Immunohistochemical BAG1 expression improves the estimation of residual risk by IHC4 in postmenopausal patients treated with anastrazole or tamoxifen: a TransATAC study.

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.

Effects of cyclin D1 gene amplification and protein expression on time to recurrence in postmenopausal breast cancer patients treated with anastrozole or tamoxifen: a TransATAC study.

INTRODUCTION: Gene amplification of CCND1 is observed in a subgroup of breast cancers with poor prognosis, whereas overexpression of the protein cyclin D1 has been linked to both worse and better clinical outcome. CCND1 amplification and protein overexpression have also been associated with resistance to treatment with tamoxifen or even to a potentially detrimental effect of tamoxifen. METHODS: To clarify these challenging and partly contrasting treatment predictive and prognostic links for cyclin D1 we analysed a large cohort of postmenopausal breast cancer patients randomised to receive either adjuvant anastrozole or tamoxifen, as part of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. The CCND1 amplification status and protein expression of cyclin D1 were assessed by chromogenic in situ hybridisation and immunohistochemistry, respectively, in 1,155 postmenopausal, oestrogen-receptor-positive breast cancer patients included in the TransATAC substudy. RESULTS: Amplification of CCND1 was observed in 8.7% of the tumours and was associated with increased risk of disease recurrence (hazard ratio = 1.61; 95% confidence interval, 1.08 to 2.41) after adjustment for other clinicopathological parameters. In contrast, nuclear expression of cyclin D1 protein was associated with decreased recurrence rate (hazard ratio = 0.6; 95% confidence interval, 0.39 to 0.92). The intensity of nuclear or cytoplasmic expression was not of prognostic value. There was no significant interaction between cyclin D1 status and treatment efficacy, ruling out any major detrimental effect of tamoxifen in CCND1-amplified postmenopausal breast cancer. CONCLUSIONS: In summary, CCND1 amplification and low nuclear expression of cyclin D1 predicted poor clinical outcome in postmenopausal breast cancer patients treated with either anastrozole or tamoxifen. TRIAL REGISTRATION: Current Controlled Trials ISRCTN18233230.

ER and HER2 expression are positively correlated in HER2 non-overexpressing breast cancer.

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.

Extreme loss of immunoreactive p-Akt and p-Erk1/2 during routine fixation of primary breast cancer.

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 characterize the differences in immunoreactivity of common biomarkers that may occur (a) due to tissue handling at surgery and (b) 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 to 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-Erk 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). 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 vs 101 and 69 vs 193, respectively; both P<0.0001 [two-sided]). This difference was significantly greater in mastectomy than lumpectomy specimens for p-Erk1/2 (P=0.01). CONCLUSIONS: The delay in fixation in core-cuts taken after post-operative 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 phospho-staining 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.

Relationship between oestrogen receptor status and proliferation in predicting response and long-term outcome to neoadjuvant chemotherapy for breast cancer.

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.

Pre-surgical study of the biological effects of the selective cyclo-oxygenase-2 inhibitor celecoxib in patients with primary breast cancer.

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.

The prognostic significance of Ki67 before and after neoadjuvant chemotherapy in breast cancer.

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.

The farnesyltransferase inhibitor R115777 (tipifarnib) in combination with tamoxifen acts synergistically to inhibit MCF-7 breast cancer cell proliferation and cell cycle progression in vitro and in vivo.

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.

BRCA1/BRCA2 mutation status and analysis of cancer family history in participants of the Royal Marsden Hospital tamoxifen chemoprevention trial.

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.

Proliferation and apoptosis as markers of benefit in neoadjuvant endocrine therapy of breast cancer.

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.

Biomarker changes during neoadjuvant anastrozole, tamoxifen, or the combination: influence of hormonal status and HER-2 in breast cancer--a study from the IMPACT trialists.

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.

Short-term changes in Ki-67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence-free survival.

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.

Comparison of the selective estrogen receptor modulator arzoxifene (LY353381) with tamoxifen on tumor growth and biomarker expression in an MCF-7 human breast cancer xenograft 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.

Phase II study of the efficacy and tolerability of two dosing regimens of the farnesyl transferase inhibitor, R115777, in advanced breast cancer.

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 > or = 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 43%; 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.

Comparison of the systemic and intratumoral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer.

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 proportional changes in Ki67 at 2 weeks with tumor volume changes and clinical response at 12 weeks were not significant (P =.09) and marginally significant (P =.04), respectively. Serum lipids did not differ between groups. Serum levels of EI, E2, and E1S were suppressed markedly by vorozole, whereas levels of SHBG increased and LH and FSH fell significantly with tamoxifen. IGF-1 levels fell significantly with tamoxifen (P =.001) compared with the nonsignificant rise with vorozole. Twelve-week CTx values fell by 19% with tamoxifen (P =.006) and rose by 11% with vorozole (P =.15). CONCLUSION: The correlation with vorozole of Ki67 with volume and clinical response supports this as an intermediate marker. The nonsignificant effects on bone and lipid metabolism by the aromatase inhibitor may be important to consider for adjuvant and potential prevention strategies.

Correlation between cyclooxygenase-2 expression and angiogenesis in human breast cancer.

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.

Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention.

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.

Molecular profiling of aromatase inhibitor-treated postmenopausal breast tumors identifies immune-related correlates of resistance.

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.

CYP2D6 and UGT2B7 genotype and risk of recurrence in tamoxifen-treated breast cancer patients.

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.