24 were reallocated to a low-risk group and 13 to a high-risk group. CONCLUSION: IHC4+C reclassifies more than half of the patients stratified as being in intermediate-risk group by the AoL and NPI. The use of IHC4+C may substantially improve decision-making on adjuvant chemotherapy..

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Genome-wide association studies (GWAS) have successfully identified common genetic variants that contribute to breast cancer risk. Discovering additional variants has become difficult, as power to detect variants of weaker effect with present sample sizes is limited. An alternative approach is to look for variants associated with quantitative traits that in turn affect disease risk. As exposure to high circulating estradiol and testosterone, and low sex hormone-binding globulin (SHBG) levels is implicated in breast cancer etiology, we conducted GWAS analyses of plasma estradiol, testosterone, and SHBG to identify new susceptibility alleles. Cancer Genetic Markers of Susceptibility (CGEMS) data from the Nurses' Health Study (NHS), and Sisters in Breast Cancer Screening data were used to carry out primary meta-analyses among ~1600 postmenopausal women who were not taking postmenopausal hormones at blood draw. We observed a genome-wide significant association between SHBG levels and rs727428 (joint β = -0.126; joint P = 2.09 × 10(-16)), downstream of the SHBG gene. No genome-wide significant associations were observed with estradiol or testosterone levels. Among variants that were suggestively associated with estradiol (P<10(-5)), several were located at the CYP19A1 gene locus. Overall results were similar in secondary meta-analyses that included ~900 NHS current postmenopausal hormone users. No variant associated with estradiol, testosterone, or SHBG at P<10(-5) was associated with postmenopausal breast cancer risk among CGEMS participants. Our results suggest that the small magnitude of difference in hormone levels associated with common genetic variants is likely insufficient to detectably contribute to breast cancer risk..

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Purpose: The majority of breast cancer patients who have estrogen receptor positive (ER+) tumors whose proliferation is reduced after estrogen deprivation by aromatase inhibitors (AI). This study investigates any link between proliferation and hypoxia, a major determinant of tumor biology, and defines the effect of estrogen deprivation on hypoxia-associated genes.Methods: Genome-wide expression profiles were obtained from tumor biopsies from 81 ER+ postmenopausal patients, before and after 2 weeks' anastrozole treatment. A hypoxia metagene was developed by identifying genes clustered with classical hypoxia-regulated genes, excluding those associated with proliferation. Proliferation was measured by Ki67 and a proliferation metagene derived from two published breast cancer data sets.Results: Hypoxia and proliferation metagenes were associated at baseline (Pearson correlation coefficient, r = 0.67, P < 10(-4)) and after 2 weeks (r = 0.71, P < 10(-4)). Hypoxia metagene at baseline was associated with 2-week Ki67 (r = 0.43, P = 0.0002) and more weakly with poor 2-week Ki67 change consistent with a weak association with AI resistance. Hypoxia metagene was significantly downregulated with AI. This downregulation was significantly associated with change in the proliferation metagene and with Ki67 but, importantly, not with the substantial change in expression of classical estrogen-dependent genes.Conclusions: Hypoxia metagene is closely associated with proliferation before and after AI treatment. The downregulation of hypoxia metagene after AI therapy is most likely the result of changes in proliferation. There may be a weak effect of hypoxia metagene on de novo resistance to AIs. These findings are important to consider in coapplication of antiproliferative agents with antiangiogenic or antihypoxia agents. Clin Cancer Res; 17(9); 3005-12. (C) 2011 AACR..

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Although normal breast tissue and breast cancer estrogens are known to be elevated compared with plasma estrogen levels, the mechanism behind this phenomenon has been an issue of debate for 2 decades. If local estrogen aromatization were to be confirmed as the main estrogen source in breast cancer tissue, tissue-specific inhibition of estrogen production, avoiding systemic side effects, would become a potentially attractive option for breast cancer treatment and prevention. Based on recent results from our groups exploring tissue estrogens, together with estrogen-synthesizing and estrogen-regulated gene expression levels, we propose a new model to explain elevated breast tissue estrogen levels. Although local estrogen production may be important, the local contribution is overruled by rapid plasma-to-tissue equilibration, including active uptake of circulating estrogens or enhanced tissue binding. As for breast cancer tissue levels, elevated levels of estradiol may be explained to a large extent by estrogen receptor binding and local conversion of estrone into estradiol. This model indicates that effective suppression of benign and malignant tissue estrogens as a treatment for ER+ breast cancer requires systemic suppression and will not be markedly affected by local enzyme targeting..

Purpose: Although most patients with estrogen receptor alpha (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped.Experimental Design: We adapted four ER+ human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy.Results: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth.Conclusions: A gene expression signature derived from ER+ breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. Clin Cancer Res; 17(7); 2024-34. (C)2011 AACR..

AIMS: To evaluate the clinicopathological associations and predictive value of the transcription factor NF-κB in a large series of breast cancer patients treated with neoadjuvant chemotherapy. METHODS: A retrospective search of a prospectively maintained database was performed to identify patients. Immunohistochemistry was used to assess the p65 subunit of NF-κB, using nuclear staining as a surrogate of activation. RESULTS: Nuclear NF-κB expression was found in 26.3% (35/133) of cases. Nuclear NF-κB staining was associated with high histological grade (p=0.05), oestrogen receptor (ER) negativity (p=0.01) and higher Ki67 index (p=0.002). Patients with nuclear NF-κB staining had a higher pathological complete response (pCR) rate than those without (26.5% vs 6.0% respectively, p=0.004); there was no significant association with clinical response or outcome. In an exploratory hypothesis-generating analysis, in the ER+/HER2- subgroup (n=43) a significantly lower clinical response rate was observed in those with nuclear NF-κB staining compared with those who had no nuclear NF-κB staining (14.3% vs 61.0%, p=0.038). There were no pCRs in ER+/ HER2- tumours. CONCLUSIONS: Nuclear NF-κB expression is associated with ER negativity, higher Ki67 index and tumour grade. It was also found to be significantly associated with increased pCR but not clinical response to neoadjuvant chemotherapy..

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Approximately 80% of human breast carcinomas present as oestrogen receptor alpha-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs = 0.67, 0.64, and 0.55 respectively, FDR < 1x10(-7)). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ER alpha antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ER alpha. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences on the recently identified relationship between SNPs in this region and breast cancer risk..

Over the last 20 years, aromatase inhibitors have been developed to become a highly effective treatment strategy for treatment of hormone receptor positive breast cancer. Despite their success, poor response and resistance limit the effectiveness of these agents in up to 50% of patients. In recent years, studies using highly sensitive hormone assays have provided insight into the source of oestrogen production for the stimulation of oestrogen receptor positive breast cancer growth, suggesting that uptake from the circulation is likely to make a significant contribution to intratumoural oestradiol. To obtain insight into how tumours become resistant to oestrogen after aromatase inhibition, long term oestrogen deprivation of cultured cells has been used to mimic acquired resistance to aromatase inhibitors. This work has aided the selection of agents to rationally combine with aromatase inhibitors to combat resistance. Molecular profiling using genome-wide approaches has shed new light on the heterogeneity of responses to oestrogen deprivation and predictors of resistance in vivo. Testing new agents and combinations in short-term pre-surgical studies using biomarkers such as Ki67 is critical for increasing the rate at which new rational combinations can be assessed for efficacy..

Background: In postmenopausal women estrogens can be formed by the aromatase pathway, which gives rise to estrone, and the steroid sulfatase (STS) route which can result in the formation of estrogens and androstenediol, a steroid with potent estrogenic properties. Aromatase inhibitors, such as anastrozole, are now in clinical use whereas STS inhibitors, such as STX64, are still undergoing clinical evaluation. STX64 was recently shown to block STS activity and reduce serum androstenediol concentrations in postmenopausal women with breast cancer. In contrast, little is known about the effects of aromatase inhibitors or anti-estrogens on STS activity or serum androgen levels. Patients and Methods: Study 1: Blood was collected from ten postmenopausal women with breast cancer before and after two-week treatment with anastrozole and serum concentrations of androstenediol and other androgens and estrogens were assessed. Study 2: Blood samples were collected from 15 breast cancer patients before and after four-week treatment with anastrozole and 10 patients before and after four-week treatment with tamoxifen. Blood was used to assess STS activity in peripheral blood lymphocytes (PBLs) and serum dehydroepiandrosterone sulfate and dehydroepiandrosterone levels. Results: Neither anastrozole nor tamoxifen had any significant effect on STS activity as measured in PBLs. Anastrozole did not affect serum androstenediol concentrations. Conclusion: Anastrozole and tamoxifen did not inhibit STS activity and serum androstenediol concentrations were not reduced by aromatase inhibition. As androstenediol has estrogenic properties, it is possible that the combination of an aromatase inhibitor and STS inhibitor may give a therapeutic advantage over the use of either agent alone..

BACKGROUND: In postmenopausal women estrogens can be formed by the aromatase pathway, which gives rise to estrone, and the steroid sulfatase (STS) route which can result in the formation of estrogens and androstenediol, a steroid with potent estrogenic properties. Aromatase inhibitors, such as anastrozole, are now in clinical use whereas STS inhibitors, such as STX64, are still undergoing clinical evaluation. STX64 was recently shown to block STS activity and reduce serum androstenediol concentrations in postmenopausal women with breast cancer. In contrast, little is known about the effects of aromatase inhibitors or anti-estrogens on STS activity or serum androgen levels. PATIENTS AND METHODS: Study 1: Blood was collected from ten postmenopausal women with breast cancer before and after two-week treatment with anastrozole and serum concentrations of androstenediol and other androgens and estrogens were assessed. Study 2: Blood samples were collected from 15 breast cancer patients before and after four-week treatment with anastrozole and 10 patients before and after four-week treatment with tamoxifen. Blood was used to assess STS activity in peripheral blood lymphocytes (PBLs) and serum dehydroepiandrosterone sulfate and dehydroepiandrosterone levels. RESULTS: Neither anastrozole nor tamoxifen had any significant effect on STS activity as measured in PBLs. Anastrozole did not affect serum androstenediol concentrations. CONCLUSION: Anastrozole and tamoxifen did not inhibit STS activity and serum androstenediol concentrations were not reduced by aromatase inhibition. As androstenediol has estrogenic properties, it is possible that the combination of an aromatase inhibitor and STS inhibitor may give a therapeutic advantage over the use of either agent alone..

P>ObjectiveLow circulating levels of testosterone and sex-hormone-binding globulin (SHBG) are associated with increased cardiovascular risk in men. This association may be partially mediated through changes in glucose metabolism, but relatively few data are available on the relationship between sex hormones and markers of long-term glycaemia. We assessed the associations of endogenous testosterone and SHBG with glycated haemoglobin (HbA(1c)) in men.Design and subjectsCross-sectional study of 1292 men from the Norfolk population of European Prospective Investigation into Cancer (EPIC-Norfolk).MeasurementsGlycated haemoglobin, total testosterone (TT) and SHBG levels were measured, and free testosterone (FT) levels were calculated. Multiple linear regression models were used to assess the associations of TT, SHBG and FT with HbA(1c).ResultsMen with diabetes had lower testosterone and SHBG levels. In non-diabetic men, HbA(1c) levels were inversely associated with TT and calculated FT independently of age, body mass index, smoking, alcohol consumption and physical activity. The adjusted change in HbA(1c) was 0 center dot 055 (95% CI 0 center dot 025; 0 center dot 085) per standard deviation (SD) decrease in TT and 0 center dot 041 (95% CI 0 center dot 010; 0 center dot 073) per SD decrease in calculated FT, respectively. SHBG levels were inversely associated with HbA(1c) after multivariable adjustment (beta = 0 center dot 038 per SD decrease (95% CI 0 center dot 004; 0 center dot 071)).ConclusionsIn middle-aged and older men, low endogenous testosterone and SHBG levels are associated with glycaemia, even below the threshold for diabetes. Further studies are needed to determine the effects of interventions that raise testosterone levels in men having increased HbA(1c) and subnormal testosterone levels..

Aromatase inhibitors (AI) have improved the treatment of oestrogen receptor positive (ER+) breast cancer. Despite the efficacy of these agents over 40% of patients relapse with endocrine resistant disease. Here we describe an in vitro model of acquired resistance to long-term oestrogen deprivation (LTED). The LTED cells retain expression of the ER and appear hypersensitive to oestrogen as a result of altered kinase activity. Furthermore analysis of temporal changes in gene expression during the acquisition of resistance highlight growth factor receptor pathways as key mediators of this adaptive process..

Estrogen receptor-alpha (ER alpha) positive breast cancer frequently responds to inhibitors of ER alpha activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ER alpha in ER alpha-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ER alpha over-expression in ER alpha-positive breast cancer cells, we over-expressed ER alpha in the MCF-7 breast cancer cell line using adenovirus gene transduction. ER alpha over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ER alpha transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ER alpha-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ER alpha remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ER alpha could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients..

Considerable heterogeneity exists amongst oestrogen receptor positive (ER+ve) breast cancer in both its molecular profile and response to therapy. Attempts to better define variation amongst breast tumours have led to the definition of four main "intrinsic" subtypes of breast cancer with two of these classes, Luminal A and B. composed almost entirely of ER+ve cancers. In this study we set out to investigate the significance of intrinsic subtypes within a group of ER+ve breast cancers treated with neoadjuvant anastrozole. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with anastrozole was analyzed on Illumina 48K microarrays. Gene-expression based subtypes and risk of relapse (ROR) scores for tumours pre- and post-treatment were determined using the PAM50 method. Amongst pre-treatment samples, all intrinsic subtypes were found to be present, although luminal groups were represented most highly. Luminal A and B tumours obtained similar benefit from treatment, as measured by the proportional fall in the proliferation marker Ki67 upon treatment (mean suppression = 75.5% vs 75.7%). Tumours classified as basal and Her2-like showed poor reductions in Ki67 upon treatment. Residual Ki67 staining after two weeks remained higher in the Luminal B group. ROR score was significantly associated with anti-proliferative response to AI and with clinical response. These results suggest that in the short-term, Luminal A and B tumours may gain similar benefit from an AI but that the higher residual Ki67 level seen in Luminal B is indicative of poorer long term outcome. (C) 2011 Elsevier Inc. All rights reserved..

Estrogen receptor (ER) positive primary breast cancers have a wide range of clinical outcomes. Prediction of the likely course of the disease aids treatment decision-making. In the translational arm of the ATAC (anastrozole or tamoxifen alone or combined) trial (TransATAC) we have assessed individual and multiparameter biomarkers for their prediction of overall and distant recurrence. None of the biomarkers identified differential benefit for anastrozole versus tamoxifen. Each of ER, PgR, HER2 and Ki67 was associated with risk of recurrence. A combination of these to create a single predictor IHC4 was as informative as the 21-gene recurrence score (RS). Integration of each of these molecular profiles with classical clinicopathologic variables provided the most accurate prediction of outcome..

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The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER. (C) 2011 Elsevier Inc. All rights reserved..

BACKGROUND: Surprisingly few data are published on the relevance of even commonly used biomarkers of response to aromatase inhibitors (AIs) in advanced breast cancer. Here, we aim to determine the effectiveness of AIs in that setting according to quantitative levels of estrogen receptor (ER), progesterone receptor (PgR) and Ki67 or human epithelial growth factor receptor-2 (HER-2) status. PATIENTS AND METHODS: ER, PgR, HER-2 and Ki67 protein expressions were centrally assessed in 177 archival formalin-fixed paraffin-embedded primary or locally recurrent breast tumours from women who subsequently received AI treatment of advanced disease. RESULTS: Among ER-positive patients (n = 146), higher PgR, but not ER, levels were associated with increased time to AI treatment failure (TTF). Higher Ki67 staining was associated with decreased TTF. ER-positive/HER-2-positive patients showed a non-significant trend for decreased TTF compared with ER-positive/HER-2-negative patients. PgR level, but not Ki67, remained a significant predictor of TTF in multivariate analysis of ER-positive patients. CONCLUSIONS: Higher PgR and Ki67 levels are significantly associated with increased and decreased TTF, respectively, in ER-positive patients receiving AI treatment of advanced disease. The higher proliferation seen in PgR-negative tumours does not explain the poorer clinical responsiveness of this subgroup..

Most estrogen receptor a (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER+ xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.SIGNIFICANCE: ER alpha retains genomic activity and drives a CDK4/E2F-dependent transcriptional program despite estrogen deprivation therapy. Combined inhibition of ER and PI3K induced regression of ER+ xenografts, supporting further development of strong ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for the treatment of antiestrogen- resistant breast cancers. Cancer Discovery; 1(4); 338-51. (C) 2011 AACR..

CONTEXT: Sex hormones play a key role in women's health, but little is known about lifestyle factors that influence their levels. OBJECTIVE: The objective of the study was to investigate the relationship between cigarette smoking habits and endogenous sex hormone levels in postmenopausal women. DESIGN AND PARTICIPANTS: This was a cross-sectional study among 2030 postmenopausal women aged 55-81 yr from the Norfolk population of the European Prospective Investigation into Cancer. All women were at least 1 yr postmenopausal and not currently using hormone replacement therapy. General linear models were used to examine the relationship between smoking habits and sex hormone levels. RESULTS: Among current smokers, the daily number of cigarettes smoked was associated with increased levels of testosterone (19-37%), free testosterone (19-34%), 17-hydroxprogesterone (17-22%), androstenedione (2-23%), SHBG (6-10%), and estradiol (-2 to 15%). Stratified analysis for body mass index revealed an interaction such that the association with SHBG was restricted to lean women, whereas a smoking-related increase in free estradiol was found only in overweight women. No clear dose-response relationship was observed for estrone, although its levels were highest in heavy smokers. Current smoking habit was associated with a larger difference in sex hormone levels than lifetime cigarette exposure as measured by pack-years. Among former smokers, sex hormones were at levels of never smokers within 1-2 yr of smoking cessation. CONCLUSIONS: Cigarette smoking is associated with higher circulating levels of androgens, estrogens, 17-hydroxprogesterone, and SHBG in postmenopausal women. The almost immediate lower levels with smoking cessation may indicate that hormone related disease risks could potentially be modified by changing smoking habits..

Development of resistance to the antioestrogen tamoxifen occurs in a large proportion of patients with oestrogen receptor-positive (ER+) breast cancer and is an important clinical challenge. While loss of ER occurs in c.20% of tamoxifen-resistant tumours, this cannot be the sole explanation for tamoxifen treatment failure. PI3K pathway activation, including by insulin-like growth factor receptor 1 (IGF1R), has been implicated in some resistance models. The primary aim was to determine whether evidence exists in clinical breast cancer for a role of IGF1R and/or the PI3K pathway, in acquisition of resistance to tamoxifen. Invasive primary and recurrent tamoxifen-resistant tumours from the same patient (n=77) were assessed for changes in ER, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), IGF1R, stathmin, PTEN expression and PIK3CA mutations where possible. ER and PgR levels were significantly reduced at recurrence with 22 and 45%, respectively, showing negative status at this time. Acquisition of HER2 overexpression occurred in 6% of cases. IGF1R expression was significantly reduced in both ER+ and ER- recurrences and stathmin levels increased. A positive association between stathmin and IGF1R emerged in recurrent samples, despite their opposing relationships with ER, suggesting some coalescence of their activities may be acquired. The data confirm loss of ER and PgR and gain of HER2 in some tamoxifen-resistant tumours. There is no evidence for IGF1R gain in tamoxifen resistance; increases in stathmin levels suggest that activation of the PI3K pathway may have contributed, but PTEN loss and PIK3CA hotspot mutations were relatively rare..

PURPOSE: The Arimidex, Tamoxifen Alone or in Combination (ATAC) study was a double-blind randomized trial in which postmenopausal women with early-stage breast cancer were assigned to receive anastrozole, tamoxifen, or the combination. We have conducted a retrospective analysis to examine the effects of comorbidities and age on treatment received, breast cancer-related mortality, and competing causes of mortality. PATIENTS AND METHODS: The current analyses were based on 10-year median follow-up data in the two monotherapy arms (anastrozole, n = 3,092; tamoxifen, n = 3,094) of the ATAC study. Baseline comorbidities and tumor and treatment characteristics were compared between women age less than 70 years and women age ≥ 70 years. The cumulative incidence of breast cancer-related and non-breast cancer-related mortality was assessed according to age and comorbidities. RESULTS: One thousand six hundred sixty-two patients (27%) were age ≥ 70 years at study entry. Older women were more likely to undergo mastectomy (odds ratio [OR], 1.92; 95% CI, 1.71 to 2.16) and less likely to receive radiotherapy (OR, 0.49; 95% CI, 0.44 to 0.55) or chemotherapy (OR, 0.24; 95% CI, 0.18 to 0.29). Women age ≥ 70 years had an increased risk of recurrence compared with women age less than 70 years (hazard ratio [HR], 1.21; 95% CI, 1.08 to 1.37) and a substantially increased risk of death without recurrence (HR, 4.13; 95% CI, 3.53 to 4.83). The risk of death without recurrence increased with comorbidity score (10-year estimates of 8.4%, 20.0%, and 30.4% for Satariano score 0, 1, and 2+, respectively; P < .001). CONCLUSION: Age influences the risk of recurrence, and age and comorbidities significantly influence the risk of death without recurrence. Formal assessment of comorbidities should be incorporated into decisions regarding adjuvant therapies..

PURPOSE: We recently reported that the mRNA-based, 21-gene Genomic Health recurrence score (GHI-RS) provided additional prognostic information regarding distant recurrence beyond that obtained from classical clinicopathologic factors (age, nodal status, tumor size, grade, endocrine treatment) in women with early breast cancer, confirming earlier reports. The aim of this article is to determine how much of this information is contained in standard immunohistochemical (IHC) markers. PATIENTS AND METHODS: The primary cohort comprised 1,125 estrogen receptor-positive (ER-positive) patients from the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial who did not receive adjuvant chemotherapy, had the GHI-RS computed, and had adequate tissue for the four IHC measurements: ER, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and Ki-67. Distant recurrence was the primary end point, and proportional hazards models were used with sample splitting to control for overfitting. A prognostic model that used classical variables and the four IHC markers (IHC4 score) was created and assessed in a separate cohort of 786 patients. RESULTS: All four IHC markers provided independent prognostic information in the presence of classical variables. In sample-splitting analyses, the information in the IHC4 score was found to be similar to that in the GHI-RS, and little additional prognostic value was seen in the combined use of both scores. The prognostic value of the IHC4 score was further validated in the second separate cohort. CONCLUSION: This study suggests that the amount of prognostic information contained in four widely performed IHC assays is similar to that in the GHI-RS. Additional studies are needed to determine the general applicability of the IHC4 score..

Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes. The Pharmacogenomics Journal (2011) 11, 348-358; doi:10.1038/tpj.2010.50; published online 15 June 2010.

The substantial reduction in risk of recurrence and mortality in premenopausal breast cancer patients of estrogen deprivation as treatment for early ER+ breast cancer is well accepted. Surgical, radiotherapeutic or medical approaches to ovarian ablation/suppression all seem to be similarly effective and appear to be at least partially additive to the reduction seen with chemotherapy. Cytotoxic treatment of premenopausal women also frequently elicits a reduction in frequency and regularity of menstruation and sometimes a complete and permanent amenorrhea as a reflection of reduced ovarian activity. While it is certain that the associated reduction in estrogenic exposure of patients contributes to the overall effectiveness of chemotherapy in some premenopausal women the degree to which this is the case is a subject of vigorous debate. Furthermore, the extent to which chemotherapy induced ovarian suppression impacts on molecular predictors of chemotherapy benefit is poorly understood and recommendations for further study of this issue are made..

Factors associated with an increased risk of breast cancer include prior breast cancer, high circulating estrogens, and increased breast density. Adjuvant aromatase inhibitors are associated with a reduction in incidence of contralateral breast cancer. We conducted a prospective, single-arm, single-institution study to determine whether use of anastrozole is associated with changes in contralateral breast density and circulating estrogens. Eligible patients included postmenopausal women with hormone receptor-positive early-stage breast cancer who had completed local therapy, had an intact contralateral breast, and were recommended an aromatase inhibitor as their only systemic therapy. Participants received anastrozole 1 mg daily for 12 months on study. We assessed contralateral breast density and serum estrogens at baseline, 6, and 12 months. The primary endpoint was change in contralateral percent breast density from baseline to 12 months. Secondary endpoints included change in serum estrone sulfate from baseline to 12 months. Fifty-four patients were accrued. At 12 months, compared with baseline, there was a nonstatistically significant reduction in breast density (mean change: -16%, 95% CI: -30 to 2, P = 0.08) and a significant reduction in estrone sulfate (mean change: -93%, 95% CI: -94 to -91, P < 0.001). Eighteen women achieved 20% or greater relative reduction in contralateral percent density at 12 months compared with baseline; however, no measured patient or disease characteristics distinguished these women from the overall population. Large trials are required to provide additional data on the relationship between aromatase inhibitors and breast density and, more importantly, whether observed changes in breast density correlate with meaningful disease-specific outcomes..

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Uncontrolled proliferation is a hallmark of cancer. In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Increasingly, Ki67 is measured in these scenarios for clinical research, including as a primary efficacy endpoint for clinical trials, and sometimes for clinical management. At present, the enormous variation in analytical practice markedly limits the value of Ki67 in each of these contexts. On March 12, 2010, an international panel of investigators with substantial expertise in the assessment of Ki67 and in the development of biomarker guidelines was convened in London by the co-chairs of the Breast International Group and North American Breast Cancer Group Biomarker Working Party to consider evidence for potential applications. Comprehensive recommendations on preanalytical and analytical assessment, and interpretation and scoring of Ki67 were formulated based on current evidence. These recommendations are geared toward achieving a harmonized methodology, create greater between-laboratory and between-study comparability, and allow earlier valid applications of this marker in clinical practice..

A tumour biomarker is a characteristic that is objectively measured and evaluated in tumour samples as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The development of a biomarker contemplates distinct phases, including discovery by hypothesis-generating preclinical or exploratory studies, development and qualification of the assay for the identification of the biomarker in clinical samples, and validation of its clinical significance. Although guidelines for the development and validation of biomarkers are available, their implementation is challenging, owing to the diversity of biomarkers being developed. The term 'validation' undoubtedly has several meanings; however, in the context of biomarker research, a test may be considered valid if it is 'fit for purpose'. In the process of validation of a biomarker assay, a key point is the validation of the methodology. Here we discuss the challenges for the technical validation of immunohistochemical and gene expression assays to detect tumour biomarkers and provide suggestions of pragmatic solutions to address these challenges..

BACKGROUND: Breast cancers can be classified by hierarchical clustering using an "intrinsic" gene list into one of at least five molecular subtypes: basal-like, HER2, luminal A, luminal B, and normal breast-like. Five different intrinsic gene lists composed of varying numbers of genes have been used for molecular subtype identification and classification of breast cancers. The aim of this study was to determine the objectivity and interobserver reproducibility of the assignment of molecular subtype classes by hierarchical cluster analysis. METHODS: Three publicly available breast cancer datasets (n = 779) were subjected to two-way average-linkage hierarchical cluster analysis using five distinct intrinsic gene lists. We used free-marginal Kappa statistics to analyze interobserver agreement among five breast cancer researchers for the whole classification and for each molecular subtype separately according to each intrinsic gene list for each breast cancer dataset. RESULTS: None of the classification systems tested produced almost perfect agreement (Kappa ≥ 0.81) among observers. However, substantial interobserver agreement (70.8% to 76.1% of the samples and free-marginal Kappa scores from 0.635 to 0.701) was consistently observed in all datasets for four molecular subtypes (luminal, basal-like, HER2, and normal breast-like). When luminal cancers were subdivided (luminal A, B, and C), none of the classification systems produced substantial agreement (Kappa ≥ 0.61) in all the datasets analyzed. Analysis of each subtype separately revealed that only two (basal-like and HER2) could be reproducibly identified by independent observers (Kappa ≥ 0.81). CONCLUSIONS: Assignment of molecular subtype classes of breast cancer based on the analysis of dendrograms obtained with hierarchical cluster analysis is subjective and shows modest interobserver reproducibility. For the development of a molecular taxonomy, objective definitions for each molecular subtype and standardized methods for their identification are required..

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PURPOSE: The 21-gene breast cancer assay recurrence score (RS) is widely used for assessing recurrence risk and predicting chemotherapy benefit in patients with estrogen receptor (ER) -positive breast cancer. Pathologic and clinical factors such as tumor size, grade, and patient age also provide independent prognostic utility. We developed a formal integration of these measures and evaluated its prognostic and predictive value. PATIENTS AND METHODS: From the National Surgical Adjuvant Breast and Bowel (NSABP) B-14 and translational research cohort of the Arimidex, Tamoxifen Alone or in Combination (TransATAC) studies, we included patients who received hormonal monotherapy, had ER-positive tumors, and RS and traditional clinicopathologic factors assessed (647 and 1,088, respectively). Individual patient risk assessments from separate Cox models were combined using meta-analysis to form an RS-pathology-clinical (RSPC) assessment of distant recurrence risk. Risk assessments by RS and RSPC were compared in node-negative (N0) patients. RSPC was compared with RS for predicting chemotherapy benefit in NSABP B-20. RESULTS: RSPC had significantly more prognostic value for distant recurrence than did RS (P < .001) and showed better separation of risk in the study population. RSPC classified fewer patients as intermediate risk (17.8% v 26.7%, P < .001) and more patients as lower risk (63.8% v 54.2%, P < .001) than did RS among 1,444 N0 ER-positive patients. In B-20, the interaction of RSPC with chemotherapy was not statistically significant (P = .10), in contrast to the previously reported significant interaction of RS with chemotherapy (P = .037). CONCLUSION: RSPC refines the assessment of distant recurrence risk and reduces the number of patients classified as intermediate risk. Adding clinicopathologic measures did not seem to enhance the value of RS alone nor the individual biology RS identifies in predicting chemotherapy benefit..

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165 Background: Weight gain is commonly reported by patients with breast cancer. Aromatase inhibitors are being tested in the preventive setting in high risk women and it is important to evaluate the association between treatment and weight change as this may effect the patient's decision to continue to take the drug. METHODS: Weight change in postmenopausal women from three large clinical trials (ATAC, IBIS-I, IBIS-II) investigating endocrine treatment for the treatment and prevention of breast cancer have been analysed. The objective of this retrospective study was to assess the effects of anastrozole and tamoxifen on weight change in postmenopausal women. RESULTS: In the ATAC trial, a mean increase of 1.4 kg was observed after 12 months of follow-up and no statistically significant differences between treatment arms (anastrozole vs. tamoxifen) were found. The majority of women kept their weight stable and only 11.2% gained more than 5 kg within this time period. Significant baseline predictors for gaining more than 5 kg of weight after 12 months of follow-up were being younger than 60 years old at entry, smoking at entry, and mastectomy. Results for the two prevention studies IBIS-I and IBIS-II were quite similar. In the IBIS-II trial, the overall mean weight change after 12 months of follow-up was 0.5 kg. In the IBIS-I study women gained an average of 0.9 kg within the first 12 months of follow up. In both studies no differences between treatment and placebo (IBIS-I: tamoxifen vs. placebo; IBIS-II: anastrozole vs. placebo) were observed. Overall, women in the adjuvant setting gained more than 5 kg of weight (11.2%) after 12 months of follow up compared to those in the preventive setting (5.3% IBIS-I, 5.7% IBIS-II), but similar overall weight gain was seen after 60 months of follow up in both settings (5.7% IBIS-I vs. 6.1% ATAC). CONCLUSIONS: All three trials have demonstrated that weight gain occurs primarily within the first 12 months of active treatment and are similar to those on placebo. Weight stabilises after 12 months of follow up and major weight gain was rare in all trials, and unrelated to treatment..

2525 Background: Many advanced ER+ breast cancer pts show intrinsic resistance to endocrine treatment with the remainder acquiring resistance. Androgenic steroids upstream of aromatase can drive steroid receptor signaling critical to tumour growth. Evidence also exists for an ERα-/AR+ subset of breast cancers transcriptionally similar to ERα+ disease. We hypothesized that AA, a cytochrome (CYP) 17 inhibitor that irreversibly inhibits androgen and estrogen synthesis, would have anti-tumour activity in ER+ or ER-/AR+ pts. METHODS: Post-menopausal women with ER+ or ER-/AR+ advanced breast cancer resistant to >2 lines of hormone therapies were treated in 6-pt cohorts with AA at doses between 250 and 2000mg daily. RESULTS: A total of 25 pts were treated in the phase I trial with 2 pts ongoing on study. Median time on treatment was 1.8 mths (range 0.7 - 11.6 mths). There were no dose limiting toxicities. The majority of adverse events (AEs) were Common Toxicity Criteria grade 1 or 2: fatigue, nausea, anorexia, dyspnoea, palpitations, dizziness and flushes. Hypokalemia was frequent, due to secondary mineralocorticoid syndrome: grade 3/4 hypokalemia occurred in 4 pts. This was effectively managed with potassium supplementation, hydrocortisone (20 mg mane, 10 mg nocte) and eplerenone (50-200 mg) with no clinical consequences. Other grade 3 AEs were neutropenia and reduced left ventricular ejection fraction (1 pt) and exercise-induced hypotension with dizziness (1 pt). At the 2000mg dose 5/5 subjects had suppression of estradiol, testosterone, DHEA and DHEAS to below the lower limit of detection of the assay. Two pts (both ER+/AR+) continued on study beyond 11 mths, one of whom achieved a radiological partial response and 80% reduction in serum CA15.3 from baseline. Pharmacokinetic data will be presented. CONCLUSIONS: AA is well tolerated in advanced breast cancer pts with preliminary evidence of antitumour activity. The predominant AEs are mechanism-based (hypokalemia) and can be managed expectantly, although careful monitoring of potassium levels is recommended..

10509 Background: Neoadjuvant chemotherapy (NAC) leads to a pathological complete response in ~20% of patients with breast cancer. The remaining patients have residual disease and are more likely to develop metastasis. In post-NAC tumors, Ki67 levels, a marker of cell proliferation, have been shown to inform odds of recurrence and death. We hypothesized that post-NAC residual cancers with a high Ki67 harbor molecular alterations that are associated with drug resistance. METHODS: We utilized Nanostring digital RNA quantification to measure the expression levels of 350 cancer-related genes in 49 post-NAC breast cancers. Gene expression data were tested for associations with post-NAC Ki67. The cohort was enriched for triple-negative breast cancer (24/49 tumors). The candidate gene DUSP4 was explored in in vitro models of breast cancer. RESULTS: The post-NAC Ki67 (range 2.4 - 99%; median: 31.6%) was statistically different among the molecular subtypes of breast cancers enrolled, being highest in BLBC (p<0.0001). Gene expression signatures of KRAS activation were enriched in BLBC. Low levels of the MAPK phosphatase DUSP4 correlated with a high Ki67 in BLBCs (r=-0.54, p=8x10(-4)) and with Ras/MAPK pathway activation in human breast tumors and cell lines. Genome-wide studies identified preferential methylation of the DUSP4 promoter in BLBC. Among BLBC cell lines, low DUSP4 expression predicted for sensitivity to MEK inhibitors and resistance to docetaxel. Inhibition of MEK with AZD6244 or overexpression of DUSP4 in DUSP4-low BLBC cell lines improved sensitivity to docetaxel and inhibited mammosphere formation. RNAi-mediated knockdown of DUSP4 in BLBC cells enhanced ETS-1 phosphorylation, mammosphere formation, and MAPK-dependent secretion of the cancer stem cell-promoting cytokines IL6 and IL8. CONCLUSIONS: Low levels of DUSP4 correlate with high tumor cell proliferation after NAC. Downregulation of DUSP4 may underlie activation of the MAPK pathway in the absence of KRAS mutations in BLBC while conferring resistance to chemotherapy and a stem cell-like phenotype that is therapeutically targetable with MAPK inhibitors..

TPS117 Background: The neoadjuvant IMPACT trial suggested Ki67 levels after 2 weeks endocrine therapy predicts long-term outcome. Major changes in gene expression have also been seen in ER+ breast cancer after aromatase inhibitor (AI) treatment. POETIC evaluates whether changes in Ki67 level after 2 weeks treatment predicts for relapse-free survival (RFS) more effectively than the baseline value. It also tests whether gene expression profile at this timepoint provides more accurate prognostic and predictive information than the pre-treatment profile. Experimental evidence suggests peri-operative endocrine therapy may improve disease outcome. This hypothesis is also addressed in POETIC. ( ISRCTN63882543 ) With a sample size of 4000, an improvement in 5 year relapse from 10% to 7% could be detected with 91% power (two sided alpha of 5%), as would a 1.3 fold difference in the ability of Ki67 to predict RFS (90% power, two sided 5% significance level). Target recruitment is 4000 patients from 100 UK hospitals over 3-4 years. METHODS: Patients are randomised in the ratio of 2:1 to perioperative AI (letrozole 2.5mg or anastrozole 1mg daily) starting 2 weeks before planned surgery until 2 weeks after surgery. FFPE and RNA-later samples are taken prior to trial entry (baseline) and at surgery. Eligible patients are postmenopausal with ER+ invasive breast cancer. Consent to take additional research tissue is sought from patients undergoing diagnostic biopsy. Consenting patients donate tumour tissue in RNA-later and/or a FFPE research sample and enter POETIC following diagnosis of ER+ breast cancer. Matching tumour tissue is taken at surgery. Where an RNA-later sample at baseline is unavailable, consenting patients may undergo a further biopsy for research tissue immediately before study entry. Where consent procedures at diagnosis present logistical challenges, sites may provide FFPE tissue left over from diagnosis only. The 1(st) patient was entered in September 2008, and by January 2011 102 UK hospitals open and 1200 patients were entered. 182 optional RNA-later samples at both timepoints are available. Current success is due to a flexible approach to tissue sample collection and overcoming local and national logistical challenges..

608 Background: Germline genotypic polymorphisms in CYP19A1 have been associated with differential benefit from treatment with AIs in patients with mBC, but validation is lacking. METHODS: We retrospectively identified patients with ER-positive mBC treated with a 3(rd) generation AI. DNA was obtained from archival paraffin-embedded tumors and genotyped for 74 polymorphisms in 20 candidate genes, including CYP19A1 using the Sequenom MassARRAY Platform. The CYP19A1 Arg264Cys ( rs700519 ) variant was analyzed by Pyrosequencing. The CYP19A1 (TTTA)n(rs60271534) and TCT Ins/Del (rs11575899) variants were genotyped by fragment analysis.Primary endpoint was time to treatment failure (TTF), analyses being undertaken using Cox's regression to estimate the hazard ratio for risk of failure per allele. RESULTS: 308 patients were included. Mean TTF was 15 months (range 1 to 58). Two sets of analyses were undertaken. The first were confirmatory analyses for the CYP19A1 variants rs4646, rs10459592 and rs4775936, previously associated with letrozole efficacy in mBC.TTF was significantly improved in patients carrying the T allele of rs4775936 compared to homozygous wild-type patients [HR=0.79 (0.66-0.95); p=0.012]. However, this association lost significance when adjusted for other prognostic factors. When the analysis was limited to letrozole treated patients (n=165), the difference was no longer significant (p=0.068). None of the 60 other SNPs with MAF ≥5%, included as an exploratory set, showed significant association with TTF. Patients with >7 TTTA repeats on either allele of CYP19A1 had lower risk of failure than those without it [HR=0.84 (0.7-0.99); p=0.04]. This association also lost significance when adjusted for clinical factors. CONCLUSIONS: In this retrospective cohort of patients with mBC treated with a 3(rd) generation AI, carriage of the minor allele of the CYP19A1 variant rs4775936 and greater than seven repeats of the TTTA polymorphism within intron 4 of CYP19A1 were significantly associated with improved AI treatment efficacy in univariate analysis and require further study. There was no significant association of any other candidate SNP with efficacy of AIs..

521 Background: Aromatase inhibitors (AIs) may promote OFR in patients with CIA. True incidence of OFR is unknown partly because estradiol (E2) assays used in the clinical setting lacking sensitivity. We studied incidence and predictors of OFR measuring E2 by a clinical assay (direct) or by a highly sensitive (indirect) immunoassay, together with other markers of ovarian reserve. METHODS: Prospective exploratory study in ER positive BC patients, who had CIA lasting at least two years and postmenopausal E2 levels, while on tamoxifen. Patients received exemestane and underwent assessments of E2 (indirect and direct assay), FSH, antimullerian hormone (AMH), and antral follicule count (AFC) at baseline and at month 1, 3 and 6. We considered OFR if: resumption of menses, or developed (i) both E2 levels inconsistent with postmenopausal women on AI (indirect: >8 pmol/L; direct >165 pmol/L) and FSH (<26 IU/L) levels), or (ii) frankly non-postmenopausal E2 values (indirect >55 pmol/L; direct >220 pmol/L). RESULTS: Fifty-three consecutive patients were enrolled; median age: 48y (41-55). By indirect assay, 15 patients (28.3%; CI95%:16.1-40.4) had OFR, (11 menses, 4 OFR without menses). By direct assay 14 had OFR (11 menses, 3 OFR without menses). Mean time to OFR by indirect assay was 5.7 months (CI95%: 3.1-8.3) and 6.4 months (CI95%: 3.7-9.1) by direct assay. There was no difference in baseline E2 levels among those who recover or not OFR. Mean age was the only baseline marker different between patients who had OFR or not (45.7 vs 49.1 years, t-test p<0.0001). In patients ≤48 years (n=26) rate of OFR was 46%. AMH at baseline was below detection limit in all patients and it did not change during exemestane. Baseline and serial values for AFC were not different among patients who had OFR or not. CONCLUSIONS: There is a high incidence (28%) of OFR, in patients with CIA treated with AIs, especially in women ≤48y. The two E2 assays showed similar incidence of OFR and time to OFR, despite having markedly different performance characteristics. AMH was not helpful in defining ovarian reserve and likelihood of OFR..

BACKGROUND: Breast cancer risk for postmenopausal women is positively associated with circulating concentrations of oestrogens and androgens, but the determinants of these hormones are not well understood.METHODS: Cross-sectional analyses of breast cancer risk factors and circulating hormone concentrations in more than 6000 postmenopausal women controls in 13 prospective studies.RESULTS: Concentrations of all hormones were lower in older than younger women, with the largest difference for dehydroepiandrosterone sulphate (DHEAS), whereas sex hormone-binding globulin (SHBG) was higher in the older women. Androgens were lower in women with bilateral ovariectomy than in naturally postmenopausal women, with the largest difference for free testosterone. All hormones were higher in obese than lean women, with the largest difference for free oestradiol, whereas SHBG was lower in obese women. Smokers of 15+ cigarettes per day had higher levels of all hormones than non-smokers, with the largest difference for testosterone. Drinkers of 20+ g alcohol per day had higher levels of all hormones, but lower SHBG, than non-drinkers, with the largest difference for DHEAS. Hormone concentrations were not strongly related to age at menarche, parity, age at first full-term pregnancy or family history of breast cancer.CONCLUSION: Sex hormone concentrations were strongly associated with several established or suspected risk factors for breast cancer, and may mediate the effects of these factors on breast cancer risk. British Journal of Cancer (2011) 105, 709-722. doi:10.1038/bjc.2011.254 www.bjcancer.com Published online 19 July 2011 (C) 2011 Cancer Research UK.

The preoperative setting is increasingly popular for the clinical investigation of hormonal agents and new biological drugs. The effectiveness of endocrine agents is well established for estrogen receptor-positive disease, and the emphasis in preoperative studies is on their combination with agents targeted at resistance mechanisms over 3 or more months. New agents are also being assessed for early evidence of clinical efficacy in shorter-term window-of-opportunity studies. The establishment of Ki67 as an intermediate marker of treatment benefit and of long-term outcome, with endocrine drugs, provides the opportunity for new trial designs with Ki67 as the primary endpoint. The PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial is randomizing (2:1) 4000 estrogen receptor-positive patients to 2 weeks presurgical treatment with a nonsteroidal aromatase inhibitor or no presurgical treatment. It provides a unique opportunity for detailed study of the determinants of response and resistance to estrogen deprivation as well as testing the role of presurgical therapy for improved biomarker-based estimates of prognosis..

Oestrogen receptor (ER) negative breast cancers are more likely to achieve a pathological complete response (pCR) to neoadjuvant chemotherapy compared to those with ER positive tumours. ER positive tumours exhibit low proliferation and ER negative cancers high proliferation. The aim of this study was to determine to what extent the better response of ER negative cancers correlates with proliferation rate. A retrospective analysis of a prospectively maintained database identified 175 neoadjuvant chemotherapy patients with tissue available for Ki67 analysis. On univariate analysis, pre-therapy Ki67 (P = 0.04), ER status (P = 0.002), HER2 status (P = 0.004) and grade (P = 0.0009) were associated with a pCR. In a multivariate model, HER2 was the only significant predictor of pCR. No significant relationship between pre-therapy Ki67 and relapse-free and overall survival was demonstrated. Ki67 is not an independent predictor of clinical CR or pCR. Aspects of ER status beyond its inverse relationship with proliferation may contribute to its predictive value for pCR..

PURPOSE To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)-positive breast cancers in postmenopausal women. Materials and METHODS Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = -0.179; P = .05; and r = -0.389; P = .0005, respectively). CONCLUSION Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer..

PURPOSE: Acquired endocrine resistance in estrogen receptor (ER)alpha+/human epidermal growth factor receptor 2-negative (HER2-) breast cancer has been associated with modest adaptive increases in HER2, although exactly how aberrant HER2 signaling affects the ERalpha pathway is poorly understood. We investigated (a) whether the epidermal growth factor receptor/HER2 inhibitor lapatinib could restore endocrine responsiveness in cell models of acquired endocrine resistance with modest increases in HER2, and (b) the nature of ERalpha-HER2 cross-talk in this process. METHODS: Combination growth studies, ERalpha transcription, immunoblot, and gene expression assays were conducted in two models of acquired resistance to (a) estrogen deprivation (long-term estrogen-deprived cells) and (b) tamoxifen (long-term tamoxifen-treated cells), and in hormone sensitive controls. Changes in ERalpha, PgR, and HER2 were assessed in samples from patients treated with tamoxifen. RESULTS: Both cell models of acquired endocrine resistance showed modest adaptive upregulation in HER2, and lapatinib restored endocrine sensitivity in both. The effect of lapatinib on ERalpha signaling varied markedly depending on the nature of the HER2/ERalpha cross-talk. In long-term estrogen-deprived cells characterized by enhanced ERalpha function, lapatinib suppressed ERalpha genomic activity (as measured by pERSer118, ERalpha transcriptional activity, and PGR gene expression). In contrast, in long-term tamoxifen-treated cells with reduced ERalpha activation, lapatinib reactivated ERalpha genomic function. Twenty percent of tamoxifen-resistant patients relapsed with modest increases in HER2 and either suppressed or enhanced ERalpha/PgR expression. CONCLUSIONS: Aberrant GFR signaling can augment or suppress ERalpha function. Regardless, interrupting the HER2/ERalpha cross-talk with lapatinib can restore endocrine sensitivity and should be investigated as a therapeutic strategy in combination with endocrine therapy in ERalpha+/HER2- patients with acquired endocrine resistance..

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PurposeTo develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor ( ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers.MethodsThe American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance.ResultsUp to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate ( false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria.RecommendationsThe Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal ( normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.This guideline was developed through a collaboration between American Society of Clinical Oncology and College of American Pathologists and has been published jointly by invitation and consent in both the Journal of Clinical Oncology and the Archives of Pathology & Laboratory Medicine. Copyright (C) 2010 American Society of Clinical Oncology and College of American Pathologists. All rights reserved. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without written permission by American Society of Clinical Oncology or College of American Pathologists..

Several adjuvant endocrine strategies exist for postmenopausal women with breast cancer. This study compared the effect of two sequences of aromatase inhibitor use [steroidal (exemestane) and non-steroidal (anastrozole)] on serological and pathological biomarkers when given in the neoadjuvant setting to postmenopausal women with breast cancer. Thirty women were assigned to receive exemestane 25 mg or anastrozole 1 mg each given for 8 weeks in a randomized sequence. The effect of this treatment on serum estrone sulfate and estradiol levels, as well as tumor changes in the proliferation biomarker Ki67 were evaluated at baseline, 8 weeks and 16 weeks. WHO clinical response criteria, patient preference, and quality of life were also assessed. Assessable data was available from 28 patients. There were no differences in concentration changes of serum estradiol or Ki67 between patients in the two arms. Overall clinical response rate was 68% (19/28 assessable patients) and clinical benefit was 93% (26/28 assessable patients). There was no significant difference in toxicity or quality of life scores. The majority of patients expressed a personal preference for anastrozole over exemestane. Results suggest that the order of steroidal and non-steroidal aromatase inhibitors has little effect on outcome. The majority of patients express clear preferences for drug treatments..

BACKGROUND: Microarray expression profiling classifies breast cancer into five molecular subtypes: luminal A, luminal B, basal-like, HER2, and normal breast-like. Three microarray-based single sample predictors (SSPs) have been used to define molecular classification of individual samples. We aimed to establish agreement between these SSPs for identification of breast cancer molecular subtypes. METHODS: Previously described microarray-based SSPs were applied to one in-house (n=53) and three publicly available (n=779) breast cancer datasets. Agreement was analysed between SSPs for the whole classification system and for the five molecular subtypes individually in each cohort. FINDINGS: Fair-to-substantial agreement between every pair of SSPs in each cohort was recorded (kappa=0.238-0.740). Of the five molecular subtypes, only basal-like cancers consistently showed almost-perfect agreement (kappa>0.812). The proportion of cases classified as basal-like in each cohort was consistent irrespective of the SSP used; however, the proportion of each remaining molecular subtype varied substantially. Assignment of individual cases to luminal A, luminal B, HER2, and normal breast-like subtypes was dependent on the SSP used. The significance of associations with outcome of each molecular subtype, other than basal-like and luminal A, varied depending on SSP used. However, different SSPs produced broadly similar survival curves. INTERPRETATION: Although every SSP identifies molecular subtypes with similar survival, they do not reliably assign the same patients to the same molecular subtypes. For molecular subtype classification to be incorporated into routine clinical practice and treatment decision making, stringent standardisation of methodologies and definitions for identification of breast cancer molecular subtypes is needed. FUNDING: Breakthrough Breast Cancer, Cancer Research UK..

AIMS: To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. METHODS: Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RESULTS: RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. CONCLUSIONS: FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies..

Purpose.-To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers.Methods.-The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance.Results.-Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria.Recommendations.-The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials. (Arch Pathol Lab Med. 2010;134:907-922).

Purpose.-To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers.Methods.-The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance.Results.-Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in pre-analytic variables, thresholds for positivity, and interpretation criteria.Recommendations.-The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials. (Arch Pathol Lab Med. 2010;134:e48-e72).

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AIM: To compare SP6 and MIB1 antibodies for Ki67 staining in breast cancer. BACKGROUND: Immunohistochemical detection of Ki67 has been widely used to assess the proliferative fraction in breast cancer. Ki67 is used prognostically and is the primary end-point for some presurgical trials. MIB1 has been the preferred antibody, but SP6 has become available, with apparently improved performance. The importance of Ki67 led us to systematically compare SP6 with MIB1. METHODS: Two sets of tissue microarrays were used. These were constructed from formalin-fixed paraffin-embedded breast cancers: (i) 177 cancers with data on response to an aromatase inhibitor for advanced disease (cohort 1); (ii) 200 mainly oestrogen-receptor-positive cancers without response data (cohort 2). Twenty-eight pairs of core-cut biopsies taken before and after aromatase inhibitor treatment were also assessed (cohort 3). Stained sections were examined either visually or by using an image analysis system (Ariol). RESULTS: There was a strong correlation between the two antibodies in all cohorts of samples scored visually (cohort 1: n=161, r=0.93, p<0.0001; cohort 2: n=194, r=0.84, p<0.0001; cohort 3: n=54, r=0.89, p<0.0001). Correlation between visual and Ariol scores was markedly better with the SP6 antibody (r=0.71 and r=0.88 for MIB1 and SP6, respectively). Ki67 related similarly with time-to-treatment failure with the two antibodies (cohort 1). Changes in Ki67 values with the two antibodies after 2 weeks of aromatase inhibitor treatment also correlated strongly. CONCLUSIONS: SP6 and MIB1 provide highly comparable measures of Ki67 that predict progression of advanced disease similarly. SP6 is substantially better suited than MIB1 to image analysis..

Cyclo-oxygenase 2 (COX-2) is implicated in the regulation of aromatase transcription in malignant breast tissue and has been considered as a potential target for tissue specific aromatase suppression. We initiated a randomised controlled pre-surgical study of celecoxib versus no treatment in women with primary breast cancer to determine the effects of COX-2 inhibition on markers of biological response. Postmenopausal women (50-80 years of age) with stage I or II, primary breast cancer, were randomised 2:1 to receive 400 mg/day celecoxib or no treatment for 14 days prior to surgery. A core biopsy was obtained pre- and post-treatment. Paired baseline and endpoint biopsies were analysed for Ki67, apoptosis, COX-2, CD31, estrogen receptor (ER) and progesterone receptor (PgR). Comparisons between the treatment groups were conducted using the Mann-Whitney test with a two-sided 5% significance. Of the 25 patients treated, 23 had evaluable data and 19 (83%) were ER positive. Overall the geometric mean change in Ki67, the primary end point, relative to baseline in the celecoxib arm was -16.6% (P = 0.056). The change in the no-treatment group was -8.1% (P = 0.24). There was no statistically significant difference in the change between the two groups. Celecoxib did not significantly affect apoptosis, COX-2, ER or PgR expression. There is only modest evidence for a biological effect of celecoxib in primary breast cancer. However, the trend towards a reduction in Ki67 in ER-positive breast cancer warrants further investigations in a larger cohort of patients..

To review the influence of sex hormones on the progression of breast, prostate, gynecologic, and colorectal cancer. The literature was reviewed in an informal manner utilizing the authors' prior knowledge to collate the current evidence for the involvement of sex hormones, particularly estrogens and androgens in the progression of a range of hormonally responsive cancers. In particular, the effect of treatment involving hormone withdrawal treatment was considered strong evidence for involvement. The impact of basal levels of endogenous steroids was considered. Data from clinical trials indicate the efficacy of therapeutic interventions that result in ablation or antagonism of host steroids for a range of cancers. Demonstration of the correlation of the completeness of withdrawal with clinical outcome together with direct evidence of progression from studies looking at the influence of tissue and circulating levels of sex hormones more recently in conjunction with gene expression profiles all provide compelling evidence for the involvement of steroids in the progression of disease. The involvement of steroids in the progression of cancer in hormone-sensitive tissues is well established and an important target for therapy..

To review the influence of sex hormones on the progression of breast, prostate, gynecologic, and colorectal cancer. The literature was reviewed in an informal manner utilizing the authors' prior knowledge to collate the current evidence for the involvement of sex hormones, particularly estrogens and androgens in the progression of a range of hormonally responsive cancers. In particular, the effect of treatment involving hormone withdrawal treatment was considered strong evidence for involvement. The impact of basal levels of endogenous steroids was considered. Data from clinical trials indicate the efficacy of therapeutic interventions that result in ablation or antagonism of host steroids for a range of cancers. Demonstration of the correlation of the completeness of withdrawal with clinical outcome together with direct evidence of progression from studies looking at the influence of tissue and circulating levels of sex hormones more recently in conjunction with gene expression profiles all provide compelling evidence for the involvement of steroids in the progression of disease. The involvement of steroids in the progression of cancer in hormone-sensitive tissues is well established and an important target for therapy..

PURPOSE To determine whether the Recurrence Score (RS) provided independent information on risk of distant recurrence (DR) in the tamoxifen and anastrozole arms of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) Trial. PATIENTS AND METHODS RNA was extracted from 1,372 tumor blocks from postmenopausal patients with hormone receptor-positive primary breast cancer in the monotherapy arms of ATAC. Twenty-one genes were assessed by quantitative reverse transcriptase polymerase chain reaction, and the RS was calculated. Cox proportional hazards models assessed the value of adding RS to a model with clinical variables (age, tumor size, grade, and treatment) in node-negative (N0) and node-positive (N+) women. RESULTS Reportable scores were available from 1,231 evaluable patients (N0, n = 872; N+, n = 306; and node status unknown, n = 53); 72, 74, and six DRs occurred in N0, N+, and node status unknown patients, respectively. For both N0 and N+ patients, RS was significantly associated with time to DR in multivariate analyses (P < .001 for N0 and P = .002 for N+). RS also showed significant prognostic value beyond that provided by Adjuvant! Online (P < .001). Nine-year DR rates in low (RS < 18), intermediate (RS = 18 to 30), and high RS (RS > or = 31) groups were 4%, 12%, and 25%, respectively, in N0 patients and 17%, 28%, and 49%, respectively, in N+ patients. The prognostic value of RS was similar in anastrozole- and tamoxifen-treated patients. CONCLUSION This study confirmed the performance of RS in postmenopausal HR+ patients treated with tamoxifen in a large contemporary population and demonstrated that RS is an independent predictor of DR in N0 and N+ hormone receptor-positive patients treated with anastrozole, adding value to estimates with standard clinicopathologic features..

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Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer..

Trials have shown superiority of aromatase inhibitors (AIs) over tamoxifen for post menopausal oestrogen receptor positive advanced breast cancer (ER + ABC) We previously reported the use of goserelin plus anastrozole (G + A) as second line endocrine therapy for pre menopausal ER + ABC We report clinical and endocrine data from G + A as first line systemic therapyThirty six patients (median age = 44 years) with metastatic (N = 28) and locally advanced disease were administered G + A for >= 6 months (unless progressed prior) Some (N = 13) received further therapy with goserelin plus another AI (steroidal), exemestane (G + E) Serial serum hormone assays (oestradiol dehydroepiandrosterone sulphate testosterone follicle stimulating hormone and luteinising hormone) were performedTwenty four patients (67%) derived clinical benefit (CB) (5% complete response 31% partial response 31% stable disease for 6 months) with median time to progression and duration of CB of 12 (2-47) and 24 + (7-78+) months respectively Ten patients were still receiving first line G + A at analysis Amongst 13 patients who went onto receive G + E 38% achieved CB with a mean duration of 13+(7-32) months Therapy was well tolerated with no withdrawalsThe combination of G + A resulted in 98% reduction (from pre treatment to 6 month) in median levels of oestradiol (from 574 5 pmol/L inter quartile range (IQR) = 209-1426 (N = 6) to 13 45 pmol/L IOQ = 5 5-31 5 (N = 4) whilst the levels of other hormones had minimal fluctuations during therapy The combinations of ovarian function suppression (using G) and AIs produce sus tamed CB and minimal side effects in pre menopausal ER + ABC with significant reduction in oestradiol levels Within the limitations of being a non randomised study they should be considered in appropriate patients with hormone sensitive ABC (C) 2010 Elsevier Ltd All rights reserved.

Breast cancer treatment has experienced several changes in the past decades due to the discovery of specific prognostic and predictive biomarkers that enable the application of more individualized therapies to different molecular subgroups. These subgroups show specific differences regarding biological clinical behavior. In addition to the classical clinical prognostic factors of breast cancer, established molecular biomarkers such as estrogen receptor and progesterone receptor have played a significant role in the selection of patients benefiting from endocrine therapy for many years. More recently, the human epidermal growth factor receptor 2 (HER2) has been validated to be not only a prognostic factor, but also a predictor of response to HER2 targeting therapy. The shift toward an earlier diagnosis of breast cancer due to improved imaging methods and screening programs highlights the need for new factors and combinations of biomarkers to quantify the residual risk of patients and to indicate the potential value of additional treatment strategies. The marker of proliferation Ki67 has recently emerged as an important marker due to several applications in neoadjuvant therapy in addition to its moderate prognostic value. With the introduction of high-throughput technologies, numerous multigene signatures have been identified that aim to outperform traditional markers: current prospective clinical trials are seeking evidence for their definitive role in breast cancer. There exist many more factors and approaches that have the potential to become relevant in the near future including the detection of single disseminating and circulating tumor cells in blood and bone marrow as well as of circulating cell-free DNA and microRNA. Careful randomized prospective testing and comparison with existing established factors will be required to select those emerging markers that offer substantial cost-effective benefit and thereby justify their routine use for breast cancer therapy decision-making..

Background The Arimidex, Tamoxifen Alone or in Combination (ATAC) trial was designed to compare the efficacy and safety of anastrozole (1 mg) with tamoxifen (20 mg), both given orally every day for 5 years as adjuvant treatment for postmenopausal women with early stage breast cancer In this analysis we assess the long term outcomes after a median follow up of 120 monthsMethods We used a proportional hazards model to assess the primary endpoint of disease free survival and the secondary endpoints of time to recurrence time to distant recurrence incidence of new contralateral breast cancer overall survival and death with or without recurrence in all randomised patients (anastrozole n=3125 tamoxifen n=3116) and hormone receptor positive patients (anastrozole n=2618 tamoxifen n=2598) After treatment completion we continued to collect data on fractures and serious adverse events in a masked fashion (safety population anastrozole n=3092, tamoxifen n=3094) This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN18233230Findings Patients were followed up for a median of 120 months (range 0-145) there were 24522 woman years of follow up in the anastrozole group and 23950 woman years in the tamoxifen group In the full study population, there were significant improvements in the anastrozole group compared with the tamoxifen group for disease free survival (hazard ratio [HR] 0 91 95% CI 0 83-0 99 p=0 04) time to recurrence (0 84 0 75-0 93 p=0 001) and time to distant recurrence (0 87 0 77-0 99 p=0 03) For hormone receptor positive patients the results were also significantly in favour of the anastrozole group for disease free survival (HR 0 86 95% CI 0 78-0 95 p=0 003), time to recurrence (0 79 0 70-0 89 p=0 0002) and time to distant recurrence (0 85 0 73-0 98, p=0 02) In hormone receptor positive patients absolute differences in time to recurrence between anastrozole and tamoxifen increased over time (2 7% at 5 years and 4 3% at 10 years) and recurrence rates remained significantly lower on anastrozole than tamoxifen after treatment completion (HR 0 81 95% CI 0 67-0 98 p=0 03) although the carryover benefit was smaller after 8 years There was weak evidence of fewer deaths after recurrence with anastrozole compared with tamoxifen treatment in the hormone receptor positive subgroup (HR 0 87 95% CI 0 74-1 02 p=0 09) but there was little difference in overall mortality (0 95,95% CI 0 84-1 06 p=0 4) Fractures were more frequent during active treatment in patients receiving anastrozole than those receiving tamoxifen (451 vs 351 OR 1 33 95% CI 1 15-1 55 p<0 0001) but were similar in the post treatment follow up period (110 vs 112 OR 0 98 95% CI 0 74-1 30 p=0 9) Treatment related serious adverse events were less common in the anastrozole group than the tamoxifen group (223 anastrozole vs 369 tamoxifen OR 0 57 95% CI 0 48-0 69 p<0 0001) but were similar after treatment completion (66 vs 78, OR 0 84,95% CI 0 60-1 19, p=0 3) No differences in non breast cancer causes of death were apparent and the incidence of other cancers was similar between groups (425 vs 431) and continue to be higher with anastrozole for colorectal (66 vs 44) and lung cancer (51 vs 34) and lower for endometrial cancer (six vs 24) melanoma (eight vs 19) and ovarian cancer (17 vs 28) No new safety concerns were reportedInterpretation These data confirm the long term superior efficacy and safety of anastrozole over tamoxifen as initial adjuvant therapy for postmenopausal women with hormone sensitive early breast cancer.

BACKGROUND: Cross-talk between receptor tyrosine kinases and the oestrogen receptor (ER) is implicated in resistance to endocrine therapy. We investigated whether AEE788 (a combined inhibitor of EGFR, HER2 and VEGFR) plus tamoxifen or letrozole enhanced the individual anti-tumour effects of these agents. METHODS: Breast cancer cell lines modelling endocrine-resistant and -sensitive disease were engineered to express aromatase (A) and examined using proliferation, western blotting and ER-alpha transcription assays. RESULTS: AEE788 enhanced the anti-proliferative effect of tamoxifen and letrozole in ER(+) cell lines (MCF-7 2A, ZR75.1 A3 and BT474 A3). This associated with an elevated G1 arrest and nuclear accumulation of p27. It is noteworthy that AEE788 alone or in combination with endocrine therapy increased the expression of progesterone receptor (PGR) and TFF1 in BT474 A3 cells. This may indicate a mechanism of resistance to AEE788 in ER(+)/HER2(+) breast cancers. In a ZR75.1 A3 xenograft, AEE788 alone or in combination with tamoxifen provided no further benefit compared with letrozole. However, letrozole plus AEE788 produced a significantly greater inhibition of tumour growth compared with letrozole alone. CONCLUSION: These data suggest that AEE788 plus letrozole in breast cancer overexpressing HER2 may provide superior anti-tumour activity, compared with single agents..

Purpose: Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor-positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone.Experimental Design: Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole.Results: In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17 beta-estradiol (E2) signaling in vivo.Conclusion: The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent. Clin Cancer Res; 16(16); 4178-87. (C) 2010 AACR..

PURPOSE: The concentration of estradiol (E(2)) in breast tumors is significantly higher than that in plasma, particularly in postmenopausal women. The contribution of local E(2) synthesis versus uptake of E(2) from the circulation is controversial. Our aim was to identify possible determinants of intratumoral E(2) levels in breast cancer patients. EXPERIMENTAL DESIGN: The expression of genes involved in estrogen synthesis, metabolism, and signaling was measured in 34 matched samples of breast tumor and normal breast tissue, and their correlation with estrogen concentrations assessed. RESULTS: ESR1 (9.1-fold; P < 0.001) and HSD17B7 (3.5-fold; P < 0.001) were upregulated in ER(+) tumors compared with normal tissues, whereas STS (0.34-fold; P < 0.001) and HSD17B5 (0.23-fold; P < 0.001) were downregulated. Intratumoral E(2) levels showed a strong positive correlation with ESR1 expression in all patients (Spearman r = 0.55, P < 0.001) and among the subgroups of postmenopausal (r = 0.76, P < 0.001; n = 23) and postmenopausal ER(+) patients (r = 0.59, P = 0.013; n = 17). HSD17B7 expression showed a significant positive correlation (r = 0.59, P < 0.001) whereas HSD17B2 (r = -0.46, P = 0.0057) and HSD17B12 (r = -0.45, P = 0.0076) showed significant negative correlations with intratumoral E(2) in all patients. Intratumoral E(2) revealed no correlation to CYP19, STS, and HSD17B1 expression. Multivariate models comprising ESR1 and plasma E(2) predicted between 50% and 70% of intratumoral E(2) variability. CONCLUSION: Uptake due to binding to the ER, rather than intratumoral estrogen synthesis by aromatase or sulfatase, is the single most important correlate and a probable determinant of intratumoral E(2). An increased expression of HSD17B7 may explain the increased ratio of E(2) to estrone (E(1)) in breast tumors compared with normal tissue..

PURPOSE: Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor-positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone. EXPERIMENTAL DESIGN: Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole. RESULTS: In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17beta-estradiol (E2) signaling in vivo. CONCLUSION: The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent..

PURPOSE: To assess the prevalence of defective homologous recombination (HR)-based DNA repair in sporadic primary breast cancers, examine the clincopathologic features that correlate with defective HR and the relationship with neoadjuvant chemotherapy response. EXPERIMENTAL DESIGN: We examined a cohort of 68 patients with sporadic primary breast cancer who received neoadjuvant anthracylcine-based chemotherapy, with core biopsies taken 24 hours after the first cycle of chemotherapy. We assessed RAD51 focus formation, a marker of HR competence, by immunofluorescence in postchemotherapy biopsies along with geminin as a marker of proliferative cells. We assessed the RAD51 score as the proportion of proliferative cells with RAD51 foci. RESULTS: A low RAD51 score was present in 26% of cases (15/57, 95% CI: 15%-40%). Low RAD51 score correlated with high histologic grade (P = 0.031) and high baseline Ki67 (P = 0.005). Low RAD51 score was more frequent in triple-negative breast cancers than in ER- and/or HER2-positive breast cancer (67% vs. 19% respectively; P = 0.0036). Low RAD51 score was strongly predictive of pathologic complete response (pathCR) to chemotherapy, with 33% low RAD51 score cancers achieving pathCR compared with 3% of other cancers (P = 0.011). CONCLUSIONS: Our results suggest that defective HR, as indicated by low RAD51 score, may be one of the factors that underlie sensitivity to anthracycline-based chemotherapy. Defective HR is frequent in triple-negative breast cancer, but it is also present in a subset of other subtypes, identifying breast cancers that may benefit from therapies that target defective HR such as PARP inhibitors..

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Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERalpha)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERalpha. In this study, we show that in ERalpha-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERalpha phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERalpha transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAM(R)-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERalpha-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERalpha-positive breast cancers and in particular in tamoxifen-resistant tumors..

PURPOSE: To conduct meta-analyses of randomized trials of aromatase inhibitors (AIs) compared with tamoxifen either as initial monotherapy (cohort 1) or after 2 to 3 years of tamoxifen (cohort 2). MATERIALS AND METHODS: Data submitted to the Early Breast Cancer Trialists' Collaborative Group were used in separate meta-analyses of two cohorts. Primary analyses involve postmenopausal women with tumors reported to be estrogen receptor positive. Log-rank P values are two-sided. RESULTS: Cohort 1 comprised 9,856 patients with a mean of 5.8 years of follow-up. At 5 years, AI therapy was associated with an absolute 2.9% (SE = 0.7%) decrease in recurrence (9.6% for AI v 12.6% for tamoxifen; 2P < .00001) and a nonsignificant absolute 1.1% (SE = 0.5%) decrease in breast cancer mortality (4.8% for AI v 5.9% for tamoxifen; 2P = .1). Cohort 2 comprised 9,015 patients with a mean of 3.9 years of follow-up. At 3 years from treatment divergence (ie, approximately 5 years after starting hormonal treatment), AI therapy was associated with an absolute 3.1% (SE = 0.6%) decrease in recurrence (5.0% for AI v 8.1% for tamoxifen since divergence; 2P < .00001) and an absolute 0.7% (SE = 0.3%) decrease in breast cancer mortality (1.7% for AI v 2.4% for tamoxifen since divergence; 2P = .02). There was no convincing heterogeneity in the proportional recurrence reduction with respect to age, nodal status, tumor grade, or progesterone receptor status and no indication of an increase in nonbreast deaths with AIs in either cohort. CONCLUSION AIs produce significantly lower recurrence rates compared with tamoxifen, either as initial monotherapy or after 2 to 3 years of tamoxifen. Additional follow-up will provide clearer information on long-term survival..

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PurposeThird-generation aromatase inhibitors have been widely used in postmenopausal women for the adjuvant treatment of hormone receptor-positive breast cancer. As aromatase inhibitors work by inhibiting the conversion of androgens to estrogens in adipose tissue, we hypothesized that anastrozole may be more effective in women with a high body mass index (BMI).Patients and MethodsThe Arimidex, Tamoxifen Alone or in Combination (ATAC) study was a double-blind randomized clinical trial in which postmenopausal women with early-stage breast cancer were randomly assigned to receive oral daily anastrozole (1 mg) alone, tamoxifen (20 mg) alone, or the combination in a double-blind fashion. Analyses were based on the 100-month median follow-up for women with hormone receptor-positive breast cancers (estrogen [ER] and/or progesterone [PgR] positive). Here, we investigate the impact of BMI on recurrence and the relative benefit of anastrozole versus tamoxifen according to baseline BMI.ResultsOverall, women with a high BMI (BMI > 35 kg/m(2)) at baseline had more recurrences than those women with a low BMI (BMI < 23 kg/m(2); adjusted hazard ratio [HR], 1.39; 95% CI, 1.06 to 1.82; P-heterogeneity = .03) and significantly more distant recurrences (adjusted HR, 1.46; 95% CI, 1.07 to 1.61; (Pheterogeneity) = .01). Overall, the relative benefit of anastrozole versus tamoxifen was nonsignificantly better in thin women compared to overweight women.ConclusionThese results confirm the poorer prognosis of obese women with early-stage breast cancer. Recurrence rates were lower for anastrozole than tamoxifen for all BMI quintiles. Our results suggest that the relative efficacy of anastrozole compared to tamoxifen is greater in thin postmenopausal women and higher doses or more complete inhibitors might be more effective in overweight women, but this requires independent confirmation..

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Introduction: Very few studies have investigated whether the time elapsed between surgical resection and tissue fixation or the difference between core-cut and excision biopsies impact on immunohistochemically measured biomarkers, including phosphorylated proteins in primary breast cancer. The aim of this study was to characterise the differences in immunoreactivity of common biomarkers that may occur (1) as a result of tissue handling at surgery and (2) between core-cuts and resected tumours.Methods: Core-cuts taken from surgical breast cancer specimens immediately after resection (sample A) and after routine X-ray of the excised tumour (sample B) were formalin-fixed and paraffin-embedded and compared with the routinely fixed resection specimen (sample C). The variation in immunohistochemical expression of Ki67, oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor 2 (HER2), p-Akt and p-Erk1/2 were investigated.Results: Twenty-one tissue sets with adequate tumour were available. Median time between collection of core-cuts A and B was 30 minutes (range, 20 to 80 minutes). None of the markers showed significant differences between samples A and B. Similarly, Ki67, ER, PgR and HER2 did not differ significantly between core-cuts and main resection specimen, although there was a trend for lower resection values for ER (P = 0.06). However, p-Akt and p-Erk1/2 were markedly lower in resections than core-cuts (median, 27 versus 101 and 69 versus 193, respectively; both P < 0.0001 [two-sided]). This difference was significantly greater in mastectomy than in lumpectomy specimens for p-Erk1/2 (P = 0.01).Conclusions: The delay in fixation in core-cuts taken after postoperative X-ray of resection specimens has no significant impact on expression of Ki67, ER, PgR, HER2, p-Akt or p-Erk1/2. However, extreme loss of phosphostaining can occur during routine fixation of resection specimens. These differences are likely attributable to suboptimal fixation and may have major repercussions for clinical research involving these markers..

Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed..

The human epidermal receptor-2 (HER-2) is overexpressed or amplified in 15% to 25% of breast cancers. Determination of HER-2 tumor status offers clinically useful information, as it selects patients who may benefit from treatment with trastuzumab, the monoclonal antibody against HER-2. Currently approved methods for HER-2 testing include immunohistochemistry or fluorescent in situ hybridization using tumor tissue. A fragment of HER-2 composed of its extracellular domain (ECD) can also be detected in the serum of some patients with breast cancer. As an easily accessible tumor marker, it could offer additional useful prognostic or predictive information. This review will briefly address the biology of the circulating HER-2 ECD and discuss the evidence to support the role, if any, for measuring HER-2 ECD levels in women with breast cancer. In particular, we focus on the value and limitations of serum ECD in both early and advanced breast cancer in the following clinical contexts: as a marker of HER-2 tumor tissue status; clinical implications of raised levels in women who have a tumor not overexpressing HER-2; as a prognostic indicator and as a predictor of response to treatment; and as a monitoring tool for early recurrence. On the basis of our review of the literature, we conclude that there is currently insufficient evidence to support the use of serum HER-2 ECD in the routine management of individual patients with breast cancer. This conclusion is in agreement with the 2007 American Society of Clinical Oncology guidelines on the use of biomarkers in breast cancer..

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Introduction Changes in proliferation as measured by Ki67 occur within 14 days of starting treatment with an aromatase inhibitor and these changes have been shown to be predictors of long term outcome. This study aimed to compare changes in proliferation following 14 days of treatment with anastrozole and letrozole. Methods Two hundred and six women with 209 estrogen receptor (ER) positive operable breast cancers (three bilateral) were randomly allocated to receive either 14 days treatment with 2.5 mg of letrozole or 1 mg of anastrozole prior to surgery. Changes in expression of estrogen (ER) and progesterone receptors (PgR) as assessed by ALLRED scores and proliferation as assessed by Ki67 were analysed. The HER2 status of each tumour was also assessed using a combination of the Hercept test and FISH. Results Both letrozole and anastrozole reduced ER expression (ALLRED score) by a mean of 0.32 (0.20-0.44), P < 0.001 and PgR fell by a mean of 2.54 (2.20-2.89) P < 0.0001. Letrozole reduced proliferation from a geometric mean of 6.37% to 0.81%, P < 0.0001 and anastrozole reduced proliferation from 5.81% to 0.77%, P < 0.0001. There was no differences between drugs in the fall in ER, PgR or proliferation. Both letrozole and anastrozole produced significant falls in proliferation in both HER2 positive and HER2 negative cancers, all P < 0.001. Discussion 14 days of both letrozole and anastrozole reduces proliferation, ER and PgR expression. No significant difference between these two drugs was identified..

Gene expression analysis of formalin-fixed paraffin-embedded (FFPE) material by quantitative real-time polymerase chain reaction is a Valuable tool for the retrospective analysis of clinical samples. The degraded nature of RNA from FFPE tissue can lead to variation in detection of gene expression between samples, hence, it is necessary to select reference genes that can reflect this variation. To identify suitable references for the normalization of target genes in FFPE breast tumors, expression of 10 potential references were Measured in 10 endocrine therapy-naive, primary invasive breast tumors. The most stably expressed reference genes were identified with the use of the freely available algorithm geNorm. Using the geometric mean of MRPL19, TBP, and TFRC as a reference factor for FFPE tissue, there was a good correlation of ERD alpha and HER2 measurements between FFPE and matched frozen tumors, indicating that this combination of reference genes successfully removed the effect of RNA degradation on quantitative real-time polymerase chain reaction analysis..

PURPOSE: To compare the prognostic significance of proliferation, as assessed by Ki67 expression, in breast cancer before and after neoadjuvant chemotherapy. METHODS: A retrospective search of a prospectively maintained clinical database was performed to identify patients treated with neoadjuvant chemotherapy at the Royal Marsden Hospital. The expression of Ki67 was assessed using immunohistochemistry in pre-therapy core-needle biopsy and post-therapy surgical excision specimens. The following factors were considered pre- and post-chemotherapy for their relationship with relapse-free and overall survival: age, menstrual status, T and N stage, pre-therapy operability, Ki67, ER, PgR, HER2, grade, histological subtype, vascular invasion, clinical response, chemotherapy regimen, type of surgery performed, adjuvant therapy, pathological tumour size and nodal involvement. RESULTS: In a matched cohort of 103 patients, on multivariate analysis of relapse-free survival, post-therapy Ki67 was the only significant independent prognostic factor. On multivariate analysis for overall survival, both pre- and excision Ki67 were significant independent predictors but the latter showed a stronger prognostic impact. The highest and lowest tertiles of excision Ki67 had different prognosis for both 5-year relapse-free (27% vs. 77%) and overall (39% and 93%) survival. In a cohort of 284 patients with only excision samples, post-therapy Ki67 was a significant independent prognostic factor on multivariate analysis. CONCLUSION: Post-chemotherapy Ki67 is a strong predictor of outcome for patients not achieving a pathological complete response..

Sex steroids have an important role in bone health, however previous studies on fracture risk have been carried out in older populations. The EPIC-Oxford study is a prospective cohort of men and women living in the UK. Five years after recruitment, participants self-reported previous fractures. Sex steroid concentrations (plasma estradiol, testosterone and sex hormone binding globulin) were measured in 436 cases (155 men, 46 premenopausal women and 235 postmenopausal women) with an incident fracture and 868 matched controls. Fracture risk was inversely related to concentrations of estradiol among men (RR for a doubling of estradiol 0.35, 95% CI 0.44-0.96) but there was no association between fracture risk and testosterone levels. There were no clear associations between fracture risk and hormone levels among postmenopausal women, however there was suggestion of an inverse association for both estradiol and testosterone as the RR in the highest compared with the lowest tertile for estradiol was 0.74 (95% CI 0.46, 1.18) and testosterone was 0.75 (95% CI 0.49, 1.16). Among premenopausal women fracture risk was inversely associated with levels of testosterone (RR for doubling of testosterone 0.46, 95% CI 0.26-0.81), with no association between estradiol and fracture risk. SHBG was not associated with risk of fracture among either men or women. In summary, this study finds evidence of an inverse association between endogenous estradiol and risk of fracture in men, and between endogenous testosterone and risk of fracture in premenopausal women but no clear associations among postmenopausal women..

PURPOSE: It has been postulated that castration-resistant prostate cancer (CRPC) commonly remains hormone dependent. Abiraterone acetate is a potent, selective, and orally available inhibitor of CYP17, the key enzyme in androgen and estrogen biosynthesis. PATIENTS AND METHODS: This was a phase I/II study of abiraterone acetate in castrate, chemotherapy-naive CRPC patients (n = 54) with phase II expansion at 1,000 mg (n = 42) using a two-stage design to reject the null hypothesis if more than seven patients had a prostate-specific antigen (PSA) decline of > or = 50% (null hypothesis = 0.1; alternative hypothesis = 0.3; alpha = .05; beta = .14). Computed tomography scans every 12 weeks and circulating tumor cell (CTC) enumeration were performed. Prospective reversal of resistance at progression by adding dexamethasone 0.5 mg/d to suppress adrenocorticotropic hormone and upstream steroids was pursued. RESULTS: A decline in PSA of > or = 50% was observed in 28 (67%) of 42 phase II patients, and declines of > or = 90% were observed in eight (19%) of 42 patients. Independent radiologic evaluation reported partial responses (Response Evaluation Criteria in Solid Tumors) in nine (37.5%) of 24 phase II patients with measurable disease. Decreases in CTC counts were also documented. The median time to PSA progression (TTPP) on abiraterone acetate alone for all phase II patients was 225 days (95% CI, 162 to 287 days). Exploratory analyses were performed on all 54 phase I/II patients; the addition of dexamethasone at disease progression reversed resistance in 33% of patients regardless of prior treatment with dexamethasone, and pretreatment serum androgen and estradiol levels were associated with a probability of > or = 50% PSA decline and TTPP on abiraterone acetate and dexamethasone. CONCLUSION: CYP17 blockade by abiraterone acetate results in declines in PSA and CTC counts and radiologic responses, confirming that CRPC commonly remains hormone driven..

Patients with human epidermal growth factor receptor-2 (HER2)+ breast cancer are eligible for trastuzumab treatment: therefore, accurate assessment of HER2 status is essential. Until recently, only 2 methods were validated for determining the HER2 status of breast tumors in the routine diagnostic selling: immunohistochemical analysis and fluorescence in situ hybridization (FISH).Recently bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) and silver-enhanced in situ hybridization (SISH), which combine features of immunohistochemical analysis and FISH, have been introduced for the determination of HER2 status. These new techniques use a peroxidase enzyme-labeled probe with chromogenic defection, instead of fluorescent-labeled probe, allowing results to be visualized by standard bright-field microscopy. Thus, the histologic features and HER2 status of a specimen can be evaluated in parallel. Moreover, signals do not decay over time. This review discusses recent publications regarding CISH and SISH testing, including results scoring and concordance between FISH and immunohistochemical analysis..

BACKGROUND: Analysis of estrogen receptor (ER), progesterone receptor (PgR) and HER2 status in early breast cancer (EBC) is increasingly being conducted in core needle biopsies (CNBs) taken at diagnosis but the concordance with the excisional biopsy (EB) is poorly documented. PATIENTS AND METHODS: Patients with EBC presenting to The Royal Marsden Hospital from June 2005 to September 2007 who had CNB and subsequent EB were included. ER and PgR were determined by immunohistochemistry (IHC) and graded from 0 to 8 (Allred score). HER2 was determined by IHC and scored from 0 to 3+. FISH analysis was carried out in HER2 2+ cases and in discordant cases. RESULTS: In all, 336 pairs of samples were compared. ER was positive in 253 CNBs (75%) for 255 EBs (76%) and was discordant in six patients (1.8%). PgR was positive in 221 CNBs (66%) and 227 (67.6%) EBs being discordant in 52 cases (15%). HER2 was positive in 41 (12.4%) of the 331 CNBs in which it was determined compared with 44 (13.3%) EBs and discordant in four cases (1.2%). CONCLUSIONS: CNB can be used with confidence for ER and HER2 determination. For PgR, due to a substantial discordance between CNB and EB, results from CNB should be used with caution..

Background: Serum concentrations of some hormones are risk factors for certain cancers, but little is known about their familial associations especially for females.Methods: We measured serum concentrations of estradiol (E-2), testosterone (T), SHBG, prolactin, and IGF-I for 645 Australian female postmenopausal twins and their sisters [182 monozygotic (MZ) and 107 dizygotic (DZ) pairs and 67 nontwin sisters] using well-established immunoassays. After suitable transformation and adjusting for age, body mass index (BMI), and time since menopause, familial correlations and proportions of variance attributed to genetic (h(2)) and nongenetic factors common to sisterships (c(2)) were estimated under the classic twin multivariate normal model using FISHER.Results: For all serum concentrations except prolactin, MZ, DZ, and sister pairs were correlated (P = 0.001). MZ correlations were in the range 0.5-0.7, and for all serum concentrations, there were no differences between DZ and sister correlations. MZ correlations were greater than DZ and sister correlations for log SHBG (P = 0.0001), IGF-I (P = 0.0002), and square-root T (P = 0.007) but not log E-2 (P = 0.3), and the respective h(2) estimates were 0.56 (SE = 0.14), 0.53 (0.17), 0.39 (0.14), and 0.14 (0.16). For log E-2 and square-root T, c(2) estimates were 0.39 (0.14) and 0.22 (0.12).Conclusion: There are strong familial correlations in postmenopausal SHBG, IGF-I, and to a lesser extent T, which are consistent with a genetic etiology. For E-2, and to a lesser extent T, correlations are consistent with substantial nongenetic familial factors. The latter might include maternal effects. (J Clin Endocrinol Metab 94: 4793-4800, 2009).

Mammographic density is strongly associated with breast cancer risk, and endogenous hormones, which are risk factors for breast cancer, may be involved in the mechanism. This cross-sectional study of 494 premenopausal women is the first to account for cyclic variations in estrogen levels, by measuring urinary estrone glucuronide (E1G) in the periovulatory and luteal phases of the menstrual cycle, and to assess the role of androgens. Computer-assisted density readings were obtained from digitized mammograms. Mean ovulatory E1G level and daily E1G load were both positively associated with percent density before adjustment for body mass index (BMI), with women in the top fourth having 10.2% (95% CI: 2.9%, 18.1%) and 8.9% (1.7%, 16.7%), respectively, higher density than those in the bottom fourth (Ptrend before/after BMI adjustment=0.006/0.11 and 0.01/0.13, respectively). Neither the peak nor luteal E1G levels were predictive of density after adjustment for E1G levels at other points in the cycle. The plasma androgens testosterone, androstenedione, and dehydroepiandrosterone sulfate were negatively associated with density. In mutually adjusted analyses, density was positively associated with insulin-like growth factor (IGF)-I and negatively with IGF-II (Ptrend=0.006 for both) but not with IGF binding protein-3. There was also weak evidence of a positive association of prolactin with density. The study supports the hypothesis that endogenous hormones affect density in premenopausal women; in particular, it shows a positive association between estrogen levels and density and suggests that the mean level throughout the cycle is the most biologically relevant measure. Most of these hormone-density associations were attenuated with further adjustment for BMI..

BACKGROUND: Incorporation of a taxane as adjuvant treatment for early breast cancer offers potential for further improvement of anthracycline-based treatment. The UK TACT study (CRUK01/001) investigated whether sequential docetaxel after anthracycline chemotherapy would improve patient outcome compared with standard chemotherapy of similar duration. METHODS: In this multicentre, open-label, phase III, randomised controlled trial, 4162 women (aged >18 years) with node-positive or high-risk node-negative operable early breast cancer were randomly assigned by computer-generated permuted block randomisation to receive FEC (fluorouracil 600 mg/m(2), epirubicin 60 mg/m(2), cyclophosphamide 600 mg/m(2) at 3-weekly intervals) for four cycles followed by docetaxel (100 mg/m(2) at 3-weekly intervals) for four cycles (n=2073) or control (n=2089). For the control regimen, centres chose either FEC for eight cycles (n=1265) or epirubicin (100 mg/m(2) at 3-weekly intervals) for four cycles followed by CMF (cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), and fluorouracil 600 mg/m(2) at 4-weekly intervals) for four cycles (n=824). The primary endpoint was disease-free survival. Analysis was by intention to treat (ITT). This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN79718493. FINDINGS: All randomised patients were included in the ITT population. With a median follow-up of 62 months, disease-free survival events were seen in 517 of 2073 patients in the experimental group compared with 539 of 2089 controls (hazard ratio [HR] 0.95, 95% CI 0.85-1.08; p=0.44). 75.6% (95% CI 73.7-77.5) of patients in the experimental group and 74.3% (72.3-76.2) of controls were alive and disease-free at 5 years. The proportion of patients who reported any acute grade 3 or 4 adverse event was significantly greater in the experimental group than in the control group (p<0.0001); the most frequent events were neutropenia (937 events vs 797 events), leucopenia (507 vs 362), and lethargy (456 vs 272). INTERPRETATION: This study did not show any overall gain from the addition of docetaxel to standard anthracycline chemotherapy. Exploration of predictive biomarker-defined subgroups might have the potential to better target the use of taxane-based therapy. FUNDING: Cancer Research UK (CRUK 01/001), Sanofi-Aventis, Pfizer, and Roche..

PURPOSE: To determine whether (1) immunohistochemical (IHC) HER2 status (ie, 2+ or 3+), (2) degree of fluorescence in situ hybridization (FISH) amplification according to (2a) HER2/CEP17 ratio or (2b) HER2 gene copy number, or (3) polysomy significantly influenced clinical outcome for patients with human epidermal growth factor receptor 2 (HER2) -positive breast cancer enrolled in the Herceptin Adjuvant trial of trastuzumab versus no trastuzumab administered after completion of chemotherapy. PATIENTS AND METHODS: IHC and/or FISH analyses were performed locally and required central confirmation as indicating HER2 positivity for trial entry. FISH data from the central HER2 analysis on patients in the 1-year trastuzumab and no trastuzumab arms were assessed in relation to disease-free survival (DFS) after a median 2 years of follow-up. RESULTS: Central FISH results were available for 2,071 (61%) of the 3,401 patients randomized to the 2 arms. Among patients with FISH-positive disease, (1) the hazard ratios for trastuzumab versus no trastuzumab were 0.56 (95% CI, 0.32 to 0.99) for locally IHC2+ cases (n = 340) and 0.80 (95% CI, 0.40 to 1.61) for centrally IHC2+ cases (n = 299). There was no significant prognostic relationship between (2a) HER2 FISH ratio, (2b) HER2 copy number, or (3) polysomy and DFS in the control arm or predictive relationship defining differential benefit from trastuzumab. CONCLUSION: There was no evidence for reduced benefit of trastuzumab in HER2 IHC2+FISH+ cases. The degree of HER2 amplification does not influence prognosis or benefit from adjuvant trastuzumab in patients treated with prior adjuvant chemotherapy..

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Introduction Sex steroids, insulin-like growth factors (IGFs) and prolactin are breast cancer risk factors but whether their effects are mediated through mammographic density, one of the strongest risk factors for breast cancer, is unknown. If such a hormonal basis of mammographic density exists, hormones may underlie ethnic differences in both mammographic density and breast cancer incidence rates.Methods In a cross-sectional study of 270 postmenopausal Caucasian and Afro-Caribbean women attending a population-based breast screening service in London, UK, we investigated whether plasma biomarkers ( oestradiol, oestrone, sex hormone binding globulin ( SHBG), testosterone, prolactin, leptin, IGF-I, IGF-II and IGF binding protein 3 (IGFBP3)) were related to and explained ethnic differences in mammographic percent density, dense area and nondense area, measured in Cumulus using the threshold method.Results Mean levels of oestrogens, leptin and IGF-I: IGFBP3 were higher whereas SHBG and IGF-II: IGFBP3 were lower in Afro-Caribbean women compared with Caucasian women after adjustment for higher mean body mass index (BMI) in the former group (by 3.2 kg/m(2) (95% confidence interval (CI): 1.8, 4.5)). Age-adjusted percent density was lower in Afro-Caribbean compared with Caucasian women by 5.4% (absolute difference), but was attenuated to 2.5% (95% CI: -0.2, 5.1) upon BMI adjustment. Despite ethnic differences in biomarkers and in percent density, strong ethnic-age-adjusted inverse associations of oestradiol, leptin and testosterone with percent density were completely attenuated upon adjustment for BMI. There were no associations of IGF-I, IGF-II or IGFBP3 with percent density or dense area. We found weak evidence that a twofold increase in prolactin and oestrone levels were associated, respectively, with an increase (by 1.7% (95% CI: 0.3, 3.7)) and a decrease (by 2.0% (95% CI: 0, 4.1)) in density after adjustment for BMI.Conclusions These findings suggest that sex hormone and IGF levels are not associated with BMI-adjusted percent mammographic density in cross-sectional analyses of postmenopausal women and thus do not explain ethnic differences in density. Mammographic density may still, however, be influenced by much higher premenopausal hormone levels..

PURPOSE: To compare the effects of anastrozole and letrozole on plasma estradiol (E2) and estrone sulfate (E1S) levels. PATIENTS AND METHODS: Fifty-four postmenopausal women with estrogen receptor-positive breast cancer receiving aromatase inhibitors (AIs) as part of their adjuvant therapy were randomly assigned to receive either 3 months of anastrozole (1 mg) followed by 3 months of letrozole (2.5 mg), both given orally once daily, or 3 months of the opposite sequence. Blood was taken at the same time and the same day of the week from each patient, before and after 3 months of each drug, and plasma levels of E2 and E1S were determined using highly sensitive radioimmunoassays. RESULTS: There were 27 patients in each group. The mean age of the patients was 63 years (range, 49 to 83 years). Baseline E2 levels ranged from 3 pmol/L to 91 pmol/L with a mean of 25.7 pmol/L. Only one of 54 (2%) patients had an E2 value >or= 3 pmol/L after receiving letrozole, versus 20 of 54 (37%) patients after receiving anastrozole (P < .001). Extrapolation revealed a mean E2 level after anastrozole treatment of 2.71 pmol/L (range, 2.38 to 3.08 pmol/L). Following letrozole, it was 1.56 pmol/L (range, 1.37 to 1.78 pmol/L). Mean residual E2 was 10.1% for anastrozole and 5.9% for letrozole. Residual E1S levels were 4.6% for anastrozole and 2.0% for letrozole (P = .001). CONCLUSION: Letrozole reduces plasma E2 and E1S levels to a significantly greater extent than anastrozole in postmenopausal women taking AIs as part of their adjuvant therapy for hormone receptor-positive breast cancer..

PURPOSE: Vascular endothelial growth factor (VEGF) is a key angiogenic factor mediating neovascularization. Soluble VEGF receptor 1 (sVEGFR-1) is an intrinsic negative counterpart of VEGF signaling and the ratio of sVEGFR-1 to VEGF has been shown to be a prognostic factor. Estrogen-bound estrogen receptor enhances VEGF expression, providing a common link between these signaling pathways that may be targeted by endocrine therapy. We investigated the effects of anastrozole and tamoxifen over time on serum VEGF and sVEGFR-1. EXPERIMENTAL DESIGN: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) trial compared the preoperative use of anastrozole with tamoxifen in postmenopausal women with estrogen receptor-positive primary operable breast cancer over 12 weeks. Circulating VEGF and sVEGFR-1 were measured by ELISA in 106 patients treated with anastrozole or tamoxifen alone at baseline and after 2 and 12 weeks of treatment. RESULTS: The increase in serum VEGF from baseline to 12 weeks was significantly different between anastrozole and tamoxifen (anastrozole versus tamoxifen, 6% versus 38%; P = 0.047). There was a significant increase in sVEGFR-1 levels after 12 weeks of anastrozole (P = 0.037). The sVEGFR-1/VEGF ratio significantly decreased in the tamoxifen arm (P = 0.013) and the change in sVEGFR-1/VEGF ratio from baseline to 12 weeks was significantly different between anastrozole and tamoxifen (anastrozole versus tamoxifen, 24% increase versus 34% decrease; P = 0.013). CONCLUSIONS: Treatment with anastrozole and tamoxifen resulted in differential effects on serum angiogenic markers. This may be related to the relative effectiveness of the treatments. These data provide further support for cross talk between estrogen receptor and VEGF..

PURPOSE: Studies indicate that castration-resistant prostate cancer (CRPC) remains driven by ligand-dependent androgen receptor (AR) signaling. To evaluate this, a trial of abiraterone acetate-a potent, selective, small-molecule inhibitor of cytochrome P (CYP) 17, a key enzyme in androgen synthesis-was pursued. PATIENTS AND METHODS: Chemotherapy-naïve men (n = 21) who had prostate cancer that was resistant to multiple hormonal therapies were treated in this phase I study of once-daily, continuous abiraterone acetate, which escalated through five doses (250 to 2,000 mg) in three-patient cohorts. RESULTS: Abiraterone acetate was well tolerated. The anticipated toxicities attributable to a syndrome of secondary mineralocorticoid excess-namely hypertension, hypokalemia, and lower-limb edema-were successfully managed with a mineralocorticoid receptor antagonist. Antitumor activity was observed at all doses; however, because of a plateau in pharmacodynamic effect, 1,000 mg was selected for cohort expansion (n = 9). Abiraterone acetate administration was associated with increased levels of adrenocorticotropic hormone and steroids upstream of CYP17 and with suppression of serum testosterone, downstream androgenic steroids, and estradiol in all patients. Declines in prostate-specific antigen >or= 30%, 50%, and 90% were observed in 14 (66%), 12 (57%), and 6 (29%) patients, respectively, and lasted between 69 to >or= 578 days. Radiologic regression, normalization of lactate dehydrogenase, and improved symptoms with a reduction in analgesic use were documented. CONCLUSION: CYP17 blockade by abiraterone acetate is safe and has significant antitumor activity in CRPC. These data confirm that CRPC commonly remains dependent on ligand-activated AR signaling..

BACKGROUND: Understanding how tumor response is related to relapse risk would help clinicians make decisions about additional treatment options for patients who have received neoadjuvant endocrine treatment for estrogen receptor-positive (ER+) breast cancer. METHODS: Tumors from 228 postmenopausal women with confirmed ER+ stage 2 and 3 breast cancers in the P024 neoadjuvant endocrine therapy trial, which compared letrozole and tamoxifen for 4 months before surgery, were analyzed for posttreatment ER status, Ki67 proliferation index, histological grade, pathological tumor size, node status, and treatment response. Cox proportional hazards were used to identify factors associated with relapse-free survival (RFS) and breast cancer-specific survival (BCSS) in 158 women. A preoperative endocrine prognostic index (PEPI) for RFS was developed from these data and validated in an independent study of 203 postmenopausal women in the IMPACT trial, which compared treatment with anastrozole, tamoxifen, or the combination 3 months before surgery. Statistical tests were two-sided. RESULTS: Median follow-up in P024 was 61.2 months. Patients with confirmed baseline ER+ clinical stage 2 and 3 tumors that were downstaged to stage 1 or 0 at surgery had 100% RFS (compared with higher stages, P < .001). Multivariable testing of posttreatment tumor characteristics revealed that pathological tumor size, node status, Ki67 level, and ER status were independently associated with both RFS and BCSS. The PEPI model based on these factors predicted RFS in the IMPACT trial (P = .002). CONCLUSIONS: Breast cancer patients with pathological stage 1 or 0 disease after neoadjuvant endocrine therapy and a low-risk biomarker profile in the surgical specimen (PEPI score 0) have an extremely low risk of relapse and are therefore unlikely to benefit from adjuvant chemotherapy..

PURPOSE: To determine the relationship between quantitative estrogen-receptor (ER) and progesterone-receptor (PgR) expression and human epidermal growth factor 2 (HER-2) status with time to recurrence (TTR) in postmenopausal women with hormone receptor-positive primary breast cancer treated with anastrozole or tamoxifen as adjuvant therapy. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tumor blocks were retrospectively collected from patients in the monotherapy arms of the Arimidex, Tamoxifen Alone or in Combination (ATAC) trial and centrally tested for ER, PgR and HER-2. ER and PgR were scored using continuous scales and HER-2 was scored as 0 to 3+ with 2+ cases being analyzed by fluorescence in situ hybridization. RESULTS: Blocks were collected from 2,006 of 5,880 eligible patients. Tissue was assessable and ER and/or PgR positivity confirmed centrally in 1,782 cases. In these, TTR was longer for anastrozole than for tamoxifen by a similar extent to that in the overall trial. None of the three biomarkers identified a set of patients with differential benefit from anastrozole over tamoxifen. Patients with low ER, low PgR, and high HER-2 expression had a poorer prognosis with either drug. Only 2.6% of patients in the highest quartile of PgR experienced recurrence after 5 years, compared with 13.2% in the lowest quartile. CONCLUSION: Quantitative expression of ER and PgR and HER-2 status did not identify patients with differential relative benefit from anastrozole over tamoxifen: TTR was longer for anastrozole than for tamoxifen in all molecular subgroups. Low ER or PgR or high HER-2 expression are associated with a high risk of recurrence with either anastrozole or tamoxifen..

Purpose: Chemotherapy can be an integral component of the adjuvant management strategy for women with early stage breast cancer. To date, no tool is available to predict or monitor the efficacy of these therapies. The aim of this proof-of-principle study was to assess whether NEUROD1 DNA methylation is able to predict the response to neoadjuvant and adjuvant chemotherapy.Experimental Design: Recently, we showed that NEUROD1 DNA is differentially methylated in neoplastic versus nonneoplastic breast tissue samples. In this study, we used MethyLight and analyzed NEUROD1 methylation in (a) 74 breast cancer tissue samples, (b) two independent sets of pretreatment core biopsies of 23 (training set) and 21 (test set) necadjuvantly treated breast cancer patients, and (c) pretherapeutic and posttherapeutic serum samples from 107 breast cancer patients treated with adjuvant chemotherapy.Results: High-grade tumors showed higher NEUROD1 methylation levels. Estrogen receptor negative breast cancers with high NEUROD1 methylation were 10.8-fold more likely to respond with a complete pathologic response following neoadjuvant chemotherapy. Patients with positive serum pretreatment NEUROD1 methylation, which persisted after chemotherapy, indicated poor relapse-free and overall survival in univariate and multivariate analyses (relative risk for relapse, 6.2; 95% confidence interval, 1.6-24; P = 0.008, and relative risk for death, 14; 95% confidence interval, 1.6-120; P = 0.02).Conclusions: These data support the view that NEUROD1 methylation is a chemosensitivity marker in estrogen receptor-negative breast cancer..

Objective: Exogenous sex hormone use is associated with many health effects. Current exogenous hormone use influences endogenous sex hormone levels, but little is known about longer term effects on endogenous hormones after cessation of use. The aim of this study was to examine the relationship between past hormone use and current endogenous hormone status.Design: This was a cross-sectional study of 1,983 postmenopausal women aged 55 to 81 years from the general community. The women were not currently using exogenous hormones. Past use of oral contraceptives (OCs) and hormone therapy (HT) as well as circulating endogenous sex hormones and sex hormone-binding globulin concentrations were evaluated.Results: Past OC users had significantly lower endogenous estradiol, estrone, androstenedione, testosterone, and sex hormone-binding globulin concentrations compared with never users independent of age, body mass index, smoking, physical activity, and reproductive factors. Past HT users had significantly lower testosterone and 17 alpha-hydroxyprogesterone concentrations. Past OC use and HT use were both independently associated with lower testosterone concentrations: - 9% (95% CI: - 16% to - 2%) for ever OC use compared with never OC use and -7% (95% CI: -17% to -2%) for ever HT use compared with never HT use. The magnitude of 5% to 10% differences in endogenous hormone concentrations was similar or greater for past OC use compared with past HT use, although OC use occurred earlier in the past.Conclusions: Past OC use and HT use seem to be related to long-term differences in endogenous sex hormones and sex hormone-binding globulin concentrations in postmenopausal women many years after cessation of use. These findings have implications for understanding the longer term effects of exogenous hormone exposures earlier in life with health and disease risk in later life..

Aromatase inhibitors play a key role in the clinical management of hormone receptor-positive breast cancer and have potential utility as chemopreventive agents. Further understanding of the molecular effects of estrogen and its deprivation in normal breast tissue may allow the development of biomarkers of risk of breast cancer and help to predict the value of chemoprevention with aromatase inhibitors. Core biopsies of normal breast tissue were taken before and after letrozole treatment from postmenopausal women in the LITMaS pilot prevention study. RNA was extracted from these samples and used for cDNA microarray analysis. Gene expression changes induced by letrozole treatment were much less extensive than observed in estrogen receptor-positive malignant tissue; however, overall, they correlated to a highly significant degree (rho = 0.511; P < 10(-20)). As well as some classically estrogen-associated genes, many genes associated with extracellular matrix remodeling were affected by estrogen deprivation in the normal breast in vivo. These data indicate for the first time that gene expression of normal breast tissue remains dependent on endogenous estrogens after the menopause. The modest degree of gene change suggests that intermediate markers of chemoprevention may be difficult to identify..

We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/z features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry..

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HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets..

INTRODUCTION: Changes in breast cancer cell biology following hormonal treatment have been claimed as promising predictor markers of clinical benefit even outperforming clinical response. From previous work we selected 10 genes showing both a well known regulation by oestrogen and a high level of early transcriptional regulation following therapy with aromatase inhibitors. Here we use an animal breast cancer model to explore the feasibility of the determination of their expression in minimally invasive samples and to further assess the magnitude of their regulation by letrozole. ANIMAL AND METHODS: Aromatase inhibitor sensitive breast cancer tumours were grown in athymic mice under supplement with androstenedione. Following initial tumour growth animals were assigned to a control group or to receive letrozole at two different dosages. Fine needle aspirates were obtained at the moment of treatment assignation and one week later. Expression of the following genes at both time points was determined: Ki-67, Cyclin D1, pS2, Trefoil Factor 3, PDZ domain containing 1, Ubiquitin-conjugating enzyme E2C, Stanniocalcin 2, Topoisomerase 2 alfa, MAN1A1 and FAS. RESULTS: Fine needles aspirates were found to be a feasible and reproducible technique for RNA extraction. Trefoil Factor 3, pS2, Cyclin D1 and Stanniocalcin 2 were significantly downregulated by letrozole. Among them pS2 appears to be most sensitive to aromatase inhibitor treatment even differentiating sub-optimal from optimal letrozole dosage. DISCUSSION: We present pre-clinical evidence to justify the exploration in clinical trials of pS2, Trefoil factor 3, Cyclin D1 and Stanniocalcin as dynamic markers of oestrogen-driven pathway activation..

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These guidelines update the previous UK HER2 testing guidelines and have been formulated to give advice on methodology, interpretation and quality assurance to ensure that HER2 testing results are accurate, reliable and timely with the expansion of testing to all patients with breast cancer at the time of primary diagnosis. The recommendations for testing are the use of immunohistochemistry but with analysis of equivocal cases by in situ hybridisation to clarify their HER2 status or the use of frontline fluorescence in situ hybridisation (FISH) testing for those laboratories wishing to do so; the inclusion of a chromosome 17 probe is strongly recommended. Laboratories using chromogenic or silver in situ hybridisation should perform an initial validation against FISH. For immunohistochemistry and in situ hybridisation there must be participation in the appropriate National External Quality Assurance scheme..

Purpose: Ovarian failure and infertility following adjuvant chemotherapy for early breast cancer are major concerns for some young women. Techniques for oocyte harvesting are associated with delay in starting treatment, potentially undesirable estrogen stimulation and a relatively low success rate. We report an audit of our experience with the luteinising hormone-releasing hormone agonist, goserelin, to achieve transient ovarian suppression during chemotherapy as a means of preserving ovarian function.Patient and methods: Pre-menopausal women were offered goserelin 3.6 mg by subcutaneous injection every 28 days during chemotherapy, starting 0-14 days prior to treatment. The primary end-point was recovery of menstruation. Serum luteinising hormone, follicle stimulating hormone and oestradiol were measured at recovery of menstruation or at first year follow-up if amenorrhoea persisted. Subsequent pregnancies were recorded.Results: Fifty-one evaluable women were audited. Amenorrhoea occurred in all but one. All received combination anthracycline-containing chemotherapy regimens with a mean cumulative cyclophosphamide dose of 3.9 g/m(2). Forty-five (90%) recovered menstruation during the first year of follow-up; mean time to recovery 5 months. Eight pregnancies in 10 women attempting this so far.Conclusion: Using goserelin concurrently with chemotherapy is associated with a high rate of ovarian function preservation..

The effect of breast cancer surgery timing during the menstrual cycle on prognosis remains controversial. We conducted a multicentre prospective study to establish whether timing of interventions influences prognosis. We report 3-year overall and disease-free survival ( OS/DFS) results for 'primary analysis' patients ( regular cycles, no oral contraceptives within previous 6 months). Data were collected regarding timing of interventions in relation to patients' last menstrual period ( LMP) and first menstrual period after surgery ( FMP). Hormone profiles were also measured. Cox's proportional hazards model incorporated LMP in continuous form. Exploratory analyses used menstrual cycle categorisations of Senie, Badwe and Hrushesky. Hormone profiles with LMP and FMP data were also used to define menstrual cycle phase. Four hundred and twelve 'primary analysis' patients were recruited. Three-year OS from first surgery was 90.7, 95% confidence interval ( CI) [ 87.9, 93.6%]. Menstrual cycle according to LMP was not statistically significant ( OS: hazard ratio ( HR) = 1.02, 95% CI [ 0.995,1.042], P = 0.14; DFS: HR = 1.00, 95% CI [ 0.980,1.022], P = 0.92). Timing of surgery in relation to menstrual cycle phase had no significant impact on 3-year survival. This may be due to 97% of patients receiving some form of adjuvant therapy. Survival curves to 10 years indicate results may remain true for longer-term survival..

BACKGROUND: We analysed the clinical features, distribution of basal markers, prevalence of oncogene amplification, and outcome of triple negative (TN) compared to those of non-TN cancers in a series of adjuvant-anthracycline treated breast cancer patients. METHODS: We examined the prognostic impact of the TN and BL phenotype in 245 breast cancer patients uniformly treated with adjuvant anthracycline-based chemotherapy following primary surgery, with regards to local relapse-free (LRFS), metastasis free (MFS), and breast cancer specific survival (BCSS). A comparative analysis of the clinicopathological characteristics, expression of basal markers (cytokeratins (Cks) 5/6, 14, 17, EGFR, and caveolin 1 and 2), MIB-1, p53 and topoisomerase II alpha, and prevalence of CCND1, MYC and TOP2A amplification in TN and non-TN breast tumours was performed. RESULTS: TN cancers were significantly associated with the expression of basal markers (all P < 0.0001). However 19.4% of TN tumours were negative for basal markers, whilst 7.3% of non-TN tumours expressed basal markers. TN phenotype was significantly associated with p53, MIB-1 and topoisomerase II alpha (all, P < 0.01) expression. No TN cancer harboured amplification of CCND1 or TOP2A. In univariate analysis, TN and BL phenotype were significantly associated with shorter MFS (both, P < 0.01) and BCSS (both, P < 0.005) but not LRFS. CONCLUSIONS: Despite treatment with standard dose anthracycline-based chemotherapy, the clinical outcome of TN and BL cancers remains poor. Alternative chemotherapeutic regimens and/or novel therapeutic approaches are warranted. Although a significant phenotypic overlap exists between TN and basal-like tumours, the TN phenotype is not an ideal surrogate marker for basal-like breast cancers..

Preoperative systemic therapy (PST) in operable breast cancer allows a small increase in breast conservation rates and has significant potential as a research platform. PST offers the ability to discern treatment effect in vivo, and may allow smaller trials targeting specific breast cancer subtypes and making more efficient use of resources. Early observations of a specific outcome of interest in individual patient subgroups may improve the design of larger definitive randomized adjuvant trials using survival as a main outcome. PST offers the potential for therapeutic adjustments midcourse, which assumes the existence of validated intermediate end points and effective alternative therapies. This article reviews critical research issues affecting the design of PST trials, including the appropriate selection of trial end points and markers for long-term outcome, baseline marker expression as a predictor of response, and statistical considerations using novel trial designs. Key issues regarding optimal tumor subtype selection for individual trials, novel approaches using nontherapeutic window trial designs, and ethical and advocacy considerations are also discussed. PST requires an experienced and cohesive multidisciplinary team for it to fulfill its potential in both research and clinical care..

Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. We used an MCF-7 breast tumor cell line (Tam(R)-1) resistant to tamoxifen to investigate this mechanism. We demonstrate that Tam(R)-1 express elevated levels of phosphorylated AKT and MAPK3/1-activated RPS6KA2 compared with the parental MCF-7 cell line (MCF-7). There was no change in the level of total ESR between the two cell lines; however, the Tam(R)-1 cells had increased phosphorylation of ESR1 ser(167). SiRNA blockade of AKT or MAPK3/1 had little effect on ESR1 ser(167) phosphorylation, but a combination of the two siRNAs abrogated this. Co-localization studies revealed an association between ERBB2 and ESR1 in the Tam(R)-1 but not MCF-7 cells. ESR1 was redistributed to extranuclear sites in Tam(R)-1 and was less transcriptionally competent compared with MCF-7 suggesting that nuclear ESR1 activity was suppressed in Tam(R)-1. Tamoxifen resistance in the Tam(R)-1 cells could be partially overcome by the ERBB2 inhibitor AG825 in combination with tamoxifen, and this was associated with re-localization of ESR1 to the nucleus. These data demonstrate that tamoxifen-resistant cells have the ability to switch between ERBB2 or ESR1 pathways promoting cell growth and that pharmacological inhibition of ERBB2 may be a therapeutic strategy for overcoming tamoxifen resistance..

Background: Circulating levels of sex hormone-binding globulin (SHBG) are inversely associated with breast cancer risk in postmenopausal women. Three polymorphisms within the SHBG gene have been reported to affect SHBG levels, but there has been no systematic attempt to identify other such variants.Methods: We looked for associations between SHBG levels in 1,134 healthy, postmenopausal women and 11 tagging single nucleotide polymorphisms (SNP) in or around the SHBG gene. Associations between SHBG SNPs and breast cancer were tested in up to 6,622 postmenopausal breast cancer cases and 6,784 controls.Results: Ten SNPs within or close to the SHBG gene were significantly associated with SHBG levels as was the (TAAAA)(n) polymorphism. The best-fitting combination of rs6259, rs858521, and rs727428 and body mass index, waist, hip, age, and smoking status accounted for 24% of the variance in SHBG levels (natural logarithm transformed). Haplotype analysis suggested that rs858518, rs727428, or a variant in linkage disequilibrium with them acts to decrease SHBG levels but that this effect is neutralized by rs6259 (D356N). rs1799941 increases SHBG levels, but the previously reported association with (TAAAA)(n) repeat length appears to be a consequence of linkage disequilibrium with these SNPs. One further SHBG SNP was significantly associated with breast cancer (rs6257, per-allele odds ratio, 0.88; 95% confidence interval, 0.82-0.95; P = 0.002).Conclusion: At least 3 SNPs showed associations with SHBG levels that were highly significant but relatively small in magnitude. rs6257 is a potential breast cancer susceptibility variant, but relationships between the genetic determinants of SHBG levels and breast cancer are complex. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3490-8).

The era of personalized medicine is likely to see an escalation in the use of biomarkers to ensure breast cancer patients receive optimal treatment. A combination of prognostic and predictive biomarkers should enable better quantification of the residual risk faced by patients and indicate the potential value of additional treatment. Established biomarkers such as estrogen receptor and progesterone receptor already play a significant role in the selection of patients for endocrine therapy. Human epidermal growth factor receptor 2 (HER2) is recognized as a strong predictor of response to trastuzumab whereas, more recently, the role of estrogen receptor and HER2 as negative and positive indicators for chemotherapy has also been explored. Ki67 has traditionally been recognized as a modest prognostic factor, but recent neoadjuvant studies suggest that on-treatment measurement may be a more effective predictor of treatment efficacy for both endocrine treatment and chemotherapy. The last decade has seen the emergence of numerous multigene expression profiles that aim to outdo traditional predictive and prognostic factors. The Oncotype DX assay and the MammaPrint profile are currently undergoing prospective clinical trials to clearly define their role. Other gene expression-based assays also show potential but are yet to be tested clinically. Rigorous comparison of these emerging markers with current treatment selection criteria will be required to determine whether they offer significant benefit to justify their use..

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For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template..

PURPOSE: Human epidermal growth factor receptor 2 (HER-2) expression is associated with increased risk of high-grade disease, nodal metastasis, and absence of estrogen receptors (ERs) in early breast cancer. We tested interactions between ER and HER-2 to determine if they may modulate breast cancer nodal metastasis and proliferation. PATIENTS AND METHODS: Tumors from the Cancer Research UK Taxotere as Adjuvant Chemotherapy phase III trial were tested for HER-2 using current diagnostic procedures. ER status, progesterone status, clinicopathologic characteristics, and patient age were included in a logistic regression analysis to identify associations with HER-2 status (positive v negative). RESULTS: A total of 841 (23.6%) of 3,565 samples were HER-2 positive (3+ by immunohistochemistry or positive by fluorescent in situ hybridization). ER-negative tumors were more likely to be HER-2 positive than were ER-positive tumors (odds ratio [OR] = 1.87, ER negative v ER positive; P < .001). For ER-positive tumors, risk of HER-2 positivity increased by grade (OR = 7.6, grade 3 v grade 1; P < .001) but not nodal status (OR = 1.3, four or more positive nodes v node negative; P = .08). Conversely, ER negative node-positive tumors were markedly more frequently HER-2 positive than node-negative cases (OR = 3.05, four or more positive nodes v node negative; P < .001) but independent of grade (OR = 0.82, grade 3 v grade 1; P = .76). CONCLUSION: In early breast cancer patients selected for cytotoxic chemotherapy, we identified significant interactions between HER-2 and ER expression that correlate with tumor pathology. In ER-positive breast cancers, HER-2 expression correlates with grade, not nodal metastasis. In ER-negative breast cancers, HER-2 expression correlates with increased nodal positivity, not grade. ER and HER-2 expression may modify tumor pathology via ER/HER-2-mediated cross talk..

PURPOSE: Increased epidermal growth factor receptor (EGFR) expression may promote breast cancer resistance to endocrine therapy. We have therefore investigated whether neoadjuvant gefitinib, an EGFR inhibitor, might overcome biologic and clinical resistance to neoadjuvant anastrozole in a phase II placebo-controlled trial. PATIENTS AND METHODS: Postmenopausal women with stage I to IIIB hormone receptor-positive early breast cancer received anastrozole 1 mg daily for 16 weeks and were randomly assigned at a ratio of 2:5:5 to receive, in addition, gefitinib 250 mg/d orally for 16 weeks: placebo 1 tablet/d orally for 2 weeks and then gefitinib for 14 weeks or placebo for 16 weeks. The primary end point was biologic change in proliferation as measured by Ki67 at 2 and 16 weeks; the main secondary end point was overall objective response (OR). RESULTS: Two hundred six women were randomly assigned. Mean changes in Ki67 with anastrozole and gefitinib versus anastrozole alone were -77.4% and -83.6%, respectively, between baseline and 16 weeks (geometric mean ratio = 1.37; 95% CI, 0.79 to 2.39; P = .26), -80.1% and -71.3% between baseline and 2 weeks (geometric mean ratio = 0.70; 95% CI, 0.39 to 1.25; P = .22) and -19.3% and -43% (geometric mean ratio = 1.42; 95% CI, 0.86 to 2.35; P = .16) between 2 and 16 weeks. ORs in the combination and anastrozole alone groups were 48% and 61% (estimated difference = -13.1%; 95% CI, -27.3% to 1.2%), respectively, with a nonsignificant trend against the combination (P = .08) and 48% versus 72% (estimated difference = -24.1%; 95% CI, -45.3% to -2.9%) in the progesterone-receptor-positive subgroup, which was significant (P = .03) and consistent with Ki67 changes. Common treatment-related adverse events included diarrhea, rash, alopecia, dry skin, and nausea. There was no evidence of a pharmacokinetic interaction. CONCLUSION: Addition of gefitinib to neoadjuvant anastrozole had no additional clinical or biologic effect, failing to support our original hypothesis..

Context: Little is known of associations between hip geometry and skeletal regulators. This is important because geometry is a determinant of both hip function and resistance to fracture.Objective: We aimed to determine the effects of sex hormone status and other candidate regulators on hip geometry and strength.Subjects and Methods: A random sample of 351 women aged 67 - 79 had two to four hip dual-energy x-ray absorptiometry scans performed over 8 yr of follow-up. Hip structural analysis software was used to measure subperiosteal diameter (PD) and the distance from the center of mass to the lateral cortical margin (d-lat) on three 5-mm-thick cross-sectional regions: narrow neck, intertro-chanter, and shaft. Section modulus (Z), bone mineral density (grams per centimeter squared), and an index of bone mineral content (cross-sectional area) were calculated as estimators of bone strength. Serum analytes measured at baseline included SHBG, estradiol, PTH, creatinine, albumin, vitamin D metabolites, and glutamate- and gamma-carboxyglutamate-osteocalcin (OC). A linear mixed model was used to model associations with predictor variables, including testing whether the predictors significantly modified the effect of aging.Results: Aging was associated with increasing PD and d-lat, and higher baseline SHBG significantly modified this effect, in the case of PD, increasing the rates of change at the narrow neck region by 19% for SHBG level 2 SD higher than population mean (P = 0.026). Higher baseline creatinine was independently associated with faster increases in PD and d-lat with aging (P < 0.041). Z declined faster with aging if baseline PTH was higher, and higher albumin had a contrary effect. Z was positively associated with free estradiol and inversely associated with SHBG and glutamate-OC.Conclusion: These results show large effects of SHBG on the regulation of proximal femur expansion and bending resistance, probably acting as a surrogate for low bioavailable estrogen. Potentially important effects for fracture resistance in old age were also revealed for PTH, markers related to renal function and the nutritional markers albumin and undercarboxylated OC..

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Sex hormones are intrinsically linked to the development and treatment of the majority of breast cancers. High oestradiol levels are associated with elevated breast cancer risk in the postmenopausal setting and are given increasing importance in determining chemoprevention target groups. This review aims to evaluate the impact of various lifestyle factors including body mass index, diet, smoking, and alcohol on normal physiological sex hormone profiles. The role of genetic polymorphisms is also discussed, and all are placed within the context of designing future epidemiological studies and breast cancer risk algorithms..

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The majority of breast cancers in male patients are hormone receptor positive. Tamoxifen has proven to be successful in both adjuvant and metastatic settings and remains the standard of care. Given the improved outcomes in female patients with aromatase inhibitors (AI), these drugs have become a potential therapeutic tool for male patients. Preliminary data show effective suppression of oestradiol levels in males treated with AI and some reports have demonstrated objective responses. Here we report a case of a male patient with metastatic breast cancer treated with letrozole who achieved clinical response associated with a decrease in blood oestradiol levels..

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Background - The relation between endogenous testosterone concentrations and health in men is controversial.Methods and Results - We examined the prospective relationship between endogenous testosterone concentrations and mortality due to all causes, cardiovascular disease, and cancer in a nested case-control study based on 11 606 men aged 40 to 79 years surveyed in 1993 to 1997 and followed up to 2003. Among those without prevalent cancer or cardiovascular disease, 825 men who subsequently died were compared with a control group of 1489 men still alive, matched for age and date of baseline visit. Endogenous testosterone concentrations at baseline were inversely related to mortality due to all causes (825 deaths), cardiovascular disease (369 deaths), and cancer (304 deaths). Odds ratios (95% confidence intervals) for mortality for increasing quartiles of endogenous testosterone compared with the lowest quartile were 0.75 (0.55 to 1.00), 0.62 (0.45 to 0.84), and 0.59 (0.42 to 0.85), respectively (P < 0.001 for trend after adjustment for age, date of visit, body mass index, systolic blood pressure, blood cholesterol, cigarette smoking, diabetes mellitus, alcohol intake, physical activity, social class, education, dehydroepiandrosterone sulfate, androstanediol glucuronide, and sex hormone binding globulin). An increase of 6 nmol/L serum testosterone (approximate to 1 SD) was associated with a 0.81 (95% confidence interval 0.71 to 0.92, P < 0.01) multivariable-adjusted odds ratio for mortality. Inverse relationships were also observed for deaths due to cardiovascular causes and cancer and after the exclusion of deaths that occurred in the first 2 years.Conclusions - In men, endogenous testosterone concentrations are inversely related to mortality due to cardiovascular disease and all causes. Low testosterone may be a predictive marker for those at high risk of cardiovascular disease..

Human epidermal growth factor receptor 2 (HER2) positivity in breast cancer is a prognostic factor regarding tumor aggressiveness and a predictive factor for response to trastuzumab (Herceptin). Early and accurate HER2 testing of all breast cancer patients at primary diagnosis is essential for optimal disease management. Routine HER2 tests, such as immunohistochemistry and fluorescence in situ hybridization (FISH), are subject to interlaboratory variation, and validation by laboratory proficiency testing is important to improve standardization. This study compared immunohistochemistry and FISH testing between five international pathology reference centers. Each center evaluated 20 immunohistochemistry and 20 FISH breast cancer specimens in five testing rounds. In each round, one center selected two sets of four different invasive tumor specimens (set A for immunohistochemistry and set B for FISH) and sent samples to the other four centers in a blinded manner, while retaining samples for its own evaluation. Results were analyzed by an independent coordinator. With immunohistochemistry, there were no differences between the five centers for any of the specimens at the level of diagnostic decision (positive or negative HER2 status). However, differences between laboratories were observed in immunohistochemistry scoring. Of the 20 specimens, four were scored as negative (0/1+) and five as positive (3+) in all centers; eight were negative or equivocal (2+), and three positive or equivocal. After FISH retesting of nine of the 11 equivocal immunohistochemistry cases, consensus was achieved in 15 of 18 (83%) specimens. FISH analysis of set B specimens resulted in consensus between centers in 16 of 20 (80%) specimens (six negative and 10 positive). All four discordant FISH specimens were scored as having HER2:CEP17 ratios within the range 1.7-2.3 by at least one center. Equivocal immunohistochemistry and borderline FISH cases are difficult to interpret, even for highly experienced and validated laboratories, highlighting the need for quality-control procedures..

Background: Short-term trials indicate that intensive physical activity may influence endogenous sex hormone concentrations. However, the relationship between usual daily physical activity and endogenous hormones in postmenopausal women in the general population is still uncertain.Objective and Methods: To determine the relationship between usual physical activity and endogenous sex hormones in postmenopausal women. A cross-sectional population-based study of 2,082 postmenopausal women ages 55 to 81 years, residing in the general community of Norfolk, United Kingdom, and not currently using hormone replacement therapy were chosen to participate. Physical activity in the past 1 year was assessed using a validated questionnaire, and endogenous sex hormone and sex hormone-binding globulin (SHBG) concentrations were determined.Results: Usual physical activity levels were inversely associated with circulating concentrations of testosterone and estradiol, testosterone/SHBG ratio, and positively associated with SHBG. These associations were only slightly attenuated after adjusting for potential covariates including body mass index, smoking status, alcohol intake, and reproductive variables. Testosterone concentrations and testosterone/SHBG ratios were 19% [95% confidence interval (95% CI), 9-27%, P < 0.001] and 24.0% (95% CI, 13-34% P < 0.001) lower, respectively, whereas estradiol concentrations were 6% (95% Cl, 0-12%; P < 0.05) lower in the highest compared with lowest activity levels, respectively. A decreasing trend for the estradiol/SHBG ratio and 17 alpha-thydroxyprogesterone concentrations was also observed. Androstenedione levels did not differ significantly according to physical activity.Conclusions: Higher usual physical activity levels among postmenopausal women seem to be related to lower endogenous testosterone and estradiol concentrations. This may be one mechanism that could partly explain the reported inverse relationship between physical activity and breast cancer risk in some studies..

BACKGROUND: Trastuzumab--a humanised monoclonal antibody against HER2--has been shown to improve disease-free survival after chemotherapy in women with HER2-positive early breast cancer. We investigated the drug's effect on overall survival after a median follow-up of 2 years in the Herceptin Adjuvant (HERA) study. METHODS: HERA is an international multicentre randomised trial that compared 1 or 2 years of trastuzumab treatment with observation alone after standard neoadjuvant or adjuvant chemotherapy in women with HER2-positive node positive or high-risk node negative breast cancer. 5102 women participated in the trial; we analysed data from 1703 women who had been randomised for treatment with trastuzumab for 1 year and 1698 women from the control group, with median follow-up of 23.5 months (range 0-48 months). The primary endpoint of the trial was disease-free survival. Here, we assess overall survival, a secondary endpoint. Analyses were done on an intent-to-treat basis. This trial is registered with the European Clinical Trials Database, number 2005-002385-11. FINDINGS: 97 (5.7%) patients randomised to observation alone and 58 (3.4%) patients randomised to 1 year of treatment with trastuzumab were lost to follow-up. 172 women stopped trastuzumab prematurely. 59 deaths were reported for trastuzumab and 90 in the control group. The unadjusted hazard ratio (HR) for the risk of death with trastuzumab compared with observation alone was 0.66 (95% CI 0.47-0.91; p=0.0115). 218 disease-free survival events were reported with trastuzumab compared with 321 in the control group. The unadjusted HR for the risk of an event with trastuzumab compared with observation alone was 0.64 (0.54-0.76; p<0.0001). INTERPRETATION: Our results show that 1 year of treatment with trastuzumab after adjuvant chemotherapy has a significant overall survival benefit after a median follow-up of 2 years. The emergence of this benefit after only 2 years reinforces the importance of trastuzumab in the treatment of women with HER2-positive early breast cancer..

Time spent in transit may affect the concentration of various constituents of collected blood samples and, consequently, results of sex hormone assays. Whole blood was collected from 46 women, and one third was processed immediately, one third was stored at ambient conditions (22 degrees C) for 1 day, and one third was stored for 2 days. Estradiol concentration increased by 7.1% [95% confidence interval (95% CI), 3.2-11.3%] after a delay in processing of 1 day and by 5.6% (95% CI, 0.2-11.4%) after a delay in processing of 2 days; the change was most apparent at lower than median concentrations. Progesterone concentrations showed no substantial change. Testosterone concentrations changed by 23.9% (95% CI, 17.8-30.3%) after a delay of 1 day but little thereafter. The sex hormone-binding globulin concentration decreased by 6.6% (95% CI, 4.6-8.6%) and 10.9% (95% CI, 8.1-13.6%), follicle-stimulating hormone increased by 7.4% (95% CI, 4.2-10.7%) and 13.9% (95% CI, 8.7-19.3%), and luteinizing hormone increased by 4.9% (95% CI, 1.3-8.5%) and 6.7% (95% CI, 2.2-11.5%) after a delay in processing of 1 and 2 days. Increases in calculated values for biologically available levels of estradiol and testosterone were greater than the increases seen in measured total hormone concentrations. Similar changes are likely when samples are delayed in transit, and evidence of etiology may be obscured unless study designs or analyses take into account processing delays..

OBJECTIVE: To gain an understanding of current attitudes among oncologists and pathologists to prospective HER2 testing in breast cancer and to gauge whether a national consensus exists regarding extent and quality of testing. DESIGN: Qualitative study, with semi-quantitative components, using emailed questionnaires and open-ended discussion documents. PARTICIPANTS: 186 relevant specialists, including 76 breast oncologists and 99 pathologists, representing all but three of the UK cancer networks. RESULTS: A strong consensus was seen in favour of universal, non-selective testing for HER2 at the point of breast cancer diagnosis. Similarly, an overwhelming majority of participants agreed that, to optimise the quality of test results, all laboratories undertaking HER2 testing should be CPA-accredited, participate in the recognised national external quality assessment scheme (UK NEQAS), and carry out a formal annual audit of its testing service. A further recommendation that testing be restricted to laboratories undertaking a minimum 250 tests per annum for immunohistochemistry and 100 tests per annum for in situ hybridisation techniques met with majority support. However, this was not a clear consensus; a significant minority of participants favoured continued use of local services falling short of these criteria. CONCLUSION: This study was successful in gauging national specialist opinion regarding the extent and quality assurance of HER2 testing in the UK..

The majority of candidates for breast cancer prevention have not accepted tamoxifen because of the perception of an unfavorable risk/benefit ratio and the acceptance of raloxifene remains to be determined. One means of improving this ratio is to identify women at very high risk of breast cancer. Family history, age, atypia in a benign biopsy, and reproductive factors are the main parameters currently used to determine risk. The most powerful risk factor, mammographic density, is not presently employed routinely. Other potentially important factors are plasma estrogen and androgen levels, bone density, weight gain, age of menopause, and fracture history, which are also not currently used in a comprehensive risk prediction model because of lack of prospective validation. The Breast Cancer Prevention Collaborative Group (BCPCG) met to critically examine and prioritize risk factors that might be selected for further testing by multivariate analysis using existing clinical material. The BCPCG reached a consensus that quantitative breast density, state of the art plasma estrogen and androgen measurements, history of fracture and height loss, BMI, and waist-hip ratio had sufficient priority for further testing. As a practical approach, these parameters could be added to the existing Tyrer-Cuzick model which encompasses factors included in both the Claus and Gail models. The BCPCG analyzed potentially available clinical material from previous prospective studies and determined that a large case/control study to evaluate these new factors might be feasible at this time..

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Improving endocrine responsiveness and preventing the development of resistance is the goal of many current strategies that are looking to combine aromatase inhibitors with novel drugs that target various pathways in estrogen receptor (ER) positive breast cancer. Pre-clinical models of acquired resistance to aromatase inhibitors have suggested an increase in several signaling pathways including peptide growth factor signaling (EGFR, HER2) and activation of the mTOR signaling pathway. These may result in associated 'cross-talk' activation of ER-dependent gene transcription, such that dual blockade of ER together with other signaling pathways has become a logical approach to improve endocrine responsivness. Clinical strategies with aromatase inhibitors are looking to prevent activation of these pathways either through combination with the selective ER downregulator fulvestrant, or with various signal transduction inhibitors (STIs) including monoclonal antibodies (trastuzumab), small molecule tyrosine kinase inhibitors (TKIs) against EGFR or HER2 (lapatinib, gefitinib) and mTOR antagonists (temsirolimus). Early clinical data have emerged this year for some of these approaches with mixed results. This article reviews the rationale for these strategies, and discusses the lessons that need to be learnt if we are to successfully integrate these new drugs with aromatase inhibitors in the clinic..

Aromatase inhibitors are now considered to be part of the endocrine treatment for most hormone receptor-positive breast cancer in post-menopausal women for both early and advanced disease. Despite the impressive efficacy of these agents, up to 50% of treated patients exhibit de novo or intrinsic resistance to aromatase inhibitors and hence identification of response predictors is essential to allow treatment to be directed towards responsive populations and for alternative or additional therapies to be offered to resistant patients. Emerging data seem to suggest a role for the conventional tumour markers of oestrogen receptor and progesterone receptor as possible predictors of response but, particularly in the adjuvant setting, the extent to which these are useful has not been fully elucidated. Data from both the neo-adjuvant and advanced disease settings suggest that response to aromatase inhibitors does not appear to be adversely affected by HER-2 overexpression. Within neo-adjuvant aromatase inhibitor studies, the proliferation marker Ki67 has shown a significant correlation with relapse-free survival, suggesting a role in prediction for measurement of Ki67 and other dynamic markers of response. Analysis of multiple gene expression changes over a short treatment period may also have potential clinical utility for prediction of response..

Cross-talk between receptor tyrosine kinases and estrogen receptor is at least partly responsible for the development of acquired resistance to endocrine therapies. Hence, targeting receptor tyrosine kinases and their downstream partners with inhibitors/antagonists may reverse this resistance. Although ras mutations are rare in breast cancer (2%), aberrant function of Ras signal transduction pathways is common. We therefore investigated the efficacy of the farnesyltransferase inhibitor (FTI) R115777 (tipifarnib) in combination with tamoxifen in MCF-7 human breast cancer models both in vitro and in vivo. There was a synergistic antiproliferative interaction between R115777 and 4-hydroxy-tamoxifen in vitro as calculated by median effect analysis. The combination resulted in a significantly greater G(1) arrest than either drug alone and this was associated with marked inhibition of cyclin D1 and induction of the cell cycle inhibitor p27(kip1). Combining R115777 with either tamoxifen or estrogen withdrawal in vivo produced a significantly greater inhibition of tumor growth and lower xenograft cell proliferation than either therapy alone. These results suggest that the combination of this FTI with endocrine therapy may be of therapeutic benefit in the treatment of breast cancer. Enhanced G1 arrest due to modulation of cell cycle regulatory proteins may be the underlying mechanism for the positive interaction between FTIs and tamoxifen..

Demonstration of the clinically significant activity of bevacizumab in breast cancer has attracted a great deal of interest. Numerous other antiangiogenic treatments are in clinical development and some established therapies including tamoxifen and trastuzumab might function, in part, by suppressing angiogenesis. In this Review, we discuss the potential of various components of the angiogenic pathway as prognostic and predictive factors in breast cancer. In addition, we describe existing clinical trials of antiangiogenic agents and the challenges facing the clinical development and optimum use of these agents for the treatment of breast cancer..

An association between physical activity and premenopausal breast cancer risk may be due, in part, to relationships with sex hormones or growth factors. Therefore, we assessed whether MET-h/week of total physical activity (moderate-to-vigorous intensity), walking, or vigorous physical activity, and h/week of standing or sitting were associated with plasma concentrations of several hormones. We examined levels of estrogens, androgens, progesterone, prolactin, sex hormone binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), IGF binding protein-3, and growth hormone (GH) in 565 premenopausal women, ages 33-52 years, from the Nurses' Health Study II (NHSII). About 87% of women had both timed follicular and luteal samples; other women had one untimed sample. In general we observed few associations between sex hormone or IGF levels and measures of physical activity or inactivity. However, free testosterone was modestly inversely associated with total physical activity (p-trend = 0.02). Luteal estradiol, free estradiol, and estrone also were inversely associated with total physical activity (p-trend = 0.10, 0.04, 0.01, respectively); however, the trend was substantially attenuated when excluding women with anovulatory cycles or irregular cycles. These cross-sectional results suggest that physical activity and inactivity have limited associations with premenopausal sex hormone and growth factor levels, except possibly luteal estrogens..

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BACKGROUND: TOP2A gene encodes topoisomerase II alpha, the direct molecular target of anthracyclines. This gene is frequently coamplified with HER2. The aims of this study were to analyse the pattern of TOP2A amplification and protein expression in relation to the molecular subgroups of breast cancers; and to define the impact of TOP2A amplification on the outcome of a series of patients homogeneously treated with adjuvant anthracyclines. METHODS: A cohort of 245 patients with early breast cancer homogeneously treated with anthracyclines in the adjuvant setting was selected. A tissue microarray containing these cancers was used to determine HER2 and TOP2A gene copy number by means of chromogenic in situ hybridization. Immunohistochemical staining of topoisomerase II alpha was also performed using a monoclonal antibody (Ki-S1). TOP2A amplification and protein expression were correlated with classical prognostic parameters, expression of immunohistochemical markers and with a gene expression profiling classification using surrogate immunohistochemical markers. Kaplan-Meier method was used to construct survival curves and results were compared with log-rank test. RESULTS: TOP2A amplification was restricted to tumours with HER2 amplification and was significantly associated with ER positivity. In the subgroup of patients with HER2 amplified tumours, TOP2A amplification predicted a better overall survival and disease free survival (P = 0.028 and 0.026, respectively). On multivariate analysis, TOP2A amplification maintained its predictive value for DFS. CONCLUSION: TOP2A amplification is likely to be a useful marker to predict the subset of patients who will benefit from anthracyclines..

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Purpose: Tibolone is a selective tissue estrogenic activity regulator, approved for the treatment of vasomotor symptoms in postmenopausal women. We have done an exploratory, double-blind, randomized, placebo-controlled pilot trial to investigate the tissue-specific effects of 2.5 mg tibolone on breast cancer in postmenopausal women, in particular on tissue proliferation (STEM, Study of Tibolone Effects on Mamma carcinoma tissue).Experimental Design: Postmenopausal women with initially stage I/II, estrogen receptor-positive (ER+) primary breast cancer, were randomly assigned to 14 days of placebo or 2.5 mg/d tibolone. Core biopsies of the primary tumor were obtained before and after treatment. Ki-67 and apoptosis index were analyzed in baseline and corresponding posttreatment specimen.Results: Of 102 enrolled patients, 95 had evaluable data. Baseline characteristics were comparable between both treatment groups. Breast cancer cases are mainly invasive (99%), stage I or II (42% and 50% respectively), and ER+ (99%). Median intratumoral Ki-67 expression at baseline was 13.0%, in the tibolone group and 17.8% in the placebo group, and decreased to 12.0% after 14 days of tibolone while increasing to 19.0% in the placebo group. This change from baseline was not significantly different between tibolone and placebo (Wilcoxon test; P = 0.17). A significant difference was observed between the treatment groups when the median change from baseline apoptosis index was compared between the treatment groups (tibolone, 0.0%; placebo, +0.3%; Wilcoxon test; P = 0.031). The incidence of adverse effects was comparable.Conclusions: In ER+ breast tumors, 2.5 mg/d tibolone given for 14 days has no significant effect on tumor cell proliferation..

Tumor expression of the proliferation antigen Ki67 is widely used to assess the prognosis of cancer patients. A change in the expression of Ki67 after short-term exposure of patients to therapeutic agents is frequently used as a pharmacodynamic marker of efficacy, particularly among breast cancer patients before undergoing surgery. To determine the clinical significance of the level of tumor cell proliferation during endocrine therapy for breast cancer, we measured the expression of Ki67 in tumor biopsy samples taken before and after 2 weeks of presurgical treatment with anastrozole or tamoxifen or the combination of anastrozole plus tamoxifen in 158 patients with hormone receptor-positive primary disease. In a multivariable analysis, we found that higher Ki67 expression after 2 weeks of endocrine therapy was statistically significantly associated with lower recurrence-free survival (P = .004) whereas higher Ki67 expression at baseline was not. Larger baseline tumor size and lower estrogen receptor level after 2 weeks of treatment were also statistically significantly associated with poorer recurrence-free survival (P < .001 and P = .04, respectively). Our data indicate that measurements of tumor Ki67 level after short-term endocrine treatment may improve the prediction of recurrence-free survival by integrating the prognostic value of Ki67 level at baseline with changes in Ki67 level that are associated with treatment benefit..

We have analysed the pedigrees of all 70 women who developed cancer in the Royal Marsden Hospital (RMH) tamoxifen chemoprevention trial, using the Claus model, to assess breast cancer susceptibility heterozygote risk (HR) and screened the entire coding regions of BRCA1 and 2 genes in 62 of these cases. We found a reduced incidence of breast cancers developing on tamoxifen in women who have a lower HR, but not in women with higher HR. There were too few BRCA1/2 mutations (4 cases) to be able to determine the efficacy of tamoxifen by BRCA status. Immunohistochemical analysis showed a significantly lower frequency of median ER (p=0.03) in the cancers developing in tamoxifen-treated patients. These results suggest that tamoxifen is less likely to be effective at reducing breast cancers which are ER negative and also in some individuals at higher HR..

Background Several clinical trials have reported an early reduction in breast cancer incidence in healthy women using tamoxifen to reduce their risk of breast cancer but have not reported longer follow-up data for the evaluation of breast cancer prevention. We report the blinded 20-year follow-up (median follow-up = 13 years) of the Royal Marsden trial to identify any long-term prevention of breast cancer associated with tamoxifen treatment.Methods We randomly assigned 2494 healthy women to oral tamoxifen (20 mg/day) or placebo for 8 years. The primary outcome was occurrence of invasive breast cancer. A secondary planned analysis of estrogen receptor (ER)-positive invasive breast cancer was also done. Survival was assessed by use of a Cox proportional hazards model in both univariate and multivariable analyses. The durability of the treatment effect was assessed by use of a Cox regression analysis. All statistical tests were two-sided.Results Among the 2471 eligible participants (1238 participants in the tamoxifen arm and 1233 participants in the placebo arm), 186 developed invasive breast cancer (82 on tamoxifen and 104 on placebo; hazard ratio [HR] = 0.78, 95% confidence interval [Cl] = 0.58 to 1.04; P = .1). Of these 186 cancers, 139 were ER positive (53 on tamoxifen and 86 on placebo; HR = 0.61, 95% Cl = 0.43 to 0.86; P=.005). The risk of ER-positive breast cancer was not statistically significantly lower in the tamoxifen arm than in the placebo arm during the 8-year treatment period (30 cancers in the tamoxifen arm and 39 in the placebo arm; HR = 0.77, 95% Cl = 0.48 to 1.23; P = .3) but was statistically significantly lower in the posttreatment period (23 in the tamoxifen arm and 47 in the placebo arm; HR = 0.48, 95% Cl = 0.29 to 0.79; P = .004). Fifty-four participants in each arm have died from any cause (HR = 0.99, 95% Cl = 0.68 to 1.44; P = .95). The adverse event profiles for both arms were similar to those previously reported and occurred predominantly during the treatment period.Conclusions A statistically significant reduction in the incidence of ER-positive breast cancer was observed in the tamoxifen arm that occurred predominantly during the post treatment follow-up, indicating long-term prevention of estrogen-dependent breast cancer by tamoxifen..

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approximately 5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11-12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21-q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3-q4 and 18p11.31 and gains of 6p25.1-p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2-11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages..

BACKGROUND: Aromatase inhibitors such as anastrozole and letrozole are highly effective suppressants of estrogen synthesis in postmenopausal women and are the most effective endocrine treatments for hormone receptor positive breast cancer in such women. Little is known of the molecular effects of these agents on human breast carcinomas in vivo. METHODS: We randomly assigned primary estrogen receptor positive breast cancer patients to treatment with anastrozole or letrozole for 2 weeks before surgery. Expression profiling using cDNA arrays was conducted on pretreatment and post-treatment biopsies. Sample pairs from 34 patients provided sufficient RNA for analysis. RESULTS: Profound changes in gene expression were seen with both aromatase inhibitors, including many classical estrogen-dependent genes such as TFF1, CCND1, PDZK1 and AGR2, but also many other genes that are likely to represent secondary responses; decrease in the expression of proliferation-related genes were particularly prominent. Many upregulated genes are involved in extracellular matrix remodelling, including collagens and members of the small leucine-rich proteoglycan family (LUM, DCN, and ASPN). No significant differences were seen between letrozole and anastrozole in terms of molecular effects. The gene changes were integrated into a Global Index of Dependence on Estrogen (GIDE), which enumerates the genes changing by at least twofold with therapy. The GIDE varied markedly between tumours and related significantly to pretreatment levels of HER2 and changes in immunohistochemically detected Ki67. CONCLUSION: Our findings identify the transcriptional signatures associated with aromatase inhibitor treatment of primary breast tumours. Larger datasets using this approach should enable identification of estrogen-dependent molecular changes, which are the determinants of benefit or resistance to endocrine therapy..

BACKGROUND: Large numbers of translational breast cancer research topics have been completed or are underway, but they differ widely in their immediate and/or future importance to clinical management. We therefore conducted an international Web-based consultation of breast cancer professionals to identify the topics most widely considered to be of highest priority. METHODS: Potential participants were contacted via two large e-mail databases and asked to register, at a Web site, the issues that they felt to be of highest priority. Four hundred nine questions were reduced by a steering committee to 70 unique issues, and registrants were asked to select the 6 questions they considered to be the most important. RESULTS: Votes were recorded from 420 voters (2,520 votes) from 48 countries, with 48% of voters coming from North America. Half of the voters identified themselves as clinicians, with the remainder being academics, research scientists, or pathologists. The highest priority was to identify molecular signatures to select patients who could be spared chemotherapy, which gained about 50% more votes than the second topic and was consistently voted top by voters in North America, Europe, and the rest of the world. Research scientists voted the determination of the role of stem cells in breast cancer development, progression, and treatment sensitivity as the most important issue, but this was considered the sixth priority for clinicians and fourth overall. CONCLUSION: This exercise may bring a greater focus of research resources onto issues voted as top priorities..

The study of changes in proliferation as a marker of treatment benefit during presurgical endocrine treatment of breast cancer has become increasingly popular, particularly using the nuclear marker Ki67, and holds the potential for prioritizing new treatments for full clinical development. There are weakly significant relationships between Ki67 change and clinical response that differ according to data handling. In the neoadjuvant Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen trial, suppression of Ki67 at both 2 and 12 weeks was greater with the aromatase inhibitor anastrozole than with either tamoxifen or the combination of anastrozole and tamoxifen. We report here that absolute values of Ki67 after 2 weeks were also significantly lower with anastrozole than with tamoxifen and the combination. This indicates that it may be possible to make such comparisons using surgical samples only. We argue that these changes in proliferation and concurrent changes in apoptosis may be expected to be more predictive of adjuvant benefit from endocrine therapy than clinical response..

PURPOSE: Aromatase inhibitors (AIs) are now established as adjuvant therapy for early hormone receptor-positive breast cancer in postmenopausal women. Their use is sometimes extended to younger women after chemotherapy-induced amenorrhoea; we have audited this in one institution's breast unit, and we propose guidelines for use in such circumstances. PATIENTS AND METHODS: The use of aromatase inhibitors as adjuvant therapy in younger women age > or = 40 with hormone receptor-positive early breast cancer and chemotherapy-induced amenorrhea has been audited clinically and biochemically. Results A total of 45 such women were identified in the audit, with a median age of 47 years (range, 39 to 52 years). Twelve women (27%) showed a return of ovarian function (10 renewed menses, one pregnancy, one biochemically premenopausal) after starting an AI. Median age at restart of ovarian function was 44 years (range, 40 to 50 years). CONCLUSION: AIs may promote recovery of ovarian function in some women with chemotherapy-induced amenorrhea and should be used with caution. Biochemical monitoring of ovarian function requires highly sensitive immunoassays. Guidelines for the selection and delivery of adjuvant endocrine therapy in such patients are proposed..

INTRODUCTION: Doxorubicin and cyclophosphamide (Adriamycin/cytoxan, AC) is a standard chemotherapy regimen for breast cancer, but de novo resistance is frequent. We hypothesized that gene expression profiles predictive of AC response may be different from our previously published patterns with docetaxel. METHODS: Core biopsies from 40 patients were obtained before treatment with AC (6 cycles, 60/600 mg/m2q3 weeks), and clinical responses recorded after treatment. Gene expression patterns were analyzed using Affymetrix U133A chips which comprise approximately 22,200 genes. RESULTS: Clinical complete responses (cCR) were observed in 22, partial responses in 7, stable disease in 11 patients. Differential expression between sensitive cCR and resistant tumors with a low false discovery rate (< 5%) was obtained. Of these 253 differentially expressed genes, pathways up-regulated in sensitive tumors included cell cycle (BUB3, CDKN1B), survival (BCL2, BAG1, BIRC1, STK39), stress response (CYP2B6, MAPK14), and estrogen-related pathways (ER, IRS1). Resistant tumors expressed gene promoting transcription (GTF3C1, ILF3), differentiation (ST14, CTNNBIP1), signal transduction (EIF1AX, EIF4EBP1), and amino acid metabolism (SRM, PLOD1, PLOD3). With leave-one-out cross validation, 67% of the samples were correctly classified, with a permutation p-value of 0.4. The previously published 92-gene molecular portrait for docetaxel sensitivity could not discriminate AC sensitivity and resistance. CONCLUSIONS: This preliminary study supports that molecular profiles for AC response are likely to exist, with unique expression patterns for individual chemotherapy regimens. Larger validation studies are necessary to define and refine patterns for different agents..

Exposure to light at night, as experienced by rotating night shift workers, has been related to lower circulating levels of melatonin, a hormone with recognized cancer protective properties. However, little is known about the relationship of other lifestyle factors or endogenous sex steroid hormones with melatonin levels. We examined cross-sectional associations of age, reproductive and menopausal factors, body mass index (BMI), alcohol consumption, smoking history, night shift work, as well as several other breast cancer risk factors, and circulating sex steroid hormone levels with creatinine-adjusted morning urinary melatonin (6-sulfatoxymelatonin, aMT6s) levels. Participants were 459 healthy, primarily premenopausal (age range 33-50 yr) women from the Nurses' Health Study II (NHS II). Using multiple linear regression, we computed least-square mean hormone levels across categories of lifestyle factors. Age was inversely related to aMT6s levels, particularly before menopause (premenopausal women, <= 39 yr versus >= 49 yr; aMT6s, 20.8 ng/mg versus 11.8 ng/mg creatinine; P for trend, 0.02). In multivariate analyses, BMI was significantly and inversely associated with aMT6s levels (P for trend, < 0.01). Higher pack-years of smoking were associated with significantly lower aMT6s levels (never smoker versus 15+ pack-years, aMT6s = 17.4 ng/mg versus 12.3 ng/mg creatinine; P for trend, 0.04). We also observed a positive association between parity and aMT6s levels (P for trend, < 0.01), but no other reproductive factors nor any of the sex hormones (estradiol, progesterone, estrone, estrone sulfate, dehydroepiandrostenedione, dehydroepiandrostenedione sulfate, testosterone, and androstenedione), as measured either in the luteal or the follicular phase of the menstrual cycle, were significantly associated with aMT6s. In conclusion, higher age, BMI, and heavy smoking were significantly related to lower levels of melatonin, whereas parity was significantly associated with higher aMT6s levels. Melatonin levels may be one mechanism through which these factors influence the development of cancer, but more studies are needed to elucidate these mechanisms definitively..

BACKGROUND: Aromatase inhibitors (AI) are increasingly used in early breast cancer and there is a growing interest in associated long-term side-effects of profound estrogen suppression. Urogenital side-effects due to atrophic vaginitis are often managed with vaginal estrogen preparations. These are generally perceived to result in minimal systemic absorption of estrogen. We followed serum estradiol, follicle stimulating hormone (FSH) and luteinising hormone (LH) levels in seven postmenopausal women using vaginal estrogen preparations whilst on AIs for breast cancer. PATIENTS AND METHODS: Serum was analysed for estradiol, FSH and LH at baseline then 2, 4, 7-10 and 12 weeks since commencement of vaginal estradiol. Estradiol was measured on an assay specifically developed for measuring low levels in postmenopausal women. RESULTS: Serum estradiol levels rose from baseline levels < or = 5 pmol/l consistent with AI therapy to a mean 72 pmol/l at 2 weeks. By 4 weeks this had decreased to < 35 pmol/l in the majority (median 16 pmol/l) although significant further rises were seen in two women. CONCLUSIONS: The vaginal estradiol tablet Vagifem significantly raises systemic estradiol levels, at least in the short term. This reverses the estradiol suppression achieved by aromatase inhibitors in women with breast cancer and is contraindicated..

Background. Higher levels of endogenous sex steroid hormones are associated with increased risks of breast cancer in postmenopausal women. Data for premenopausal women are sparse, in part because of the complexity of measuring hormone levels that vary cyclically. We prospectively evaluated associations between plasma sex hormone levels and breast cancer risk among premenopausal women in a case-control study nested within the Nurses' Health Study II. Methods: From 1996 to 1999, blood samples were collected from 18 521 premenopausal women during the early follicular and mid-luteal phases of their menstrual cycles. A total of 197 cases of breast cancer were diagnosed among these women after blood collection and before June 1, 2003; these case subjects were matched to 394 control subjects. Logistic regression models, controlling for breast cancer risk factors, were used to calculate relative risks (RRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. Results: Women in the highest (versus the lowest) quartiles of follicular total and free estradiol levels had statistically significantly increased risks of breast cancer (RR = 2.1 [95% CI = 1.1 to 4.1], P-trend = .08, and RR = 2.4 [95% CI = 1.3 to 4.5], P-trend = .01, respectively); the associations were stronger for invasive breast cancer and for estrogen and progesterone receptor-positive (ER+/PR+) tumors. Luteal estradiol levels were not associated with breast cancer risk. Higher levels of total and free testosterone and androstenedione in both menstrual cycle phases were associated with modest, non-statistically significant increases in overall risk of breast cancer and with stronger, statistically significant increases in risks of invasive and ER+/PR+ cancers (e.g., RR of invasive cancers for the top [versus bottom] quartile of luteal total testosterone levels = 2.0 [95% CI = 1.1 to 3.6], P-trend = .05, and RR of ER+/PR+ cancers = 2.9 [95% CI = 1.4 to 6.9] P-trend = .02). Levels of estrone, estrone sulfate, progesterone, and sex hormone-binding globulin were not associated with breast cancer risk. The absolute number of cases observed over 3 years were 30 among women in the lowest 25% of follicular total estradiol levels and 50 among women in the highest 25%. Conclusions: Levels of circulating estrogens and androgens may be important in the etiology of premenopausal breast cancer..

BACKGROUND: Most women with oestrogen receptor (ER) positive primary breast cancer receive adjuvant tamoxifen after surgery. The measurement of tumour biomarkers should allow better selection of patients for such treatment or for therapies such as aromatase inhibitors. PATIENTS AND METHODS: Histopathological blocks of primary breast cancer patients who had been randomized to receive 2-years tamoxifen or no adjuvant therapy in two mature randomised clinical trials were retrieved. Immunohistochemical staining for ER, progesterone receptor (PgR), HER2 and epidermal growth factor receptor (EGFR) was undertaken. The primary endpoint was relapse free survival. RESULTS: 813 patients were included in the study. Benefit from tamoxifen was seen in ER-positive patients [Relative risk (rr) 0.77, ci 0.63-0.93]. ER-negative patients also showed a strong trend to benefit from tamoxifen (rr 0.73, ci 0.52-1.02) which was largely confined to the PgR-positive group. Amongst the ER-positive group, PgR-positive and PgR-negative patients showed similar benefit (rr 0.81; ci 0.65-1.02 and 0.70; ci 0.49-0.99, respectively). Patients positive for HER2 did not benefit significantly (rr 1.14; ci 0.75-1.73) but this group was small. CONCLUSIONS: Measurement of PgR status in ER-negative patients defines a group of patients that benefit from tamoxifen but would be excluded from tamoxifen therapy on the basis of ER status alone. The data are consistent with HER2 positive tumours being resistant to tamoxifen..

Concentrations of adrenal androgens are positively associated with postmenopausal breast cancer risk; however, results in premenopausal women are conflicting. Therefore, we conducted a prospective nested case-control study within the Nurses' Health Study II cohort to examine the relationship of DHEA and DHEA sulfate (DHEAS) with breast cancer risk in predominantly premenopausal women. Blood samples were collected from 1996 to 1999. The analysis included 317 cases of breast cancer diagnosed after blood collection and before June 1, 2003; for each case, two controls were matched on age, fasting status, time of day and month of blood collection, race/ethnicity, and timing of blood draw within the menstrual cycle. No associations were observed between DHEA or DHEAS levels and breast cancer risk overall [in situ and invasive; DHEA relative risk (RR), top versus bottom quartile, 1.2; 95% confidence interval (95% CI), 0.8-1.8, P-trend = 0.53; DHEAS RR, 1.3; 95% CI, 0.9-2.0; P-trend = 0.07]. However, both DHEA and DHEAS were positively associated with estrogen receptor-positive/progesterone receptor-positive breast cancer (DHEA RR, 1.6; 95% CI, 0.9-2.8, P-trend = 0.09; DHEAS RR, 1.9; 95% CI, 1.1-3.3, P-trend = 0.02). We observed a significant interaction by age, with an RR for DHEAS of 0.8 (95% CI, 0.4-1.5, P-trend = 0.62) for women < 45 years old and 2.0 (95% Cl, 1.2-3.5, P-trend = 0.003) for women >= 45 years old; results were similar for DHEA. Our results suggest that adrenal androgens are positively associated with breast cancer among predominately premenopausal women, especially for estrogen receptor-positive/progesterone receptor-positive tumors and among women over age 45 years..

Conflicting results on the prevalence of cyclin D1 ovexpression and its correlation with CCND1 amplification and outcome of breast cancer patients have been reported. Owing to limited sensitivity and specificity of most antibodies against cyclin D1, evaluation of cyclin D1 immunoexpression is reported to be problematic. The aims of this study were to assess the prevalence of cyclin D1 expression in breast carcinomas using the SP4 rabbit monoclonal antibody; to correlate cyclin D1 expression with amplification, assessed using chromogenic in situ hybridisation (CISH); and to analyse the relationship between CCND1 amplification and overexpression with clinicopathological parameters and outcome in a tissue microarray containing replicate tumour samples from 245 breast cancer patients. Immunohistochemistry for cyclin D1 was performed using the SP4 and the results were scored according to the Allred scoring system. CISH was carried out using the Zymed CCND1 SpotLight probe. CISH signals were counted in 60 morphologically unequivocal neoplastic cells. Amplification was defined as >5 signals per nucleus in more than 50% of cancer cells, or when large gene copy clusters were seen. Strong cyclin D1 expression and CCND1 amplification were found in 67.4 and 14.5% of the cases, respectively. A strong correlation between cyclin D1 overexpression and CCND1 amplification was demonstrated (P<0.0001). Cyclin D1 expression showed a positive correlation with hormone receptor expression (both ER and PgR, P<0.0001). An inverse correlation was observed between an immunohistochemical panel of 'basal-like' markers and both cyclin D1 overexpression (P<0.0001) and CCND1 amplification (P<0.0001). On univariate analysis cyclin D1 expression showed a correlation with longer overall survival (OS). Neither cyclin D1 nor CCND1 were independent prognostic factors for disease-free survival or OS. The results of this study confirm the association between cyclin D1 overexpression and positivity for hormone receptors and the lack of CCND1 amplification in basal-like breast carcinomas..

Mammographic density and serum sex hormone levels are important risk factors for breast cancer, but their associations with one another are unclear. We studied these phenotypes, together with single nucleotide polymorphisms (SNP) in genes related to sex hormone metabolism, in a cross-sectional study of 1,413 postmenopausal women from the European Prospective Investigation into Cancer and Nutrition-Norfolk. All women were > 1 year postmenopausal and had not taken hormone replacement therapy for > 3 months before sampling. Serum levels of 7 sex hormones [estradiol, testosterone, sex hormone-binding globulin (SHBG), androstenedione, 17-OH-progesterone, estrone, and estrone sulfate] and 15 SNPs in the CYP17, CYP19, EDH17B2, SHBG, COMT, and CYP1B1 genes were studied. Mammograms nearest in time to the blood sampling were identified through the national breast screening program and visually assessed by three radiologists using the Boyd six-category and Wolfe four-category scales. We found a weak positive association between mammographic density and SHBG levels (P = 0.09) but no association with any other hormones. None of the SNPs, including those shown previously to be associated with estradiol or SHBG, showed significant associations with density. We conclude that mammographic density is largely independent of postmenopausal steroid hormone levels, indicating that these risk factors have, to a large extent, an independent etiology and suggesting that they may be independent predictors of breast cancer risk..

Aromatase inhibitors reduce estrogen levels in postmenopausal women with breast cancer. Residual estrogen is an important determinant of bone turnover. Adjuvant anastrozole was associated with significant BMD loss and increased bone remodeling, whereas tamoxifen reduced bone marker levels..

Aromatase inhibitors (AIs) have a proven role in the treatment of early and metastatic breast cancer. The success of tamoxifen in reducing the relative risk of developing hormone-sensitive breast cancer in chemoprevention trials has been hampered by their long-term toxicity profile. AIs have the potential to further reduce rates of breast cancer in high-risk postmenopausal women. This article reviews the evidence to support the potential efficacy of AIs in the chemoprevention setting. It particularly focuses on a discussion of novel concepts of utilising AIs, so that they reduce breast cancer risk while minimising systemic toxicity, and highlights the importance of accurately developing risk prediction algorithms..

Context: Accurate measurement of low serum estradiol (E-2 < 30 pg/ml or < 110 pmol/liter) is needed to study relationships between endogenous E2 and risks of diseases in older women.Objective: The objective of this study was to determine whether an extraction-based ( indirect) assay or a non-extraction-based (direct) assay correlates better with mass spectrometry and body mass index (BMI).Design/Setting: In a pilot study of 40 postmenopausal women, endogenous E2 measurements from three indirect and four direct assay methods and gas chromatography-tandem mass spectrometry (GCMS/ MS) were compared. A confirmatory study compared an indirect and a direct assay, selected among those in the pilot study, to GCMS/ MS; this study was conducted in 374 postmenopausal women not taking hormone therapy from the Ultra Low-dose TRansdermal estrogen Assessment (ULTRA) trial.Main Outcomes: Pearson correlation coefficients among E-2 measurements by assay methods and BMI, and their confidence intervals, by bias-corrected bootstrap method, were used.Results: In the pilot study, E2 by three indirect assays correlated better (P < 0.03) with GC-MS/MS and with BMI than measurements by four direct assays. In the confirmatory study, the indirect assay correlated better (P < 0.01) with GC-MS/MS and BMI than the direct assay. Measurements by the indirect and direct assays were overestimated, but deviations in direct assay measurements were less precise. Mean E2 by the indirect and direct assays were higher ( by 14 and 68%, respectively) and less reproducible than by GC-MS/MS.Conclusion: Until mass spectrometry is practical for wide use, extractionbased indirect assays may be preferable for measuring low postmenopausal serum E2..

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Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation. of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R=0.3967, p=0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors. (c) 2006 Elsevier Ireland Ltd. All rights reserved..

Breast cancer accounts for 1 in 4 of all female cancers worldwide; approaching 13,000 women dying per year in the UK alone. Seventy five per cent of all diagnosed breast cancers are oestrogen receptor (ER) positive. Ovarian synthesis of oestrogens ceases at menopause and as breast cancer is more prevalent in postmenopausal women the non-ovarian sources of oestrogen are important in disease progression. There is now considerable evidence that associates increased breast cancer risk with prolonged exposure to oestrogens hence greater attention is now being given to determining whether the measurement of plasma oestrogen may assist in identifying chemoprevention target groups. Studies suggest that in most postmenopausal patients the intra-tumoural concentrations of oestrogens are up to 20-fold higher than those present in the plasma however, while the extent of biosynthesis of oestrogens within breast tissue is a major determinant of local exposure, plasma levels are a useful indicator of overall metabolism in peripheral tissues. As such it is important to understand factors that influence these measurements. This review summarises the impact of lifestyle such as body mass index, together with the role of genetic polymorphisms placed within the context of designing future epidemiological studies and breast cancer risk algorithms..

The association of birthweight and body size throughout life with premenopausal breast cancer risk may be due, in part, to relationships with sex hormones. Therefore, we assessed whether birthweight, body shape at ages 5 and 10, body mass index (BMI) at age 18 and adulthood, adult waist circumference and waist-to-hip ratio (WHR), and attained height were associated with the plasma concentrations of estrogens, androgens, progesterone, prolactin, and sex hormone-binding globulin (SHBG) in 592 premenopausal women, ages 33 to 52 years old, from the Nurses' Health Study II. About 85% of women provided blood samples during follicular and luteal menstrual phases; other women had a single untimed sample. We observed few associations between sex hormone levels and birthweight or body shape in childhood. However, adult BMI was inversely associated with SHBG (P trend < 0.001) and positively associated with free testosterone (P trend < 0.001) concentrations. Adult BMI was not associated with follicular or luteal free estradiol. levels (P trend : 0.15) because it was inversely associated with total estradiol levels (P trend < 0.001 for follicular and luteal estradiol levels). Testosterone, androstenedione, and progesterone were inversely associated with BMI. Comparing women with a BMI of >= 30 versus < 20 kg/m(2), levels were higher by 53% for free testosterone and lower by 51% for SHBG, 39% for follicular estradiol, 20% for luteal estradiol, 14% for androstenedione, 13% for testosterone, and 20% for progesterone. We observed no clear associations between BMI at age 18, waist circumference, WHR, or height, and sex hormone concentrations. Our results suggest that effects on premenopausal sex hormone levels may be one mechanism through which adult adiposity, but not birthweight or childhood body size, affects premenopausal breast cancer risk..

Studies to identify common genetic variants contributing to breast cancer risk often yield inconsistent results. Breast cancer is a complex disease involving both genetic and environmental determinants. Dietary isoflavones are thought to reduce breast cancer risk by stimulating circulating sex hormone-binding globulin (SHBG) levels. The SHBG gene contains a D356N polymorphism and the N variant is associated with reduced SHBG clearance compared with the D variant. In this study, we show a significant gene-environment interaction between SHBG D356N polymorphism and dietary isoflavone exposure on circulating SHBG levels in 1,988 postmenopausal women. SHBG levels were positively associated with isoflavones in women carrying the N variant (eta(2)(p) = 1.9%; P = 0.006) but not in women carrying only the D variant (eta(2)(p) = 0.0%; P = 0.999; P-interaction = 0.019). This finding shows that the subtle effects of some genetic variants may be magnified and only become detectable in the presence of certain exposures. This gene-environment interaction might explain heterogeneity in studies associating SHBG gene variants and soy consumption with breast cancer risk in Far East population exposed to high isoflavone levels compared with populations with lower levels..

Long-term endocrine therapy with either aromatase inhibitors (AIs) or tamoxifen may lead to endocrine resistance and disease progression. Recent years have seen advances in our understanding of the complex biological mechanisms associated with resistance. Growth factor signaling pathways appear to be upregulated in hormone-resistant tumours and interact with oestrogen-receptor (ER) signaling, which remains functional even after long-term endocrine deprivation. Signaling through the human epidermal and insulin-like growth-factor receptor (HER and IGFR, respectively) pathways may promote ligand-independent ER gene transcription and stimulate growth factor signaling. Therapeutic agents that inhibit these signal transduction pathways, when combined with AIs, may offer breast cancer patients new hope for more robust, longer-term remissions. Preliminary data from phase II studies of combination therapies are encouraging. There is a large programme of ongoing randomised, controlled trials, the results of which should pave the way for integrating combination therapies into clinical practice. To identify which patients will respond best to particular combinations of treatments, biomarkers and gene expression profiles are being investigated as predictors of sensitivity or resistance. In time, breast cancer treatment will become truly individualised because physicians will be able to match patients with a variety of disease phenotypes to optimal combination therapies..

Background. Prospective studies have shown an association between increased serum levels of estradiol and testosterone and breast cancer risk in postmenopausal women. Raloxifene has been shown to reduce breast cancer risk more in women with high estradiol levels than in those with lower levels. The purpose of this study was to determine whether sex hormone levels were associated with breast cancer risk and with response to tamoxifen in a high-risk population. Methods: Using a case-cohort design, we studied 135 women with postmenopausal breast cancer and 275 postmenopausal women without breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project Cancer Prevention Trial (P-1) and who had been treated with tarnoxifen or placebo for 69 months. We measured estradiol, testosterone, and sex hormone-binding globulin by using radioimmunoassay in baseline plasma samples. Relative risks (RRs) and 95% confidence intervals (CIs) for invasive breast cancer were estimated for each quartile of sex hormone level using Cox proportional hazards models. All statistical tests were two-sided. Results: Median plasma levels of estradiol, testosterone, and sex hormone-binding globulin were similar between the case and cohort groups. The relative risk of breast cancer for. women in the placebo group was not associated with sex hormone levels (risk of estrogen receptor-positive breast cancer in women by quartile of estradiol: Q1 [lowest], RR = 1.0; Q2, RR = 1.16, 95% CI = 0.49 to 2.7; Q3, RR = 1.08, 95% CI = 0.45 to 2.61; and Q4, RR = 1.29, 95% C1 = 0.59 to 2.82). The reduced risk of invasive breast cancer in tamoxifen-treated women compared with placebo-treated women was not associated with sex hormone levels. Conclusions: These data do not support the use of endogenous sex hormone levels to identify women who are at particularly high risk of breast cancer and who are most likely to benefit from chernoprevention with tamoxifen..

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552 Background: Letrozole (L) is a more potent aromatase inhibitor in vitro than anastrozole (A). One study in 12 patients showed that in patients L provides more complete inhibition of whole body aromatase and suppression of estrone sulphate levels than A but L did not significantly suppress E2 levels more than A possibly because of the small size of the study and the difficulty of measuring E2 in postmenopausal women (Geisler et al, JCO, 20, 751, 2002). The current aim was to conduct a much larger study to determine whether 2.5mg L suppresses E2 significantly more than 1mg A and if so in what proportion of patients this is the case. METHODS: 54 postmenopausal women with invasive estrogen receptor positive breast cancer were randomised as part of their adjuvant hormone therapy to receive either: 12 weeks of L followed by 12 weeks of A (L→A) or 12 weeks of A followed by 12 weeks of L (A→L). Blood for hormones were taken at the same time of the day before and after 12 weeks of each drug. E2 was measured by a highly sensitive radioimmunoassay (Dowsett et al, Cancer Res 1987;47:1957-61) with a formal detection limit of 3pmol/l. In this study we also quantified values by extrapolation below this limit. RESULTS: 27 patients had L→A and 27 A→L. Baseline E2 levels varied from 3 to 91 pmol/l with a median of 26 pmol/l. Only 1of 54 (2%) patients had an E2 value ≥ 3pmol/l after L compared with 20 of 54 (37%) after A(p <0.000005). After extrapolation, mean E2 level after A was 2.91 (SEM 0.18) pmol/l with a median of 2.70 pmol/l and after L was 1.76 (SEM 0.10) pmol/l with a median of 1.70 pmol/l (p<0.0001). Mean residual estradiol was 9.2% of baseline with A and 5.6% with L. CONCLUSIONS: This study has demonstrated unequivocally that the more complete inhibition of aromatase achieved by 2.5mg of letrozole than 1mg of anastrozole results in a greater degree of suppression of E2, the most bioactive oestrogen. [Table: see text]..

1014 Background: Aromatase inhibitors (AIs) are important agents for the treatment of hormone receptor positive breast cancer in post menopausal women. They are being assessed as chemopreventives of breast cancer in large clinical trials, but there are known issues of tolerability notably affecting bone. It is possible that decreasing, but not abolishing, circulating estradiol levels may reduce breast cancer risk without such side effects. We have undertaken a novel pilot study to establish whether healthy volunteers on an AI could be delivered very low dose estradiol replacement to a predictable serum level of 10-20 pmol/L using a transdermal patch, and whether this would reverse changes in the bone resorption biomarker CTx. METHODS: Ten healthy postmenopausal women with normal estradiol levels received letrozole 2.5mg/day for 16 weeks. From weeks 6-16 they were also given a quarter of Estraderm MX25 transdermal patch (6.25 μg of estradiol) replaced, twice weekly. Fasting blood samples were taken at baseline, 6, 10, 12, 14 and 16 weeks for estradiol, FSH, LH, SHBG and CTx. RESULTS: Serum estradiol levels fell to <3pmol/L at 6 weeks consistent with AI therapy. The quarter patch successfully replaced estradiol levels to a median of 12pmol/L although there was marked inter and intrasubject variability. FSH rose significantly with AI therapy with no appreciable change in SHBG or LH. There is encouraging evidence that CTx levels which increased with AI (p=0.076) can be corrected with low dose estradiol replacement although this did not reach statistical significance in this initial group. We continue to collect data on a further 8 subjects already recruited into the study. CONCLUSIONS: A quarter of Estraderm MX25 transdermal patch can be used to provide a serum estradiol level of 10-20 pmol/l in postmenopausal women given an AI. Further data will confirm whether this novel approach to chemoprevention can reverse the negative impact of AIs on bone metabolism and be suitable for extending to a full chemoprevention trial. [Table: see text] No significant financial relationships to disclose..

515 Background: Gefitinib, an EGFR-tyrosine kinase inhibitor, reduces breast cancer cell growth and potentiates endocrine therapy in model systems. This double-blind multicentre study compared anastrozole 1 mg/day alone with anastrozole + gefitinib 250 mg/day as neoadjuvant therapy for breast cancer in a novel design aiming to assess additional benefit from gefitinib in individual patients. METHODS: Postmenopausal women with stage I-IIIB breast cancer and ER and/or PgR+ tumours received anastrozole for 16 wks and were randomised (2:5:5 ratio) to: combination with gefitinib for 16 wks (AG); placebo for 2 wks then gefitinib for 14 wks (A:AG, to test for additional Ki67 suppression); placebo for 16 wks (A alone). Biopsies were taken at baseline, 2 and 16 wks. Primary comparison was change in Ki67 by 16 wks. Secondary comparison was objective tumour response rate (ORR) using UICC/WHO criteria at 16 wks. RESULTS: 206 patients (pts) were randomised: (31 AG, 90 A:AG, 85 A alone); demography was well balanced between the groups. 109 pts were evaluable for Ki67: 59 AG + A:AG; 50 A alone. Change in Ki67 levels at 16 wks was not significantly different in those pts who received gefitinib + anastrozole versus anastrozole alone (p=0.257). The addition of gefitinib after 2 weeks of anastrozole did not further suppress Ki67 levels (p=0.164). 188 pts were evaluable for ORR (109 AG + A:AG; 79 A alone). The ORR was 48% in pts who received gefitinib + anastrozole and 61% in pts treated with anastrozole alone (p=0.067). CONCLUSIONS: Neither the biological nor the clinical activity of anastrozole was enhanced by the addition of gefitinib; although non-significant, both endpoints unexpectedly suggested a trend against the combination in this patient population. Molecular investigations of signal transduction pathways are underway to understand the significance of these findings. [Table: see text] [Table: see text]..

INTRODUCTION: The aim of this study was to examine the effect of the cellular composition of biopsies on the error rates of multigene predictors of response of breast tumours to neoadjuvant adriamycin and cyclophosphamide (AC) chemotherapy. MATERIALS AND METHODS: Core biopsies were taken from primary breast tumours of 43 patients prior to AC, and subsequent clinical response was recorded. Post-chemotherapy (day 21) samples were available for 16 of these samples. Frozen sections of each core were used to estimate the proportion of invasive cancer and other tissue components at three levels. Transcriptional profiling was performed using a cDNA array containing 4,600 elements. RESULTS: Twenty-three (53%) patients demonstrated a 'good' and 20 (47%) a 'poor' clinical response. The percentage invasive tumour in core biopsies collected from these patients varied markedly. Despite this, agglomerative clustering of sample expression profiles showed that almost all biopsies from the same tumour aggregated as nearest neighbours. SAM (significance analysis of microarrays) regression analysis identified 144 genes which distinguished high- and low-percentage invasive tumour biopsies at a false discovery rate of not more than 5%. The misclassification error of prediction of clinical response using microarray data from pre-treatment biopsies (on leave-one-out cross-validation) was 28%. When prediction was performed on subsets of samples which were more homogeneous in their proportions of malignant and stromal cells, the misclassification error was considerably lower (8%-13%, p < 0.05 on permutation). CONCLUSION: The non-tumour content of breast cancer samples has a significant effect on gene expression profiles. Consideration of this factor improves accuracy of response prediction by expression array profiling. Future gene expression array prediction studies should be planned taking this into account..

Introduction Biological markers that reliably predict clinical or pathological response to primary systemic therapy early during a course of chemotherapy may have considerable clinical potential. This study evaluated changes in Ki-67 labeling index and apoptotic index ( AI) before, during, and after neoadjuvant anthracycline chemotherapy.Methods Twenty-seven patients receiving neoadjuvant FEC (5-fluorouracil, epirubicin, and cyclophosphamide) chemotherapy for operable breast cancer underwent repeat core biopsy after 21 days of treatment. Tissue from pre-treatment biopsy, day 21 and surgery was analysed for Ki-67 index and AI.Results The objective clinical response rate was 56%. Eight patients (31%) achieved a pathological response by histological criteria; two patients had a near-complete pathological response. A reduction in Ki-67 index was observed in 68% of patients at day 21 and 72% at surgery; Ki-67 index increased between day 21 and surgery in 54%. AI decreased in 50% of tumours by day 21, increased in 45% and was unchanged in one patient; 56% demonstrated rebound increases in AI by the time of surgery. Neither pre-treatment nor post-chemotherapy median Ki-67 index nor median AI at all three time points or relative changes at day 21 and surgery differed significantly between clinical or pathological responders and non-responders. Clinical responders had lower median Ki-67 indices at day 21 (11.4% versus 27.0%, p = 0.02) and significantly greater percentage reductions in Ki-67 at day 21 than did non-responders (- 50.6% versus - 5.3%, p = 0.04). The median day-21 Ki-67 was higher in pathological responders (30.3% versus 14.1%, p = 0.046). A trend toward increased AI at day 21 in pathological responders was observed (5.30 versus 1.68, p = 0.12). Increased day-21 AI was a statistically significant predictor of pathological response ( p = 0.049). A strong trend for predicting pathological response was seen with higher Ki-67 indices at day 21 and AI at surgery ( p = 0.06 and 0.06, respectively).Conclusion The clinical utility of early changes in biological marker expression during chemotherapy remains unclear. Until further prospectively validated evidence confirming the reliability of predictive markers is available, clinical decision-making should not be based upon individual biological tumour marker profiles..

Cross-sectional studies investigating the relationship between phytoestrogens in diet, urine, or blood with plasma estradiol and sex hormone binding globulin (SHBG) have been inconclusive. We investigated the relationship among phytoestrogen exposure, polymorphisms in the ESR1, COMT, CYP19, and SHBG genes, and plasma estradiol and SHBG levels in 125 free-living postmenopausal women taking part in a cohort study (European Prospective Investigation of Cancer and Nutrition-Norfolk) using three different markers: dietary, urinary, and serum phytoestrogens. Phytoestrogen levels (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) in spot urine and serum were analyzed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, respectively. Plasma estradiol and SHBG were measured by immunoassays. Adjusting for age and body mass index, urinary daidzein, genistein, glycitein, and serum daidzein and glycitein were negatively correlated with plasma estradiol (R = -0.199 to -0.277, P < 0.03), with particularly strong associations found in the 18 women with CC crenoty f genotype or ESRI PuvII polymorphism (R = -0.597 to -834, P < 0.03). The negative correlations observed between isoflavones and estradiol in women as a whole became no longer significant when we excluded women with ESR1 PuvII CC genotype, indicating that the correlations observed %vere due mainly to this group of women. There Was no relationship between dietary isoflavones and plasma estradiol and no association was found between any of the dietary, urinary, and serum phytoestrogen and plasma SHBG or between these factors and polymorphisms in CYP19, SHBG, and COMT. We conclude that higher isoflavone exposure is associated with lower plasma estradiol in postmenopausal women and that this preliminary study is suggestive of the indvolvement of diet-gene interactions..

PURPOSE: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined With Tamoxifen (IMPACT) trial was designed to test the hypothesis that the clinical and/or biologic effects of neoadjuvant tamoxifen compared with anastrozole and with the combination of tamoxifen and anastrozole before surgery in postmenopausal women with estrogen receptor (ER) -positive, invasive, nonmetastatic breast cancer might predict for outcome in the Arimidex, Tamoxifen Alone or in Combination (ATAC) adjuvant therapy trial. PATIENTS AND METHODS: Postmenopausal women with ER-positive, invasive, nonmetastatic, and operable or locally advanced potentially operable breast cancer were randomly assigned to neoadjuvant tamoxifen (20 mg daily), anastrozole (1 mg daily), or a combination of tamoxifen and anastrozole for 3 months. The tumor objective response (OR) was assessed by both caliper and ultrasound. Comparisons were also made of clinical response with ultrasound response, actual and feasible surgery with feasible surgery at baseline, OR in human epidermal growth factor receptor 2 (HER2)-positive cancers, and tolerability. RESULTS: There were no significant differences in OR in the intent-to-treat population between patients receiving tamoxifen, anastrozole, or the combination. In patients who were assessed as requiring mastectomy at baseline (n = 124), 44% of patients received breast-conserving surgery (BCS) after anastrozole compared with 31% of patients after tamoxifen (P = .23); this difference became significant for patients who were deemed feasible for BCS by their surgeon (46% v 22%, respectively; P = .03). The OR for patients with HER2-positive cancer (n = 34) was 58% for anastrozole compared with 22% for tamoxifen (P = .18). All treatments were well tolerated. CONCLUSION: Neoadjuvant anastrozole is as effective and well tolerated as tamoxifen in ER-positive operable breast cancer in postmenopausal women, but the hypothesis that clinical outcome might predict for long-term outcome in adjuvant therapy was not fulfilled..

Molecular markers have been extensively investigated with a view to providing early and accurate information on long-term outcome and prediction of response to treatment of early breast cancer. Proliferation is a key feature of the progression of tumors and is now widely estimated by the immunohistochemical assessment of the nuclear antigen Ki-67. The expression of Ki-67 correlates with other measurements of proliferation, including S-phase and bromodeoxyuridine uptake. High Ki-67 is a sign of poor prognosis associated with a good chance of clinical response to chemotherapy, but its independent significance is modest and does not merit measurements in most routine clinical scenarios. However, its application as a pharmacodynamic intermediate marker of the effectiveness of medical therapy holds great promise for rapid evaluation of new drugs..

The standard adjuvant endocrine treatment for postmenopausal women with hormone-receptor-positive localised breast cancer is 5 years of tamoxifen, but recurrences and side-effects restrict its usefulness. The aromatase inhibitor anastrozole was compared with tamoxifen for 5 years in 9366 postmenopausal women with localised breast cancer. After a median follow-up of 68 months, anastrozole significantly prolonged disease-free survival (575 events with anastrozole vs 651 with tamoxifen, hazard ratio 0.87, 95% CI 0.78-0.97, p=0.01) and time-to-recurrence (402 vs 498, 0.79, 0.70-0.90, p=0.0005), and significantly reduced distant metastases (324 vs 375, 0.86, 0.74-0.99, p=0.04) and contralateral breast cancers (35 vs 59, 42% reduction, 12-62, p=0.01). Almost all patients have completed their scheduled treatment, and fewer withdrawals occurred with anastrozole than with tamoxifen. Anastrozole was also associated with fewer side-effects than tamoxifen, especially gynaecological problems and vascular events, but arthralgia. and fractures were increased. Anastrozole should be the preferred initial treatment for postmenopausal women with localised hormone-receptor-positive breast cancer..

There is an increasing rationale for effective combinations of endocrine therapy with novel drugs that target aberrant signal transduction pathways in estrogen receptor (ER) positive breast cancer. Prolonged endocrine therapy can be associated with an acquired increase in peptide growth factor signaling (EGFR, HER2), together with cross-talk activation of ER-dependent gene transcription and cell growth that leads to endocrine resistance. Current approaches to target these pathways include both the selective ER downregulator fulvestrant, and various signal transduction inhibitors (STIs). Fulvestrant can overcome resistance to tamoxifen (TAM-R) and long-term estrogen deprivation (LTED-R) in experimental models by reducing ER expression, and represents a current option for post-menopausal women with endocrine resistant ER+ve breast cancer. Emerging data suggest that fulvestrant's effect may be greater when combined with estrogen deprivation, and several phase III trials are assessing fulvestrant combined with aromatase inhibitors (AIs). Small molecule STIs such as tyrosine kinase inhibitors (TKIs), farnesyltransferase inhibitors (FTIs) and mTOR antagonists are also active in breast cancer. Pre-clinical data suggest that combined endocrine/STI therapy may result in greater growth inhibition than either therapy alone, and thus delay emergence of resistance. Several clinical trials are now examining STIs combined with AIs both in the tamoxifen-resistant and first-line advanced breast cancer setting, while pre-surgical studies are investigating the efficacy of combined endocrine/STI therapy utilising biological primary endpoints. This article reviews the pre-clinical rationale for this strategy and the clinical trials in this area..

Aromatase inhibitors are rapidly becoming the first choice for hormonal treatment of steroid receptor positive breast cancer in postmenopausal women. An understanding of the resistance mechanisms to these agents is, therefore, important for the appropriate delivery of treatment to responsive patients and the rational development of new agents targeted at the resistance pathways. De novo resistance appears to be a quantitative rather than qualitative phenomenon with virtually all oestrogen receptor positive tumours showing an anti-proliferative response to the aromatase inhibitor anastrozole. While the expression of type 1 growth factor receptors reduces response to tamoxifen this appears to have little detrimental effect on response to aromatase inhibitors. Studies of acquired resistance in vitro have indicated that acquisition of hypersensitivity to oestrogenic stimulation is a key mechanism that is dependent on enhanced cross-talk of growth factor and oestrogen signaling pathways. Collection of resistant biopsy tissues from patients is important to determine if this mechanism is clinically relevant..

The knowledge that steroids play a pivotal role in the development of breast cancer has been exploited clinically by the development of endocrine treatments. These have sought to perturb the steroid hormone environment of the tumour cells, predominately by withdrawal or antagonism of oestrogen. Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumours to circumvent the need for steroid hormones, whilst in most cases, retaining the nuclear steroid receptors. The mechanisms involved in resistance to estrogen deprivation are of major clinical relevance for optimal treatment of breast cancer patients and the development of new therapeutic regimes. We have shown that long-term culture of MCF7 cells in medium depleted of oestrogen (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of both ERalpha phosphorylated on Ser(118) and ERK1/ERK2. Our data suggest elevated ERK1/ERK2 activity results wholly or in part from enhanced ERBB2 expression in the LTED cells. These cells showed greater sensitivity to the tyrosine kinase inhibitor ZD1839 in both ERalpha-mediated transcription and growth assays compared with the wt-MCF7. Similarly the MEK inhibitor U0126 decreased basal ERalpha-mediated transcription and proliferation in the LTED cells by 50% and reduced their sensitivity to the proliferative effects of E2 10-fold, whilst having no effect on the wild type (wt). However, complete suppression of ERK1/ERK2 activity in the LTED cells did not inhibit ERalpha Ser(118) phosphorylation suggesting that the cells remained ligand-dependent. This was further confirmed by the increased sensitivity of the LTED cells to the growth suppressive effects of ICI 182,780 and suggested that the LTED cells remained wholly or partially dependent on oestrogen receptor (ER)/oestrogen responsive elements directed growth. These findings suggest that treatments targeted at growth factor signalling pathways may be useful in patients acquiring resistance to oestrogen deprivation with aromatase inhibitors and that the pure anti-oestrogen ICI 182,780 may also be effective by blocking or destabilizing ER and hence disrupting cross-talk..

De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors..

The ratio of estrogen receptor beta (ER beta) to ER alpha can alter the estrogen-like properties of tamoxifen. Transient transfection of ER beta cDNA into cells can decrease the estrogen-like properties of the ER alpha:tamoxifen complex, whereas an increase in the amount of ER beta is associated with tamoxifen-resistant breast cancer. We have addressed each of these hypotheses by examining well characterized laboratory models. We determined whether changes in endogenous ER beta are responsible for the estrogen-like or antiestrogenic properties of tamoxifen or raloxifene in MDA-MB-231 cells transfected with cDNAs for ER alpha or mutants D351G, D351Y. We found that the amount of ER beta inRNA in separate, stable transfectants of mutant ERa cDNA was always < 2% of ERa. Since at least a 50:50 mixture of ER alpha:ER beta is needed to silence the tamoxifen:ER alpha complex, we conclude that insufficient ER beta mRNA is available for selective ER modulation in stable transfectants of D351G and D351Y ER alpha. Similarly, to test the hypothesis that ER beta is up-regulated and plays an important role during the development of tamoxifen-stimulated tumor growth, we quantitatively analyzed ER beta and ER alpha mRNA ill tamoxifen-naive (MCF-7:E-2, ECC1:E-2) and tamoxifen-stimulated tumors (MCF-7:TAM, EnCa 101:TAM). We found that ER beta mRNA levels were not significantly elevated in tamoxifen-stimulated tumors and the ER alpha inRNA remained over 99% out of all ER species for all the tumors tested. The same results were also obtained when mRNA levels of ER beta and ER alpha in a series of tamoxifen-naive and tamoxifen-resistant breast cancer was analyzed. We conclude that endogenous ER beta may not play a dominant role in the modulation of the tarnoxifen ER alpha complex, or in the development of tamoxifen-stimulated resistant minor growth..

PURPOSE: To investigate the relationships between biomarker changes in breast cancer during neoadjuvant (preoperative) endocrine therapy. PATIENTS AND METHODS: The IMPACT trial compared the preoperative use of tamoxifen with anastrozole alone or in combination in postmenopausal women (n = 330) with primary breast cancer. Biomarkers were measured in tumor biopsy specimens taken at baseline, and after 2 and 12 weeks of treatment. RESULTS: 52 (93%) of 56, 46 (85%) of 54, and 37 (84%) of 44 patients in the anastrozole, tamoxifen, and combination groups, respectively. There was a significantly greater suppression of Ki67 in the anastrozole-treated group than in the tamoxifen- or combination-treated groups, which is parallel to the greater efficacy seen for anastrozole over these two treatments in the Arimidex, Tamoxifen, Alone or in Combination adjuvant trial. A positive relationship was noted between estrogen-receptor level and Ki67 suppression in all patients. Ki67 was reduced to a greater extent in progesterone receptor-positive tumors compared with progesterone receptor-negative tumors. HER-2-negative tumors tended to show a greater reduction in Ki67 compared with HER-2-positive tumors, but the difference was only significant in the tamoxifen group after 2 weeks, and in the anastrozole group after 12 weeks. CONCLUSION: These results confirm the value of Ki67 as a molecular marker, and provide information regarding the relationships between treatment-induced changes in Ki67 and other important biomarkers. Studies such as this should help integrate agents targeted at growth factor signaling with endocrine agents in breast cancer..

BACKGROUND: Estrogen has beneficial effects on lipid metabolism and bone preservation. The IMPACT trial evaluated neoadjuvant therapy with anastrozole or tamoxifen alone, or a combination. The comparative effects of these treatments on serum lipids and bone resorption were assessed. PATIENTS AND METHODS: Non-fasting clotted blood samples were taken from 176 postmenopausal patients at baseline, 2 and 12 weeks for assessment of serum levels of estradiol, the bone resorption marker CTx and lipid profiles [total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and non-HDL cholesterol (N-HDL-C)]. RESULTS: After 12 weeks, tamoxifen was associated with a significant increase in HDL-C (26.5%), and a decrease in TC (6.5%) and N-HDL-C (12.3%). Anastrozole was associated with a significant increase in HDL-C (11.2%), and a non-significant increase in TC (2.9%) and N-HDL-C (3.4%), both of which were significantly different from tamoxifen. The combination was associated with a significant increase in HDL-C (9.4%), and a decrease in TC (10.9%) and N-HDL-C (13.9%). For tamoxifen and the combination, there were non-significant decreases in CTx compared with a significant increase (45.6%) with anastrozole. No correlation between serum estradiol and CTx was seen in any of the treatment groups. CONCLUSION: Anastrozole did not have a detrimental effect on lipid profiles following 3 months of therapy. There was a significant increase in CTx with anastrozole in contrast to tamoxifen..

PurposeTo evaluate growth factor receptor cross talk with the estrogen receptor (ER) in paired clinical breast cancer specimens and in a xenograft model before tamoxifen and at tumor progression as a possible mechanism for tamoxifen resistance.MethodsSpecimen pairs from 39 patients were tissue arrayed and stained for ER, progesterone receptor (PgR), Bcl-2, c-ErbB2 (HER-2), and phosphorylated (p) p38 mitogen-activated protein kinase (MAPK), p-ERK1/2 MAPK, and p-Akt. Xenograft MCF-7 tumors before and after tamoxifen resistance were assessed for levels of p-p38.ResultsPretreatment, there were strong correlations between ER, PgR, and Bcl-2, and an inverse correlation between ER and HER-2. These correlations were lost in the tamoxifen-resistant tumors and replaced by strong correlations between ER and p-p38 and p-ERK. ER expression was lost in 17% of resistant tumors. Three (11%) of the 26 tumors originally negative for HER-2 became amplified and/or overexpressed at resistance. All ER-positive tumors that overexpressed HER-2 originally or at resistance expressed high levels of p-p38. In the pretreatment and tamoxifen-resistant specimens, there were strong correlations between p-p38 and p-ERK. In the tamoxifen-resistant xenograft tumors, like the clinical samples, there was a striking increase in p-p38.ConclusionThe molecular pathways driving tumor growth can change as the tumor progresses. Crosstalk between ER, HER-2, p38, and ERK may contribute to tamoxifen resistance and may provide molecular targets to overcome this resistance..

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Phytoestrogens have been hypothesized to protect against prostate cancer via modulation of circulating androgen concentrations. We conducted a cross-sectional study of 267 men in the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort with 2 aims: first, to investigate the association between phytoestrogen exposure (measured from diet, urine, and serum) and plasma concentrations of sex hormone-binding globulin (SHBG), androstanediol glucuronide, testosterone and Free Androgen Index (FAI); and second, whether the association may be modified by polymorphisms in CYP19 and SHBG genes. Dietary daidzein and genistein intakes were obtained from food diaries and computed using an in-house food composition database. Urinary and serum concentrations of 3 isoflavones (daidzein, genistein, glycitein), 2 daidzein metabolites O-desmethylangolensin (O-DMA) and 2 lignan metabolites (enterodiol and enterolactone) were measured using mass spectrometry. There was no association between dietary, urinary, and serum phytoestrogens and plasma SHBG concentrations. Enterolactone was positively associated with plasma androstanediol glucuronide concentrations (urinary enterolactone: r = 0.127, P = 0.043; serum enterolactone: r = 0.172, P = 0.006) and FAI (urinary enterolactone: r = 0.115 P = 0.067; serum enterolactone: r = 0.158, P = 0.011). Both urinary and serum equol were associated with plasma testosterone (urinary equol: r = 0.332, P = 0.013; serum equol: r = 0.318, P = 0.018) and FAI (urinary equol: r = 0.297, P 0.027; serum equol: r = 0.380, P = 0.004) among men with the TT genotype but not the CC or CT genotypes (r = -0.029 to -0.134, P = 0.091 - 0.717) for the CYP19 3'untranslated region (UTR) T-C polymorphism. Urinary and serum enterolactone showed similar genotype-dependent associations with testosterone but not with FAI. In this first study on phytoestrogen-gene associations in men, we conclude that enterolactone and equol are positively associated with plasma androgen concentrations, and interactions with CYP19 gene may be involved..

Recognition of the role of oestrogens in the stimulation of breast tumour growth has led to the development of several therapies based on endocrine intervention. Endocrine agents are currently used as a treatment for steroid receptor-positive breast cancer and more recently as a preventative measure in high-risk populations. Accurate quantification of resulting steroid hormonal profiles is essential to the understanding of the biological action and efficacy of these agents. In premenopausal women GnRH agonists suppress ovarian oestrogen synthesis and reduce oestradiol to close to postmenopausal levels. GnRH agonists used in combination with an aromatase inhibitor suppress levels even further. In contrast, tamoxifen can lead to markedly enhanced levels. In postmenopausal women aromatase inhibitors can achieve an almost complete inhibition of the aromatase enzyme..

Long-term culture of MCF-7 wild-type (wt) cells in steroid-depleted medium (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of ER alpha and enhanced growth factor signalling. In this study, we aimed to compare the effects of the pure anti-oestrogen ICI 182,780 (ICI) with the competitive anti-oestrogen tamoxifen (TAM) on oestrogen and IGF signalling in these cells. Wt MCF-7 and LTED cells were treated with a log 7 concentration range of E2, TAM or ICI. Effects on cell growth, ER alpha transactivation, expression of ER alpha, ER beta and components of the IGF pathway were measured with and without insulin. In the presence of insulin, growth of LTED cells was refractory to TAM but inhibited by ICI and E2. In the absence of insulin, LTED cells showed persistent hypersensitivity to E2, and remained inhibited by ICI but were largely unaffected by TAM. ICI but not TAM inhibited ER-mediated gene transcription and treatment with ICI resulted in a dose-dependent reduction in ER alpha levels whilst having no effect on ER beta expression. IGF-I receptor and insulin receptor substrate 2 levels were increased in LTED versus the Wt MCF-7 cells, and ICI but not TAM reduced their expression in a dose-dependent fashion. Thus IGF signalling as well as ER alpha expression and function are enhanced during LTED. While the resultant cells are resistant to TAM, ICI down-regulates ER alpha, reducing IGF signalling and cell growth. These results support the use of ICI in women with ER-positive breast cancer who have relapsed on an aromatase inhibitor..

This study compares the sensitivities and specificities of three techniques for the detection of circulating epithelial cells in the blood of patients with breast cancer. The number of circulating epithelial cells present in the blood of 40 patients with metastatic breast cancer and 20 healthy volunteers was determined by: immunomagnetic separation (IMS) and laser scanning cytometry (LSC), cell filtration and LSC and a multimarker real-time RT-PCR assay. Numbers of cytokeratin-positive cells identified and expression of three PCR markers were significantly higher in the blood of patients with breast cancer than in healthy volunteers. Using the upper 95% confidence interval of cells detected in controls to determine positive patient samples: 30% of patients with metastatic breast cancer were positive following cell filtration, 48% following IMS, and 60, 45 and 35% using real-time RT-PCR for cytokeratin 19, mammaglobin and prolactin-inducible peptide. Samples were significantly more likely to be positive for at least one PCR marker than by cell filtration (83 vs 30%, P<0.001) or IMS (83 vs 48%, P<0.001). The use of a multimarker real-time RT-PCR assay was therefore found to be the most sensitive technique for the detection of circulating epithelial cells in the blood of patients with breast cancer..

PURPOSE: Neoadjuvant (preoperative) therapy for breast cancer may allow for the development of intermediate markers of treatment benefit, thereby circumventing the need for efficacy trials of adjuvant therapy, which require much larger patient numbers and longer follow-up. The aim of this study--as part of the Immediate Preoperative "Arimidex" (anastrozole), Tamoxifen, or Arimidex Combined with Tamoxifen (IMPACT) trial (n = 330)--was to test the hypotheses that changes in Ki-67 after 2 weeks and/or 12 weeks: (i) differed between treatments, (ii) predicted clinical tumor response, and/or (iii) may predict long-term outcome differences between treatments in adjuvant therapy. EXPERIMENTAL DESIGN: The Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial compared these same agents in the adjuvant setting. Biomarkers were measured in biopsy specimens taken before and after 2 and 12 weeks of treatment. RESULTS: Suppression of the proliferation marker Ki-67 after 2 and 12 weeks was significantly greater with anastrozole than with tamoxifen (P = 0.004 and P < 0.001) but was similar between tamoxifen and the combination (P = 0.600 and P = 0.912). This result closely parallels that seen for the relative recurrence-free survival with the treatments after a median follow-up of 31 months in the ATAC trial in 9,366 patients. Against expectations, apoptosis was not increased in any of the treatment arms. CONCLUSIONS: The data indicate that short-term changes in proliferation in the neoadjuvant setting may be able to predict outcome during adjuvant use of the same treatments. If this can be confirmed, these findings could lead to a profound change in approaches to drug development in breast cancer. The data indicate that estrogen is not an important survival factor for human breast cancer cells..

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BACKGROUND:Trastuzumab, a recombinant monoclonal antibody against HER2, has clinical activity in advanced breast cancer that overexpresses HER2. We investigated its efficacy and safety after excision of early-stage breast cancer and completion of chemotherapy.METHODS:This international, multicenter, randomized trial compared one or two years of trastuzumab given every three weeks with observation in patients with HER2-positive and either node-negative or node-positive breast cancer who had completed locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy.RESULTS:Data were available for 1694 women randomly assigned to two years of treatment with trastuzumab, 1694 women assigned to one year of trastuzumab, and 1693 women assigned to observation. We report here the results only of treatment with trastuzumab for one year or observation. At the first planned interim analysis (median follow-up of one year), 347 events (recurrence of breast cancer, contralateral breast cancer, second nonbreast malignant disease, or death) were observed: 127 events in the trastuzumab group and 220 in the observation group. The unadjusted hazard ratio for an event in the trastuzumab group, as compared with the observation group, was 0.54 (95 percent confidence interval, 0.43 to 0.67; P<0.0001 by the log-rank test, crossing the interim analysis boundary), representing an absolute benefit in terms of disease-free survival at two years of 8.4 percentage points. Overall survival in the two groups was not significantly different (29 deaths with trastuzumab vs. 37 with observation). Severe cardiotoxicity developed in 0.5 percent of the women who were treated with trastuzumab.CONCLUSIONS:One year of treatment with trastuzumab after adjuvant chemotherapy significantly improves disease-free survival among women with HER2-positive breast cancer..

PURPOSE: Arimidex, tamoxifen alone, or in combination (ATAC) trial of anastrozole (Arimidex) versus tamoxifen or a combination of the two in 9,366 postmenopausal patients with primary breast cancer found a significant improvement in disease-free survival and time to recurrence (TTR) for anastrozole compared with tamoxifen, that was restricted to patients with hormone receptor-positive (ie, estrogen receptor-positive [ER+] and/or progesterone receptor-positive [PgR+]) disease, the target population for these therapies. We retrospectively tested the hypothesis that this benefit might differ according to PgR status. PATIENTS AND METHODS: TTR was compared between the three treatment groups for subgroups defined by ER and PgR status using Cox's proportional hazards model, with and without adjustment for baseline variables. RESULTS: The unadjusted hazard ratio (HR) for anastrozole versus tamoxifen for TTR was 0.74 (95% CI, 0.64 to 0.87) for women with either ER+ or PgR+ tumors. In the ER+/PgR+ subgroup (n = 3,834) the HR was 0.84 (95% CI, 0.69 to 1.02) compared with 0.43 (95% CI, 0.31 to 0.61) in the ER+/PgR-negative (PgR-) subgroup (n = 880). In the adjusted model the HRs were 0.83 and 0.45, respectively. CONCLUSION: Time to recurrence was longer for anastrozole- than tamoxifen-treated patients in both ER+/PgR+ and ER+/PgR- subgroups, but the benefit was substantially greater in the PgR- subgroup. As this was an "exploratory" analysis, this effect should be considered as hypothesis generating and assessed prospectively in other trials comparing the adjuvant use of an aromatase inhibitor with tamoxifen..

The third-generation non-steroidal aromatase inhibitors (AIs) are challenging tamoxifen as treatments of choice for early and advanced breast cancer in postmenopausal women with estrogen receptor (ER)-positive disease. However, patients who initially respond to AIs eventually develop resistance to treatment and experience disease progression. To establish the optimal endocrine therapy following AI resistance, it is essential to understand the mechanisms that contribute to the loss of response. Data from in vitro models have suggested that acquired AI resistance is due to enhanced sensitization to low estrogen levels during long-term estrogen deprivation (LTED). Cross-talk between the ER and various growth-factor-receptor signaling pathways, including human epidermal growth factor receptor 2, and the insulin-like growth factor pathway, may also be implicated. Therefore, endocrine therapies that abolish estrogen signaling via removal of the ER could be effective in patients with AI-resistant disease. Fulvestrant ('Faslodex') is a new ER antagonist with no agonist effects that binds, blocks and degrades the ER. Due to its unique mode of action and lack of cross-resistance with existing treatments, fulvestrant is an effective therapeutic agent for use in sequential endocrine regimens. Fulvestrant has established efficacy in tamoxifen-resistant disease and there is a growing body of evidence demonstrating its efficacy in patients with AI-resistant disease. In preclinical models, MCF-7 cells undergoing LTED are refractory to tamoxifen but sensitive to fulvestrant, suggesting fulvestrant is a more appropriate choice following AI resistance. The steroidal AI, exemestane is also an option in non-steroidal AI-resistant disease. Clinical trials are underway to compare fulvestrant with exemestane as an appropriate therapy following the onset of AI resistance..

The determination of plasma oestradiol has numerous applications in epidemiology, reproductive medicine and breast cancer management. Commercially available analytical methods, which measure the hormone levels without prior purification, have been successfully developed for measuring oestradiol in premenopausal women. The application of these methodologies to the quantification of the very low levels of oestradiol in postmenopausal women is more problematic in terms of accuracy and interpretation. The importance of using appropriate methodology is discussed and illustrated with data demonstrating the disparity in the results obtained when low levels of oestradiol were quantified using direct and indirect methods..

Understanding the underlying mechanisms responsible for endocrine resistance remains a challenge in improving the treatment of breast cancer. The discovery that growth factor and estrogen receptor (ER) signaling pathways interact in endocrine resistant breast cancer has provided a rationale for disrupting these signaling cascades in ER-positive, endocrine-resistant tumors. In postmenopausal women, the ER signaling pathway may be targeted using fulvestrant ('Faslodex'), a new type of ER antagonist with no agonist effects. Fulvestrant binds, blocks and causes degradation of the ER, culminating in complete abrogation of estrogen-sensitive gene transcription. This unique mechanism of action may result in a lack of cross-resistance with other endocrine agents. Preclinical studies have confirmed the potential of fulvestrant to inhibit the growth of tamoxifen-resistant, as well as tamoxifen-sensitive, human breast cancer cell lines. Clinical studies have demonstrated that fulvestrant is an effective treatment option in postmenopausal women with advanced breast cancer who have progressed on prior endocrine therapy. Furthermore, preclinical studies indicate that combining fulvestrant with growth factor targeted agents, such as the epidermal growth factor receptor (EGFR/HER1) tyrosine kinase inhibitor gefitinib (IRESSA) or the anti-human HER2 monoclonal antibody trastuzumab ('Herceptin'), may result in greater anti-tumor activity than either agent alone. A range of clinical trials are now ongoing to determine whether the combination of growth factor-targeting agents with fulvestrant will delay the onset of endocrine resistance and so provide new strategy for women with hormone receptor-positive advanced breast cancer..

Introduction Breast Cancer Prevention Trial (BCPT) and Multiple Outcomes of Raloxifene ( MORE) data have been interpreted to indicate that tamoxifen reduces the risk of ER+ but not ER- breast carcinogenesis. We explored whether these data also support an alternative hypothesis, that tamoxifen influences the natural history of both ER+ and ER- cancers, that it may be equally effective in abrogating or delaying ER- and ER+ carcinogenesis, and place selection pressure, in some cases, for the outgrowth of ER- cancers.Methods BCPT and MORE data were used to investigate whether: first, tamoxifen could reduce equally the emergence of ER- and ER+ tumors; and second, tamoxifen could select a fraction of emerging ER+ cancers and promote their transformation to ER- cancers. Assuming that some proportion, Z, of ER+ tumors becomes ER- after tamoxifen exposure and that the risk reduction for both ER- and ER+ tumors is equal, we solved for both the transformation rate and the risk reduction rate.Results If tamoxifen equally reduces the incidence of ER+ and ER- tumors by 60%, the BCPT results are achieved with a transformation of approximately Z = 20% of ER+ to ER- tumors. Validation with MORE data using an equal risk reduction of 60% associated with tamoxifen produces an almost identical transformation rate Z of 23%.Conclusion Data support an alternative hypothesis that tamoxifen may promote ER- carcinogenesis from a precursor lesion that would otherwise have developed as ER+ without tamoxifen selection..

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By use of modern immunological and molecular analytical techniques, cells with the characteristics of tumour cells can be detected in the blood of many patients with breast cancer. The ability to detect and characterise such cells routinely could have a profound influence on the early diagnosis of breast cancer, risk stratification in the adjuvant setting, early detection of relapse, and the development of new targeted strategies. In this review we discuss current techniques to detect circulating breast-cancer cells and the limitations of these approaches. We also review the clinical studies in breast cancer and discuss the potential relevance of this research to the future management of the disorder..

Background: Isoflavone phytoestrogen therapy has been proposed as a natural alternative to hormone replacement therapy (HRT). HRT has a beneficial effect on bone, but few trials in humans have investigated the effects of isoflavones on bone.Objective: The objective of the study was to determine the effect on bone density of a red clover-derived isoflavone supplement that provided a daily dose of 26 mg biochanin A, 16 mg formononetin, I mg genistein, and 0.5 mg daidzein for 1 y. Effects on biochemical markers of bone turnover and body composition were also studied.Design: Women aged 49-65 y (n = 205) were enrolled in a double-blind, randomized, placebo-controlled trial; 177 completed the trial. Bone density, body composition, bone turnover markers, and diet were measured at baseline and after 12 mo.Results: Loss of lumbar spine bone mineral content and bone mineral density was significantly (P = 0.04 and P = 0.03, respectively) lower in the women taking the isoflavone supplement than in those taking the placebo. There were no significant treatment effects on hip bone mineral content or bone mineral density, markers of bone resorption, or body composition, but bone formation markers were significantly increased (P = 0.04 and P = 0.01 for bone-specific alkaline phosphatase and N-propeptide of collagen type 1, respectively) in the intervention group compared with placebo in postmenopausal women. Interactions between treatment group and menopausal status with respect to changes in other outcomes were not significant.Conclusion: These data suggest that, through attenuation of bone loss, isoflavones have a potentially protective effect on the lumbar spine in women..

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This paper serves to update previously published guidance on rationale and methodology for HER2 laboratory testing following the recommendation for the use of HER2 targeted treatment in the management of advanced breast cancer in the UK. Emphasis is placed on the standardisation of methodology and assessment and strategies to achieve high quality performance. A two phase testing algorithm based on first line immunocytochemistry evaluation and second line fluorescence in situ hybridisation assessment of borderline cases is recommended. To ensure maintenance of expertise, an annual caseload volume of at least 250 cases is recommended for laboratories providing a testing service..

HER-2 is over-expressed in around 25% of human breast cancers, and is associated with poor outcome. We examined the incidence of HER-2 status in inflammatory breast cancer (IBC). Forty-nine newly diagnosed IBCs were studied. Formalin-fixed paraffin-embedded pre-treatment tissue biopsies were examined immunohistochemically for the over-expression of the HER-2 protein and gene using the HercepTest and FISH assay. Clinical outcome was compared between the HER-2 positive (HercepTest score 3 + and FISH positive) and negative groups. Fifty-two per cent of the IBCs examined were HER-2 positive. The HER-2 positive group were demographically comparable to the HER-2 negative group. Ninety-six per cent of the HER-2 positive patients responded to primary chemotherapy compared to 76% of the HER-2 negative (P = 0.09). No significant differences in outcome emerged between the two groups. In conclusion, this study found the incidence of HER-2 protein over-expression in IBC is higher than previously reported in non-IBC. Early HER-2 directed therapy (such as the monoclonal antibody trastuzumab) as a part of multimodal treatment may improve outcome in this poor prognosis cancer..

Subjects of this study consisted of 333 women (aged 4575 years) drawn from a large United Kingdom prospective study of diet and cancer, the European Prospective Investigation of Cancer and Nutrition-Norfolk study. Using newly developed gas chromatography/mass spectrometry and liquid chromatograghy/mass spectrometry methods incorporating triply C-labeled standards, seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in 114 spot urines and 97 available serum samples from women who later developed breast cancer. Results, were compared with those from 219 urines and 187 serum samples from healthy controls matched by age and date of recruitment. Dietary levels were low, but even so, mean serum levels of phytoestrogens were up to 600 times greater than postmenopausal estradiol levels. Phytoestrogen concentrations in spot urine (adjusted for urinary creatinine) correlated strongly with that in serum, with Pearson correlation coefficients > 0.8. There were significant relationships (P < 0.02) between both urinary and serum concentrations of isoflavones across increasing tertiles of dietary intakes. Urinary enterodiol and enterolactone and serum enterolactone were significantly correlated with dietary fiber intake (r = 0.13-0.29). Exposure to all isoflavones was associated with increased breast cancer risk, significantly so for equol and daidzein. For a doubling of levels, odds ratios increased by 20-45% [log(2) odds ratio = 1.34 (1.06-1.70; P = 0.013) for urine equol, 1.46 (1.05-2.02; P = 0.024) for serum equol, and 1.22 (1.01-1.48; P = 0.044) for serum daidzein]. These estimates of risk are similar to those established for estrogens and androgens in postmenopausal breast cancer but need confirmation in larger studies..

The anti-oestrogen tamoxifen is the most commonly used treatment for patients with oestrogen-receptor (ER)-positive breast cancer. Although many patients benefit from tamoxifen in the adjuvant and metastatic settings, resistance is an important clinical problem. The target of tamoxifen in vivo is the ER. Over the last decade many advances have been made in our understanding of the biology of the ER which may help to explain how resistance to tamoxifen develops. Such mechanisms may include changes in the expression of ERalpha or ERbeta, alterations in co-regulatory proteins, and the influences of cellular kinase signal transduction pathways. The experimental and clinical evidence supporting these mechanisms of tamoxifen resistance are discussed in this review..

Overexpression of erbB2 in breast tumours can predict resistance to tamoxifen therapy. We conducted a small trial to determine if erbB2 status correlates with tumour response and biochemical changes in postmenopausal women receiving neoadjuvant therapy with the aromatase inhibitor, anastrozole. Twenty-four postmenopausal women with oestrogen receptor (ER)-rich, large, operable breast tumours received three months of neoadjuvant anastrozole, 1 or 10 mg daily, then surgery, followed by another five years of anastrozole I mg daily. Response to the treatment was based on changes in clinical and ultrasound measurements of tumour volume and changes in tumour proliferation and progesterone receptor (PgR) status. After follow-up for a median duration of four years therapy, there was no apparent difference between erbB2 0/1 + and erbB2 3 + tumours in clinical response or changes in proliferation and PgR expression. In conclusion, anastrozole appears to be an effective endocrine option in this patient population, irrespective of the erbB2 status. (C) 2004 Elsevier Ltd. All rights reserved..

A series of three dose escalating studies were conducted to investigate the ability of the 17alpha-hydroxylase/C(17,20)-lyase inhibitor abiraterone acetate, to cause maximum suppression of testosterone synthesis when delivered to castrate and noncastrate males with prostate cancer. Study A was a single dose study in castrate males. Study B was a single dose study in noncastrate males and study C was a multiple dose study in noncastrate males. The drug was given orally in a once-daily dose and blood samples taken to assess pharmacokinetic (PK) parameters and hormone levels in all patients. The study drug was well tolerated with some variability in PKs. Suppression of testosterone levels to <0.14 nmol l(-1) was seen in four out of six castrate males treated with a single dose of 500 mg. At 800 mg given days 1-12 in noncastrate males, target suppression was achieved in three out of three patients, but a two- to three-fold increase of Luteinising Hormone (LH) levels in two out of three patients overcame suppression within 3 days. All patients in the multiple dose study developed an abnormal response to a short Synacthen test by day 11, although baseline cortisol levels remained normal. This is the first report of the use of a specific 17alpha-hydroxylase/(17,20)-lyase inhibitor in humans. Repeated treatment of men with intact gonadal function with abiraterone acetate at a dose of 800 mg can successfully suppress testosterone levels to the castrate range. However, this level of suppression may not be sustained in all patients due to compensatory hypersecretion of LH. The enhanced testosterone suppression achieved in castrate men merits further clinical study as a second-line hormonal treatment for prostate cancer. Adrenocortical suppression may necessitate concomitant administration of replacement glucocorticoid..

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537 Background: Aromatase inhibitors and tamoxifen are key agents for endocrine treatment of postmenopausal breast cancer (BrCa). Understanding of their molecular/biological effects in BrCa is needed for their rational application alone or in combination with novel therapeutics. METHODS: 330 postmenopausal patients with invasive BrCa (>/=2cm) were randomised to 12wks neoadjuvant therapy with A, T or C in a placebo-controlled double-blind trial (IMPACT). Biopsies were available from a total of 292 patients pre-treatment and at 2 and 12 wks. Immunohistochemical (IC) studies were performed for Ki67, apoptosis (TUNEL), ER, PgR, EGFR (pre) and HER2 (pre). The growth index (GI: Ki67/apoptosis) was calculated. Plasma estradiol (E2) levels were measured. RESULTS: The %age changes in the biomarkers from baseline are shown in the table. IC results for T and C were similar in all respects. The greater reductions in Ki67 in A vs T have been reported. There were greater reductions in GI for A vs T despite the decreases in apoptosis seen with A and profound differences between the treatments in their effect on PgR. After 2wks the reduction in PgR for A, and the increase in PgR for T were correlated with the fall in Ki67 (p=0.003 and 0.027, respectively). After 12wks there was a significantly lower reduction in Ki67 in HER2+ vs HER2- tumours (geo mean: 73% vs 48%) but no significant difference between the treatment groups. CONCLUSIONS: Changes in both Ki67 and GI parallel the greater reduction in relapse rate for A vs T and C in the ATAC adjuvant trial. This suggests short-term biomarker changes may predict for long-term outcome in the adjuvant setting but this needs confirmation. The decrease in apoptosis seen with A suggests that estrogen is not an important survival factor in BrCa cells. Early increases in PgR with T are not associated with a poor antiproliferative effect. [Figure: see text] [Table: see text]..

675 Background: Estrogen has a beneficial effect on lipid metabolism and decreased estrogen levels have been associated with a decrease in bone mineral density. The IMPACT trial evaluated neoadjuvant therapy with A, T or C in postmenopausal women with estrogen receptor-positive breast cancer. During the study an assessment was made of the comparative effects of these treatments on serum lipids and bone resorption. METHODS: Non-fasting clotted blood samples were taken at baseline and at 3 months. Total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) were measured using a timed end-point method on a Synchron CX9. Non-HDL cholesterol levels (N-HDL-C) were calculated. Bone resorption was measured using serum CrossLaps (CTX) One Step ELISA. Estradiol was measured by radioimmunoassay (RIA). RESULTS: Percentage changes in parameters from baseline (using (†) arithmetic mean; (††) geometric mean) are given in the table. CONCLUSIONS: The beneficial effects of T on lipids were as expected. Following 3 months treatment with A there was a significant increase in HDL-C, but there was no significant effect on TC. A increased bone resorption, in contrast to T, but there was no significant correlation between the mean % change (from baseline to 3 months) of estradiol and CTX in any of the treatment groups. This suggests either that estrogen levels are only one of the factors affecting bone metabolism in postmenopausal women, or that 3 months is too short a period to assess bone markers. [Figure: see text] [Table: see text]..

Background: Reports suggest a relationship between circulating sex hormone levels and breast cancer risk, but genetic association studies have been inconclusive. We investigated the association between levels of sex hormones and single nucleotide polymorphisms (SNPs) in genes coding for the enzymes that regulate them. Methods: We assayed circulating levels of estradiol, testosterone, estrone, androstenedione, 17alpha-hydroxyprogesterone, and sex hormone-binding globulin (SHBG) in 1975 normal postmenopausal women. Fifteen SNPs in the CYP17, CYP19, EDH17B2, SHBG, COMT, and CYP1B1 genes were genotyped in these post menopausal women and in a breast cancer case-control study. Associations of genotypes with breast cancer risk were evaluated in the case-control study and with hormone levels in the postmenopausal women using multiple linear regression with assay batch, body mass index, parity, peri- or postmenopausal status, and age band as covariates. Results: CYP19 SNPs (rs10046 and [TCT]+/-) were associated with differences in estradiol level (P =.0006 and P =.0003, respectively) and the estradiol:testosterone ratio (P =.000001 and P =.002). SNP rs10046 explained 1.6% of the variance (r(2)) in the estradiol: testosterone ratio. SHBG SNPs (5' untranslated region [5'UTR] g-a and D356N) were associated with both SHBG levels (p< 10(-6) and P =.005) and the estradiol: SHBG ratio (P =.000008 and P =.01). These SNPs explained 2.4% and 0.6% of the variance in SHBG levels, respectively. SNPs in the other genes were not associated with differences in any hormone levels, and none were statistically significantly associated with breast cancer risk. Conclusion: Genetic variation in CYP19 and SHBG contributes to variance in circulating hormone levels between postmenopausal women, but low r(2) values may explain why these genes have given inconclusive results in breast cancer case-control studies. [J Natl Cancer Inst 2004;96:936-45].

Advances in our understanding of the molecular mechanisms underlying breast cancer have identified a considerable number of new molecular targets that may have significant roles in tumor behavior. The rapid and effective transition of these novel, preclinical scientific observations through into the clinic is described as translational research. Tumor markers are an important area of translational research, since they introduce the possibility of identifying groups of patients who possess particular diagnostic or prognostic characteristics. It is important that advances in translational breast cancer research are reviewed in order to determine how they may influence treatment. As part of this process, it is particularly important to identify how these advances may be rationally evaluated through the design of clinical trials, thereby ensuring that the resulting data are sufficiently robust in order to inform and direct clinical decision making. In this way, the treatments available should be efficiently and actively revised to incorporate new concepts of diagnosis, prognosis and treatment, to the benefit of women with breast cancer..

cDNA arrays and proteomic analyses have allowed the rapid identification of specific genes and proteins implicated in multiple tumor types. These molecules must then be validated as clinically relevant prognostic and predictive markers, and this translational research is best conducted in the context of clinical trials. Outcomes data and clinical specimens collected in the 'Arimidex', Tamoxifen, Alone or in Combination (ATAC) study, for example, can now be used to compare the expression of biomarkers with clinical outcomes. In this study, adjuvant tamoxifen and anastrozole ('Arimidex') were compared alone and in combination in more than 9000 women with breast cancer. Anastrozole was found to be superior to tamoxifen in terms of disease-free survival, time to recurrence, and reduction in the incidence of contralateral tumors. Importantly, tissue specimens from surgical excision, local relapse, and contralateral breast cancer were collected and paraffin-embedded for storage. In the TA01 (TransATAC) program, these specimens will be studied (after obtaining patient consent) using tissue microarrays; tissue biopsy cores 0.6 mm in diameter will be removed from donor blocks and placed on recipient blocks, which will be sectioned to allow the simultaneous analysis of the same samples for multiple biomarkers. These analyses can help determine differential benefits of treatment with anastrozole or tamoxifen, depending on the expression of particular biomarkers in tumor cells. This research also should clarify de novo and acquired resistance mechanisms, and the validation of relevant molecular pathways could guide the development of new drugs. Ultimately, the TA01 program has the potential to favorably impact treatment choices for breast cancer..

Translational research is intended to efficiently and accurately validate the most promising preclinical biomarkers and ensure that their diagnostic or prognostic potential is rapidly adopted in the clinic. Translational research therefore needs to take into account the biological complexity that is inherent in all cellular pathways, such as the estrogen and progesterone receptor pathways, and yet produce conclusions that will have sufficient statistical power to define and shape clinical practice. This will be achieved through the incorporation of new concepts of trial design, including neoadjuvant or preoperative hypothesis-generating studies, and the increasing use of high-throughput proteomic and genomic approaches. Clinical trials will be increasingly designed to incorporate biomarker analyses, and ongoing trials may employ the retrospective collection of archival tissues to allow the use of prospectively planned biomarker analyses. This approach is being utilized in the TA01 program, which is based upon the 'Arimidex', tamoxifen, alone or in combination (ATAC) trial. The resulting incorporation of new biomarkers and revised trial designs should promote the future development of more precise and increasingly active interventions directed towards the treatment of breast cancer..

Introduction: Isoflavones are hypothesized to protect against breast cancer, but it is not clear whether they act as oestrogens or anti-oestrogens in breast tissue. Our aim was to determine the effects of taking a red clover-derived isoflavone supplement daily for 1 year on mammographic breast density. Effects on oestradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), lymphocyte tyrosine kinase activity and menopausal symptoms were also assessed.Methods: A total of 205 women ( age range 49 - 65 years) with Wolfe P2 or DY mammographic breast patterns were randomly assigned to receive either a red clover-derived isoflavone tablet (26 mg biochanin A, 16 mg formononetin, 1 mg genistein and 0.5 mg daidzein) or placebo. Change in mammographic breast density, serum oestradiol, FSH, LH, menopausal symptoms and lymphocyte tyrosine kinase activity from baseline to 12 months were assessed.Results: A total of 177 women completed the trial. Mammographic breast density decreased in both groups but the difference between the treatment and placebo was not statistically significant. There was a significant interaction between treatment group and oestrogen receptor (ESR1) PvuII polymorphism for the change in estimated percentage breast density (mean +/- standard deviation): TT isoflavone 1.4 +/- 12.3% and TT placebo - 9.6 +/- 14.2%; CT isoflavone - 5.2 +/- 12.0% and CT placebo - 2.8 +/- 10.3%; and CC isoflavone - 3.4 +/- 9.7% and CC placebo - 1.1 +/- 9.5%. There were no statistically significant treatment effects on oestradiol, FSH, or LH ( assessed only in postmenopausal women), or on lymphocyte tyrosine kinase activity. Baseline levels of menopausal symptoms were low, and there were no statistically significant treatment effects on frequency of hot flushes or other menopausal symptoms.Conclusion: In contrast to studies showing that conventional hormone replacement therapies increase mammographic breast density, the isoflavone supplement did not increase mammographic breast density in this population of women. Furthermore, there were no effects on oestradiol, gonadotrophins, lymphocyte tyrosine kinase activity, or menopausal symptoms..

Endocrine therapies act by either blocking or downregulating the oestrogen receptor or by reducing oestrogen concentrations around and within the cancer cell. In postmenopausal women, oestrogen suppression is achieved by inhibition of the enzyme aromatase by aromatase inhibitors (AIs). Modern AIs ( anastrozole, letrozole and exemestane) are more potent than earlier ones and suppress oestradiol levels in plasma to virtually undetectable concentrations. Recent comparisons of AIs with the most widely used oestrogen receptor blocking drug tamoxifen indicate that, in general, AIs result in increased response rates and greater durations of response. Here, we summarize data supporting the difference between the two types of treatment and attempt to account for the underlying mechanisms that favour AIs..

Purpose : R115777 is an orally active farnesyl transferase inhibitor that specifically blocks farnesylation of proteins involved in growth-factor-dependent cell-signal-transduction pathways. We conducted a phase II study in 76 patients with advanced breast cancer.Patients and Methods: Two cohorts of patients were recruited sequentially. The first cohort (n = 41) received a continuous dosing [CD] regimen of R115777 400 or 300 mg bid. The second cohort (n = 35) received 300 mg bid in a cyclical regimen of 21 days of treatment followed by 7 days of rest (intermittent dosing [ID]).Results: In the CD cohort, four patients (10%) had a partial response (PR) and six patients (15%) had stable disease at greater than or equal to 24 weeks (SD). In the ID cohort, five patients (14%) had a PR and three patients (9%) had prolonged SD. The first six patients in the CD cohort treated at 400 mg bid all developed grade 3 to 4 neutropenia, so the subsequent 35 patients were treated at 300 mg bid. The incidence of hematologic toxicity was significantly lower in the ID than in the CD (300-mg bid) cohort: grade 3 to 4 neutropenia (14% v 439/6, P = .016) and grade 3 to 4 thrombocytopenia (3% v 26%, P = .013). One patient in the ID cohort developed grade 2 to 3 neurotoxicity compared with 15 patients in the CD cohort (3% v 37%; P = .0004).Conclusion: The farnesyl transferase inhibitor R115777 has demonstrated clinical activity in patients with metastatic breast cancer, and the ID regimen has a significantly improved therapeutic index compared with the CD regimen. (C) 2003 by American Society of Clinical Oncology..

Recent evidence suggests that common molecular adaptations occur during resistance to both tamoxifen and estrogen deprivation that use various signal transduction pathways, often involving cross-talk with a retained and functional estrogen receptor (ER) protein. There appear to be several different levels at which this cross-talk may occur, including peptide growth factor signaling via the type 1 tyrosine kinase growth factor receptor family [epidermal growth factor receptor (EGFR) and HER2], which may become up-regulated during endocrine treatment, ultimately being harnessed by cells to allow them hormone-independent growth. ER may remain involved in cell growth with ligand-independent phosphorylation and activation via different intracellular mitogen-activated protein kinases. ER may also become involved in non-nuclear estrogen-dependent signaling via interaction with the phosphatidylinositol 3'-kinase/Akt cell survival pathway or may interact with the stress-activated protein kinase/c-Jun-NH(2)-terminal kinase pathway. Understanding these mechanisms will permit the optimal integration of new signal transduction inhibitors (STIs) into breast cancer therapy. Preclinical approaches that have shown promise include the use of EGFR tyrosine kinase inhibitors for hormone-resistant breast cancer cells that are dependent on either EGFR or HER2 signaling. Likewise, farnesyl transferase inhibitors, mitogen-activated protein kinase inhibitors, and cell cycle inhibitors have all shown activity in experimental breast cancer models. Emerging data suggest that STIs may be more effective when given in combination with endocrine therapy either to overcome resistance or to prevent/delay emergence of the resistance phenotype. Clinical trials are in progress to determine the safety and optimal schedule for each of the various STIs, and studies of STIs in combination with aromatase inhibitors have commenced in breast cancer to see whether the therapeutic response to endocrine therapy can be enhanced further..

There are essentially two approaches to presurgical therapy. The first strategy is one in which the therapy is given to downstage the disease over a period of some 3-4 months. In these circumstances, biological studies can be conducted that can be associated with clinical response. The second strategy involves the institution of medical therapy before surgery with no delay to that surgery. This is essentially incidental and is not given with the aim of having a therapeutic benefit but rather for the biological study of the particular therapeutic approach. In the incidental therapy scenario, we have conducted a number of studies to evaluate the biological effects of raloxifene, idoxifene, fulvestrant (in comparison with tamoxifen), and the aromatase inhibitor 4-hydroxyandrostenedione. Significant reductions in proliferation were noted in all, as was down-regulation of estrogen receptor levels. The changes were most profound in those estrogen receptor-positive tumors that were also progesterone receptor positive, consistent with the greater clinical effect of these therapies in this population. This setting is particularly valuable for treatments in which there is no particular evidence for clinical benefit but in which the therapy is known to be safe. It is possible to evaluate potential resistance mechanisms by associating changes in Ki67 or apoptosis with the expression of the putative determinant of resistance in the short-term presurgical model..

Arzoxifene (ARZ) is a selective estrogen receptor (ER) modulator with greater bioavailability than raloxifene which is being developed as treatment for breast cancer. We have used an in vivo model of hormone-sensitive breast cancer to study the growth-inhibitory and pharmacodynamic effects of ARZ in comparison with the most widely used antiestrogen, tamoxifen (TAM). We compared the abilities of ARZ and TAM to antagonize the estrogen (E2)-dependent growth of MCF-7 human breast cancer xenografts in oophorectomized athymic mice. At four different time points over 28 days, we studied their time-related pharmacodynamic effects on biomarkers of tumor growth (cell proliferation/death measured by Ki-67 and apoptosis scores), cell cycle activity (cyclin D1 and p27(kip1)), and hormone-regulated gene expression (ERalpha, progesterone receptor, and pS2). Although maximal growth inhibition was seen after E2 withdrawal, ARZ and TAM induced significant and similar inhibition of E2-stimulated tumor growth. Inhibition of growth was reflected by changes in the tumor growth index (ratio posttreatment/pretreatment Ki-67/apoptosis scores). ARZ and TAM resulted in a significant (P < 0.001) increase in ER expression and reduction in progesterone receptor expression, whereas changes in cyclin D1 score were inversely related to p27(kip1) score. A significant but delayed biological effect was observed with a 10-fold lower dose of ARZ. These results show that ARZ is an effective antagonist of E2-stimulated breast cancer growth with similar growth-inhibitory and pharmacodynamic effects to TAM in this model..

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Knowledge of HER2 status is a prerequisite when considering a patient's eligibility for Herceptin (trastuzumab) therapy. Accurate assessment of HER2 status is essential to ensure that all patients who may benefit from Herceptin are correctly identified. There are several assays available to determine HER2 status: the most common in routine clinical practice are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Various factors can affect the results achieved with these assays, including the assay antibody/probe, the methodology and the experience of personnel. Many countries have implemented national testing guidelines in an attempt to standardize testing procedures and make results more accurate. These guidelines vary In the level of detail and the number of recommendations. This review looks at areas of consensus between the different national testing guidelines and highlights where errors may arise during the testing procedure. The key point underlined by this review is that whatever method is used to test for HER2 status, the technology must be validated first, and there must be regular internal and external quality control and quality assurance procedures..

Accurate diagnostic assessment of HER-2 is essential for the appropriate application of the humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) to the treatment of patients with metastatic breast cancer. The diagnostic test needs to be applicable to archival, fixed tissue removed at excision, in many cases several years earlier. We compared the assessment of HER-2 by immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH) in 426 breast carcinomas from patients being considered for trastuzumab therapy. The tumours were tested in three reference centres having been sent in from 37 hospitals. Only 2/270 (0.7%) IHC 0/1+ tumours were FISH positive. Six of 102 (5.9%) IHC 3+ tumours were FISH negative. Five of the six had between 1.75 and 2.0 HER-2 gene copies per chromosome 17 and the sixth had multiple copies of chromosome 17. Thirteen per cent of tumours were IHC 2+ and overall 48% of these were FISH positive but this proportion varied markedly between the centres. Sixty IHC-stained slides selected to be enriched with 2+ cases were circulated between the three laboratories and scored. There were 20 cases in which there was some discordance in scoring. Consideration of the FISH score in these cases led to concordance in the designation of positivity/negativity in 19 of these 20 cases. These data support an algorithm in which FISH testing is restricted to IHC 2+ tumours in reference centres. The results may not extrapolate to laboratories with less experience or using different methodologies..

Estrogen-targeted therapies such as administration of tamoxifen or aromatase inhibitors are among the most important treatment strategies in the modern management of breast cancer. Despite initial responses in the metastatic setting and prolonged disease-free intervals in the adjuvant setting, many patients subsequently become resistant to these agents. Human epidermal growth factor receptor-2 (HER2) is a transmembrane glycoprotein receptor that is overexpressed in 13%-30% of human breast cancers. There are experimental data suggesting an important role for HER2 in de novo and acquired resistance to endocrine therapies. These experimental data are discussed in this article, as are clinical data addressing the role of HER2 in resistance to endocrine therapy in the adjuvant, neoadjuvant, and metastatic settings. Responses and benefit from tamoxifen appear to be impaired in patients in whom HER2 is overexpressed. In contrast, early data from the neoadjuvant setting suggest that responses to aromatase inhibitors may be maintained in patients with HER2 overexpression..

PURPOSE: Cyclooxygenase (COX)-2 is overexpressed in breast cancer and may have a role in regulating tumor growth via effects on angiogenesis, cell proliferation, or apoptosis. This study aimed to derive data from human breast carcinomas to help substantiate or refute these relationships. EXPERIMENTAL DESIGN: We performed immunohistochemical analysis of a set of 86 breast tumors for COX-2, estrogen receptor (ER), progesterone receptor (PGR), HER-2, Ki67 (a marker of proliferation), and CD31 (an endothelial cell marker of angiogenesis). RESULTS: COX-2 protein expression was detected in 79% of all tumors studied, ER was detected in 79% of all tumors studied, PGR was detected in 73% of all tumors studied, and HER-2 was detected in 16% of all tumors studied. COX-2 protein expression did not significantly correlate with tumor size, grade, axillary lymph node status, or the presence of vascular invasion. A significant negative correlation (P < 0.001) was observed between ER and Ki67. COX-2 expression showed a significant linear correlation with CD31 staining (P < 0.001). No significant correlations were observed between COX-2 and ER, PGR, or HER-2. CONCLUSIONS: This study demonstrates a novel relationship between COX-2 expression and the neovasculature of human breast adenocarcinomas. If this is a functional relationship, it provides support for a potential therapeutic role of COX-2 inhibitors in human breast cancer tissue via their antiangiogenic properties..

Aromatase inhibitors (AIs) are now under investigation for the treatment of early stage breast cancer and for disease prevention as alternatives to standard treatment with tamoxifen. Currently identified genetic risk factors of breast cancer include BRCA-1/BRCA-2 mutations, ATM mutations, and history of high estrogen levels, as evidenced by plasma analyses and/or dense bones. To date, estrogen receptor (ER) and progesterone receptor (PgR) status has predictive value for determining response to therapy in patients with hormone receptor-positive breast cancer (ER+ and/or PgR+ tumors). Recent studies have shown AIs to be safer and more effective than tamoxifen in postmenopausal women with advanced disease. Some data suggest that letrozole may be a more effective treatment than tamoxifen for patients with ER+ and/or PgR+ early breast cancers expressing ErbB-1 and/or ErbB-2. Changes in cell proliferation markers (e.g., S-phase fraction and Ki67 antigen), plasma lipid levels, and the bone resorption marker C-terminal peptide are biomarkers that have been evaluated for preventive and prognostic value in breast cancer patients and normal volunteers. Results from biomarker screens can be used to define inclusion criteria for clinical trials and eventually to individualize treatment. Gene expression profiling (microarray analysis), i.e., genomic and proteomic studies, will probably advance the discovery of new biomarkers for breast cancer prevention and treatment..

Estrogen is critical in the progression and probably also in the initiation of breast tumors. Two classes of pharmacologic agents currently used in the treatment of estrogen receptor-positive breast tumors are the selective estrogen receptor modulators such as tamoxifen that block the interaction of estrogen with its cognate receptor, and the aromatase inhibitors (AIs) that block the final enzymatic step in the estrogen biosynthetic pathway. Recent data from first-line phase III trials in postmenopausal patients with hormone receptor-positive or -unknown metastatic breast cancer suggest that AIs are at least equivalent to or more effective than tamoxifen in this setting. As a result AIs are being evaluated in the adjuvant setting and may eventually displace tamoxifen as the endocrine therapy of choice for postmenopausal patients with breast cancer. Among the side effects of therapy that were observed in metastatic trials, AIs have shown less vascular and uterine adverse events than tamoxifen. Some concern exists, however, regarding their effect on lipid metabolism and bone turnover. To be acceptable for adjuvant treatment, AIs will have to show not only an advantage in efficacy, but also that any concerns from potential side effects may be managed appropriately. This article reviews the safety profile of AIs, discussing events related to their chemical structure, their specificity, and the effects of estrogen deprivation on target organs..

Third generation aromatase inhibitors have excellent specificity. Some reports indicate that letrozole may have a minor effect on cortisol synthesis but these were not confirmed: valid comparisons with other aromatase inhibitors requires randomised study. The putative use of a third generation inhibitor as a single agent in premenopausal women has been investigated using YM511. It was hypothesised that in this situation site-specific suppression of estrogens in breast carcinomas, without systemic effects, may lead to a down-regulation of tumour proliferation. Plasma levels of androstenedione and testosterone were significantly increased by 2 weeks treatment with YM511. Mean plasma estrone levels were suppressed, but some plasma estradiol levels were abnormally high and others abnormally low. These differential effects of YM511 on circulating estrogens supported the concept that peripheral synthesis of estrogens might be suppressed while ovarian production remained high. However, YM511 did not demonstrate anti-proliferative effects in hormone sensitive breast carcinomas. Consideration of the pharmacology of the estrogen receptor during tamoxifen therapy indicates that tamoxifen effectively saturates the receptor (>99.94% occupancy) in postmenopausal women. The addition of an aromatase inhibitor in this situation would be very unlikely to affect the biological activity of the estrogen receptor. This provides a possible explanation why the clinical efficacy of tamoxifen combined with an aromatase inhibitor appears to be equivalent to that of tamoxifen alone..

BACKGROUND: Epithelial cells may be detected in the circulation of the majority of patients with metastatic breast cancer. Quantification of such presumptive cancer cells might allow for the monitoring of patients with early or late stage disease as an early index of relapse. Additionally, biomarker analysis may allow a more rational approach to therapeutics. We have developed a new method for the detection and characterisation of these cells. METHODS: Blood was filtered through polycarbonate membranes containing cylindrical pores, 8 microm in diameter. All the red cells and a large majority of the white blood cells passed through the filter while the larger epithelial cells were trapped. Cells on the membrane were fixed in ethanol, stained with propidium iodide and anti-pan-cytokeratin-FITC (to identify epithelial cells). The filters were then examined by laser scanning cytometry (LSC), which allowed enumeration and localisation of cells. RESULTS: With normal blood spiked with cells from breast carcinoma cell lines, 99.9% of the leukocytes passed through the membrane, while close to 100% of the epithelial cells were trapped, with a detection limit of less than one epithelial cell/ml of blood. All of 20 samples from patients with widespread metastatic disease contained cytokeratin-positive cells with the morphological characteristics of carcinoma cells, the number of cells ranging from 0.2 to 5.7/ml of blood. CONCLUSIONS: Cell filtration-LSC is a viable technique for detecting and studying breast carcinoma cells in peripheral blood..

Recent clinical trials indicate that the third-generation aromatase inhibitors may be more effective than tamoxifen as first line endocrine therapy in ER+ metastatic breast cancer in postmenopausal women. This review will focus exclusively on the pharmacology of the non-steroidal inhibitor letrozole. Aromatase derived from a variety of sources is inhibited at low nM concentrations of the drug. In non-cellular systems, letrozole is 2-5 times more potent than anastrozole and exemestane in its inhibition of aromatase, whilst in cellular systems it is 10-20 times more potent. Anti-tumour effects of letrozole have been demonstrated in several animal models. In postmenopausal women, letrozole commonly suppresses circulating concentrations of estrone and estradiol to below the sensitivity limit of the assays used to measure them. In a recent randomized cross-over study, letrozole (2.5mg daily) achieved a significantly greater suppression of the plasma concentrations of both estrone and estrone sulphate than anastrozole (1mg daily) and a greater inhibition of in vivo aromatization. Letrozole appears to have a small effect on adrenal steroidogenesis such that a small number of patients exhibit an abnormal response to synthetic ACTH during letrozole therapy. This is unlikely to have any clinical significance. In short-term studies letrozole has been shown to increase markers of bone resorption indicating the need to monitor bone integrity when the drug is used for extended periods of time. A consistent effect of letrozole on serum lipids has not been demonstrated..

BACKGROUND: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates from human breast carcinomas. Previously, we demonstrated the use of laser scanning cytometry (LSC) for the measurement of Ki67, ER, and PgR in a human breast carcinoma cell line, MCF7 (21). In the present study, we investigated the expression of Ki67 in breast tumour fine needle aspirates (FNAs) using LSC and compared the results to the data obtained using conventional immunocytochemistry (ICC). METHODS: A total of 11 sets of cytospins of FNAs taken from breast tumours at various stages of tamoxifen treatment were used. For LSC, the cytospins were stained for Ki67 with fluorescein using immunofluorescence; the nuclei were counterstained with propidium iodide. A parallel set of cytospins was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy. RESULTS: Values for Ki67 obtained using the LSC were generally lower than ICC scores. The changes in Ki67 measured by the LSC were almost all parallel to those obtained by manual scoring of immunocytochemical stains. CONCLUSIONS: It should be possible to use LSC for the routine measurement of Ki67 marker in FNAs from human breast carcinomas..

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INTRODUCTION: Objectives were to characterise the relationship of the proliferation marker Ki67 with response to systemic treatment in early breast cancer and to assess its clinical utility, using fine needle aspirates. MATERIALS AND METHODS: Hundred and six women were treated with primary tamoxifen (n = 33), chemotherapy (n = 33) or chemotherapy and tamoxifen (n = 40). Treatment was not randomised and response was assessed clinically. Ki67 was evaluated prior to treatment and at Day 14 or 21 after commencing treatment. To assess reproducibility, Ki67 was evaluated in repeat FNAs taken from 37 untreated patients. RESULTS: The percentage change in Ki67 in first 21 days was different between responders and non-responders for patients treated with tamoxifen (p = 0.007) and chemotherapy (p = 0.005) but not for chemoendocrine treatment (p = 0.062). The reproducibility study indicated that a decrease to 36% or less of the pre-treatment Ki67 value in an individual patient was required for it to be regarded as a statistically significant change. A significant decrease in Ki67 was seen in responding patients treated with chemotherapy (p = 0.026) and chemoendocrine treatment (p = 0.041). Positive and negative predictive values for response were 85 and 59% for chemotherapy patients and 88 and 54% for chemoendocrine patients, respectively. CONCLUSION: Ki67 is unlikely to be useful as a predictive marker in individual patients. Further molecular markers that predict lack of response continue to be required..

BACKGROUND: The first analysis of the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial (median follow-up, 33 months) demonstrated that in adjuvant endocrine therapy for postmenopausal patients with early-stage breast cancer, anastrozole was superior to tamoxifen in terms of disease-free survival (DFS), time to recurrence (TTR), and incidence of contralateral breast cancer (CLBC). In the current article, the results of the first efficacy update, based on a median follow-up period of 47 months, are reported along with the results of an updated safety analysis, performed 7 months after the first analysis (median duration of treatment, 36.9 months). METHODS: DFS, TTR, CLBC incidence, and safety were assessed in the same patient group as in the first analysis of the ATAC trial. RESULTS: DFS estimates at 4 years remained significantly more favorable (86.9% vs. 84.5%, respectively) for patients receiving anastrozole compared with those receiving tamoxifen (hazard ratio [HR], 0.86; 95% confidence interval [CI], 0.76-0.99; P = 0.03). The benefit generated by anastrozole in terms of DFS was even greater in patients with hormone receptor-positive tumors (HR, 0.82; 95% CI, 0.70-0.96; P = 0.014). The HR for TTR also indicated a significant benefit for patients receiving anastrozole compared with those receiving tamoxifen (HR, 0.83; 95% CI, 0.71-0.96; P = 0.015), with additional benefit for patients with hormone receptor-positive tumors (HR, 0.78; 95% CI, 0.65-0.93; P = 0.007). CLBC incidence data also continued to favor anastrozole (odds ratio [OR], 0.62; 95% CI, 0.38-1.02; P = 0.062), and statistical significance was achieved in the hormone receptor-positive subgroup (OR, 0.56; 95% CI, 0.32-0.98; P = 0.042). The updated safety analysis also confirmed the findings of the first analysis, in that endometrial cancer (P = 0.007), vaginal bleeding and discharge (P \ensuremath< 0.001 for both), cerebrovascular events (P \ensuremath< 0.001), venous thromboembolic events (P \ensuremath< 0.001), and hot flashes (P \ensuremath< 0.001) all occurred less frequently in the anastrozole group, whereas musculoskeletal disorders and fractures (P \ensuremath< 0.001 for both) continued to occur less frequently in the tamoxifen group. These results indicated that the safety profile of anastrozole remained consistent. CONCLUSIONS: After an additional follow-up period, anastrozole continues to show superior efficacy, which is most apparent in the clinically relevant hormone receptor-positive population. Furthermore, anastrozole has numerous noteworthy advantages in terms of tolerability compared with tamoxifen. These findings suggest that the benefits of anastrozole are likely to be maintained in the long term and provide further support for the status of anastrozole as a valid treatment option for postmenopausal women with hormone-sensitive early-stage breast cancer. Copyright 2003 American Cancer Society..

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Important issues that underpin efforts to link new biological data on breast cancer to clinical research are considered. While animal models have been helpful in drug development, the changes in some key markers (oestrogen receptor and Ki67) in response to hormonal therapy are very different in patients. To predict that benefit of therapy in biomarker-defined subgroups requires the parallel study of a randomized control arm. Biomarker analyses have reduced statistical power compared with whole trials. Neoadjuvant therapy is an advantageous scenario to link biological and clinical research..

The mechanisms involved in resistance to estrogen deprivation are of major importance for optimal patient therapy and the development of new drugs. Long term culture of MCF-7 cells in estrogen (E2)-depleted medium (long term estrogen deprivation; LTED) results in hypersensitivity to E2 coinciding with elevated levels of estrogen receptor (ER) alpha phosphorylated on Ser118 and MAPK, together with several of its downstream targets associated previously with ERalpha phosphorylation. Our data suggest elevated MAPK activity results from enhanced ERBB2 expression in the LTED cells versus the wild-type (wt), and treatment with the tyrosine kinase inhibitor ZD1839 revealed increased sensitivity in both transcription and proliferation assays. Similarly the MEK inhibitor U0126 decreased transcription and proliferation in the LTED cells and reduced their sensitivity to the proliferative effects of E2, while having no effect on the wt. However, the complete suppression of MAPK activity in the LTED cells did not inhibit ERalpha Ser118 phosphorylation suggesting that ER activity remained ligand-dependant. The LTED cells also expressed elevated levels of insulin-like growth factor-1R, and inhibition of phosphatidylinositol 3-kinase activity with LY294002 reduced basal ERalpha transactivation by 70% in the LTED cells compared with the wt. However, LY294002 had no effect on ERalpha Ser118 phosphorylation. These data suggest that although elevated levels of MAPK occur during LTED and influence the phenotype, this is unlikely to be the sole pathway operating to achieve adaptation..

Patients undergoing primary chemotherapy for invasive breast cancer consented to a core biopsy of the invasive breast primary pre- and 24 h postchemotherapy. The resulting tissue was analysed for apoptosis, Ki67, ER and HER-2 using immunohistochemical techniques. These data were then used to evaluate the relationship between these biological markers and response to chemotherapy and overall survival. Response rate to chemotherapy in this group was 86%, 16 patients (25%) achieved a clinical complete response and 41 (63%) a partial response. Prechemotherapy there was a significant correlation between Ki67 and apoptotic index (AI), r=0.6, (P<0.001). A significant rise in AI (P<0.001), and fall in Ki67 (P=0.002) was seen 24 h following chemotherapy. No relationship was seen between pretreatment AI and clinical response, but higher Ki67 and growth index (Ki67/AI ratio, GI) did correlate with clinical response (both r=0.31, P<0.025). No correlation was seen between the change in AI or Ki67 at 24 h and clinical response or survival. Significant changes in apoptosis and proliferation can be demonstrated 24 h following chemotherapy, but these changes do not relate to clinical response or outcome in this study. Pretreatment proliferation and GI are however predictive of response to chemotherapy in breast cancer..

Background: Obesity is associated with increased breast cancer risk among postmenopausal women. We examined whether this association could be explained by the relationship of body mass index (BMI) with serum sex hormone concentrations. Methods: We analyzed individual data from eight prospective studies of postmenopausal women. Data on BMI and prediagnostic estradiol levels were available for 624 case subjects and 1669 control subjects; data on the other sex hormones were available for fewer subjects. The relative risks (RRs) with 95% confidence intervals (CIs) of breast cancer associated with increasing BMI were estimated by conditional logistic regression on case-control sets, matched within each study for age and recruitment date, and adjusted for parity. All statistical tests were two-sided. Results: Breast cancer risk increased with increasing BMI (P-trend = .002), and this increase in RR was substantially reduced by adjustment for serum estrogen concentrations. Adjusting for free estradiol reduced the RR for breast cancer associated with a 5 kg/m(2) increase in BMI from 1.19 (95% CI = 1.05 to 1.34) to 1.02 (95% CI = 0.89 to 1.17). The increased risk was also substantially reduced after adjusting for other estrogens (total estradiol, non-sex hormone-binding globulin-bound estradiol, estrone, and estrone sulfate), and moderately reduced after adjusting for sex hormone-binding globulin, whereas adjustment for the androgens (androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone) had little effect on the excess risk. Conclusion: The results are compatible with the hypothesis that the increase in breast cancer risk with increasing BMI among postmenopausal women is largely the result of the associated increase in estrogens, particularly bioavailable estradiol..

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PURPOSE: Our aims in this pilot study were to determine whether fine needle aspirates (FNAs) provide a sufficient quantity of mRNA for cDNA microarray analysis, produce a set of quality control criteria to accept individual arrays, and determine whether gene expression profiles obtained from FNAs were representative of the source tumor. EXPERIMENTAL DESIGN: Twenty-seven women with breast cancer for treatment with primary surgery had a FNA before and at the time of surgery, and a portion of excised tumor was taken for array analysis. Control experiments were performed using two Ewing's sarcoma xenograft models. mRNA was extracted from the samples and hybridized with the reference (MCF7 cell line) on cDNA microarrays. Statistical methods were applied to identify acceptability criteria for the arrays. RESULTS: Statistical analyses demonstrated that an adequate array could be identified by calculating the SD of the log of fluorescence intensities from the arrays. Using this criterion, only 4 of the 27 patients (15%) had FNA samples suitable for array analysis. Gene expression profiles from the FNAs closely resembled that of the corresponding source tumors and were clearly distinguished from FNAs derived from the xenografts. CONCLUSIONS: SD is a useful quality index for the clinical application of cDNA microarrays. This "proof of principle" study demonstrates that FNAs from primary breast cancers can be used for microarray analysis, although without amplification, it is feasible in only a small proportion of patients. For this to be clinically useful, validated amplification techniques for FNA samples are probably required..

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Interest in chemoprevention in oncology using suppressants of prostaglandin (PG) synthesis has been stimulated by epidemiological observations that the use of aspirin and other non-steroidal inflammatory drugs (NSAIDs) is associated with reduced incidence of some cancers, including cancer of the breast. The main target of NSAID activity is the cyclooxygenase (COX) enzyme. Two isoforms of COX have been identified: COX-1, the constitutive isoform; and COX-2. the inducible form of the enzyme. COX-2 can undergo rapid induction in response to many factors such as bacterial lipopolysaccharides, growth factors, cytokines and phorbol esters. COX-2 is overexpressed in some malignancies including carcinoma of the breast. It has been suggested that such enhanced expression may lead to increased angiogenesis such that the inhibition of COX-2 might have a general anticancer effect via decreased blood vessel formation. In addition, an association between COX-2, its main product PGE2 and aromatase activity in human breast cancer suggests that such inhibitors might have an additional, specific prophylactic mechanism for this tumour. New COX-2 inhibitors are already licensed for use in the treatment of arthritis and are well tolerated. Their potential role in chemoprevention of mammary carcinogenesis in rats has already been investigated. What remains to be seen is if these findings can be extrapolated to human studies..

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The aromatase enzyme converts androgens to estrogens and is the therapeutic target for aromatase inhibitors in postmenopausal patients with estrogen receptor-positive metastatic breast cancer. Third-generation inhibitors such as letrozole are being considered as potential prophylactic agents for breast cancer. The rationale for their preventive application would be aided by knowledge of their effects on the normal breast and on other estrogen-dependent processes such as bone and lipid metabolism. Thirty-two women without active breast disease were recruited to 3-month treatment with letrozole (2.5 mg/day). Core-cut biopsies from the breast and blood samples were collected before and at the end of treatment. Plasma estradiol levels were markedly suppressed in all but two patients, who were excluded from the efficacy assessment. There was no significant change in the proliferation marker Ki67 (mean change, -23%; 95% confidence interval, -50% to +23%) or estrogen receptor in breast epithelial cells with treatment. Similarly, there were no significant changes in plasma levels of insulin-like growth factor I or lipid profiles. However, there was a significant increase (25%) in the levels of the bone resorption marker C-telopeptide crosslinks (CTx). We conclude that any prophylactic effect of letrozole is not likely to be dependent on antiproliferative effects on normal breast. Studies in healthy patients will need to recognize the potential for enhanced bone resorption..

PURPOSE: Induction of apoptosis is a key factor in the response of tumors to chemotherapy. Laboratory studies have established many of the factors that regulate and execute apoptosis, but the significance of these in human tumors is poorly understood. Therefore, the relationship between key components of this machinery was examined in primary human breast carcinomas before and 24 h after the initiation of chemotherapy. EXPERIMENTAL DESIGN: Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, and proliferation was assessed using the anti-Ki67 antibody MIB-1. Monospecific polyclonal antibodies were used for immunohistochemical detection of Bcl-2, Bax, XIAP, activated (cleaved) caspase 3 and 6, and cleaved DNA Fragmentation Factor-40 (DFF40) using paraffin-embedded tissues. RESULTS: Before treatment, a significant correlation was found between apoptosis and proliferation (r = 0.64, P < 0.0001), between caspases 3 and 6 (r = 0.49, P = 0.004) and between cleaved DFF40 and active caspases 3 (r = 0.66, P < 0.0001) or 6 (r = 0.47, P = 0.006). Before treatment, expression of inhibitor of apoptosis protein, XIAP, also correlated positively with cleaved caspase 3 (r = 0.64, P < 0.0001), caspase 6 (r = 0.36, P = 0.04), and DFF40 (r = 0.61, P = 0.0001). At 24 h after chemotherapy, significant increases in apoptosis and decreases in proliferation were observed, with the degree of increase in apoptosis inversely associated with decrease in proliferation. Chemotherapy-induced increases in Bax were correlated with increases in cleaved DFF40 (r = 0.54, P = 0.0008), but no other variables showed significant change at 24 h after initiation of chemotherapy. CONCLUSION: The pretreatment biomarker relationships suggest parallel cleavage and activation of these executioner proteins in breast cancer and that XIAP may maintain cell survival in the face of caspase activation. The findings provide in vivo evidence in human breast cancer that chemotherapy induces an apoptotic program characterized by up-regulation of Bax and cleavage of caspase substrate DFF40..

Background: This study was performed to determine whether drug action on breast tumours could also be analysed using a cell turnover index (CTI), a composite measurement Of both proliferation and apoptosis. Materials and Methods: Data were obtained from a randomized, placebo-controlled trial comparing three single doses (50, 125 and 250 mg) of intramuscular fulvestrant (Faslodex(TM), previously known as ICI 182,780) with oral tamoxifen 20 mg/day for 14-21 days in women with early, operable, ER-positive breast cancer. CTI was calculated as the ratio of the proliferation index to the apoptotic index. Results: Fulvestrant 250 mg significantly reduced CTI compared with both placebo (p = 0.0003) and tamoxifen (p = 0.026). The effect on CTI with tamoxifen was not significantly different from that with placebo. Conclusion: This study suggests that CTI may be a useful indicator of drug action on breast tumour growth. A significant reduction in growth was found with fulvestrant 250 mg compared with tamoxifen..

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10(-9)M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10(-13)M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10(-9)M E2 at 10(-12) to 10(-7)M. Tamoxifen (10(-7) to 10(-6)M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERbeta expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ERalpha..

Neoadjuvant/pre-surgical medical therapy of breast cancer provides a unique opportunity to derive biological information related to tumour response. Large clinical trials of neoadjuvant chemotherapy have established that pathological complete remission is an independent predictor of improved disease-free survival. Clinical response has been found to parallel substantial reductions in the proliferation of breast cancer cells. Increased apoptosis also occurs, but it is not closely associated with response. Numerous biological markers such as p53, bcl-2, oestrogen receptor (ER) and HER2 have been assessed for their possible role in chemoresistance/response, but the data are not clear at this stage. Continuing work using cDNA microarrays may yield new, more reliable indices of likely response and an improved insight into biological processes related to chemotherapeutic response..

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PURPOSE: To determine biologic differences, if any, between presurgical endocrine treatment with an aromatase inhibitor (vorozole) and tamoxifen in patients with postmenopausal primary breast cancer. PATIENTS AND METHODS: Randomization was to 12 weeks of 2.5 mg of vorozole per day or 20 mg of tamoxifen per day, both orally. Clinical response was assessed monthly together with serum sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogens (E1, E2, and E1S), lipids, insulin-like growth factor-1 (IGF-1), and bone metabolites (CrossLaps CTx). Tissue samples for Ki67, apoptotic index (AI), estrogen receptor, and progesterone receptor were collected at 0, 2, and 12 weeks. RESULTS: Ki67 fell by 58% and 43% (means) at 2 weeks in the vorozole and tamoxifen patients, respectively (P =.13). In the vorozole group, the correlations of proportiona