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.

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.

Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance.

Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ~30% of patients with breast cancer. However, many patients have residual cancer after chemotherapy, which correlates with a higher risk of metastatic recurrence and poorer outcome than those who achieve a pCR. We hypothesized that molecular profiling of tumors after NAC would identify genes associated with drug resistance. Digital transcript counting was used to profile surgically resected breast cancers after NAC. Low concentrations of dual specificity protein phosphatase 4 (DUSP4), an ERK phosphatase, correlated with high post-NAC tumor cell proliferation and with basal-like breast cancer (BLBC) status. BLBC had higher DUSP4 promoter methylation and gene expression patterns of Ras-ERK pathway activation relative to other breast cancer subtypes. DUSP4 overexpression increased chemotherapy-induced apoptosis, whereas DUSP4 depletion dampened the response to chemotherapy. Reduced DUSP4 expression in primary tumors after NAC was associated with treatment-refractory high Ki-67 scores and shorter recurrence-free survival. Finally, inhibition of mitogen-activated protein kinase kinase (MEK) synergized with docetaxel treatment in BLBC xenografts. Thus, DUSP4 downregulation activates the Ras-ERK pathway in BLBC, resulting in an attenuated response to anti-cancer chemotherapy.

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.

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.

Endocrine therapy, new biologicals, and new study designs for presurgical studies in breast cancer.

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.

Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with the Genomic Health recurrence score in early breast cancer.

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.

Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.

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.

Predictive algorithms for adjuvant therapy: TransATAC.

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.

Association between breast cancer subtypes and response to neoadjuvant anastrozole.

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.

Close and stable relationship between proliferation and a hypoxia metagene in aromatase inhibitor-treated ER-positive breast cancer.

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.

Nuclear NF-κB/p65 expression and response to neoadjuvant chemotherapy in breast cancer.

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.

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.

A marker of homologous recombination predicts pathologic complete response to neoadjuvant chemotherapy in primary breast cancer.

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.

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.

Comparative validation of the SP6 antibody to Ki67 in breast cancer.

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.

Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

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.

Prediction of risk of distant recurrence using the 21-gene recurrence score in node-negative and node-positive postmenopausal patients with breast cancer treated with anastrozole or tamoxifen: a TransATAC study.

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.

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.