Marleen Kok

Oestrogen receptor–co‐factor–chromatin specificity in the transcriptional regulation of breast cancer

The complexity of oestrogen receptor α (ERα)‐mediated transcription is becoming apparent, but global insight into the co‐regulatory proteins that assist ERα transcription is incomplete. Here, we present the most comprehensive chromatin‐binding landscape of ERα co‐regulatory proteins to date. We map by ChIP‐seq the essential p160 co‐regulators (SRC1, SRC2 and SRC3), and the histone acetyl transferases p300 and CBP in MCF‐7 breast cancer cells. We find a complex network of co‐regulator binding, with preferential binding sites for each co‐regulator. Unlike previous suggestions, we find SRC recruitment almost exclusively following ligand treatment. Interestingly, we find specific subsets of genes regulated by ligand‐dependent and ‐independent co‐regulator recruitment. Co‐factor‐binding profiles were integrated with expression data from cell lines and primary tumour cohorts, to reveal specific transcriptional networks that influence clinical outcome. Genes that are bound by SRC3, but not other p160 proteins, have predictive value in cohorts of breast cancer patients. By generating a robust and global view of co‐factor‐binding properties, we discover new levels of co‐regulator complexity, but also reveal specific gene networks that may influence endocrine response.

Microarray-Based Determination of Estrogen Receptor, Progesterone Receptor, and HER2 Receptor Status in Breast Cancer

Purpose: The level of estrogen receptor (ER), progesterone receptor (PR), and HER2 aids in the determination of prognosis and treatment of breast cancer. Immunohistochemistry is currently the predominant method for assessment, but differences in methods and interpretation can substantially affect the accuracy, resulting in misclassification. Here, we investigated the association of microarray-based mRNA expression levels compared with immunohistochemistry. Experimental Design: Microarray mRNA quantification of ER, PR, and HER2 was done by the developed TargetPrint test and compared with immunohistochemical assessment for breast tumors from 636 patients. Immunohistochemistry was done in a central laboratory and in an independent reference laboratory according to American Society of Clinical Oncology/College of American Pathologists guidelines for 100 cases. For HER2 immunohistochemistry 2+ cases, additional chromogenic in situ hybridization (CISH) was used to determine the final status. Results: ER concordance between microarray and central immunohistochemistry was 93 [95 confidence interval (95 CI), 91-95]. Only 4 of immunohistochemistry-positive samples were classified negative using microarray, whereas 18 of immunohistochemistry-negative samples showed a positive microarray ER status. Concordance for PR was 83 (95 CI, 80-86) and 96 of all samples showed an identical classification of HER2 status by microarray and immunohistochemistry/CISH (95 CI, 94-98). Nine percent of immunohistochemistry HER2-positive samples showed a negative microarray classification. Detailed review of 11 cases with discordant classifications by American Society of Clinical Oncology/College of American Pathologists and central immunohistochemistry indicated that microarray assessment was likely to add additional information in 5 cases. Conclusion: Microarray-based readout of ER, PR, and HER2 shows a high concordance with immunohistochemistry/CISH and provides an additional, objective, and quantitative assessment of tumor receptor status in breast cancer. (Clin Cancer Res 2009;15(22):700311)

Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer.

Phosphorylation of oestrogen receptor α at serine 305 (ERαS305‐P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERαS305‐P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n = 248) and patients with advanced disease (n = 129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence‐free survival (RFS) for ERαS305‐P‐negative tumours (multivariate HR = 0.53, 95% CI 0.32–0.86, p = 0.010), but not for ERαS305‐P‐positive tumours (multivariate HR = 1.01, 95% CI 0.33–3.05, p = 0.99) (interaction p = 0.131). Notably, ERαS305‐P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR = 0.64, 95% CI 0.30–1.37, p = 0.248), indicating that ERαS305‐P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERαS305‐P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERαS305‐P‐positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup. Copyright

Estrogen receptor-alpha phosphorylation at serine-118 and tamoxifen response in breast cancer.

Although estrogen receptor-alpha (ER) [corrected] is a marker used to identify breast cancer patients most likely to benefit from endocrine therapy, approximately 50% of ER-positive [corrected] breast carcinomas are resistant to tamoxifen. Preclinical studies have shown that phosphorylation of ER [corrected] at serine-118 (ER alpha S118-P) is required for tamoxifen-mediated inhibition of ER-induced [corrected] gene expression. We evaluated the association between recurrence-free survival after tamoxifen treatment and ER alpha S118-P expression by use of Cox proportional hazards regression. Data were from 239 premenopausal patients with breast cancer who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment vs no systemic treatment. ER alpha S118-P expression was assessed by immunohistochemistry and categorized by use of the Allred score (low expression = score of 0-6; high expression = score of 7-8). All statistical tests were two-sided. Compared with systemically untreated patients, we found evidence of a benefit from adjuvant tamoxifen among patients whose tumors had high ER alpha S118-P expression (23.7 recurrences per 1000 person-years versus 72.2 recurrences per 1000 person-years, hazard ratio [HR] of recurrence = 0.36, 95% confidence interval [CI] = 0.20 to 0.65) but not among patients whose tumors had low expression (51.0 recurrences per 1000 person-years versus 57.0 recurrences per 1000 person-years, HR of recurrence = 0.87, 95% CI = 0.51 to 1.48), a statistically significant difference (P for interaction = .037). ER alpha 118-P was not associated with recurrence-free survival among untreated patients. Thus, ER alpha S118-P expression appears to be associated with response to tamoxifen. [corrected]

The CYP2C19*2 genotype predicts tamoxifen treatment outcome in advanced breast cancer patients

AIMS Tamoxifen is metabolized by cytochrome P450s, with an important role for CYP2D6. Recently, we demonstrated in 80 patients that CYP2C19*2 is associated with increased survival in breast cancer patients using tamoxifen. Here, we aimed to confirm this in a large group of 499 patients. MATERIALS & METHODS A total of 499 estrogen receptor-positive primary breast tumor specimens of advanced disease patients treated with first-line tamoxifen were genotyped for CYP2C19*2 and *17 variant alleles, with primary end point time-to-treatment failure (TTF). Effects of CYP2C19, independent of treatment, were analyzed in 243 primary systematic untreated patients. RESULTS CYP2C19*2 hetero- and homozygote patients combined showed significantly longer TTFs (hazard ratio [HR]: 0.72; 95% CI: 0.57-0.90; p = 0.004). In multivariate analysis, including CYP2D6*4 status, CYP2C19*2 remained independently associated with TTF (HR: 0.73; 95% CI: 0.58-0.91; p = 0.007). In untreated patients, the CYP2C19*17 allele was significantly associated with a longer disease-free interval (HR: 0.66; 95%CI: 0.46-0.95; p = 0.025). CONCLUSION CYP2C19 genotyping is potentially important for tamoxifen therapy for advanced disease and for breast cancer prognosis.

Mammosphere-derived gene set predicts outcome in patients with ER-positive breast cancer.

Tumourigenic subpopulations with stem cell‐like features have been identified in breast tumours and breast cancer cell lines. The hormone receptor status, molecular characteristics and clinical significance of these cells are still matters of debate. Enrichment for tumourigenic cells without the requirement of surface markers can be achieved by the in vitro mammosphere culture assay. Here we compared the hormone receptor status and genome‐wide gene expression profiles of mammospheres derived from four oestrogen‐receptor (ER) positive breast cancer cell lines with those of the respective parental cells. Immunohistochemistry and gene expression profiling revealed a significant reduction in the expression of progesterone receptor, proliferation and cell cycle regulated genes in mammospheres when compared to parental cell lines. The 200 most differentially expressed genes between mammospheres and parental cell lines were used to generate a ‘mammosphere‐derived’ gene set. Hierarchical clustering of gene expression profiles of two independent cohorts of primary ER‐positive cancers based on the ‘mammosphere‐derived’ gene set revealed that the subgroup of breast cancers with profiles similar to those of mammospheres has a significantly longer overall survival. In conclusion, tumour‐initiating breast cancer cells grown in mammospheres seem to reside in a quiescent state. ER‐positive breast cancers with expression profiles similar to those of mammospheres have a better outcome, providing evidence in support of the concept that outcome of patients with ER‐positive disease is for a large part determined by cell cycle and proliferation activity. Copyright

Targeting the programmed cell death-1 pathway in breast and ovarian cancer.

Purpose of review Immune checkpoint blockade is changing cancer therapy. Targeting the programmed cell death-1 (PD-1) pathway releases T cells from inhibitory signals within the tumor microenvironment, thereby activating a latent antitumor immune response. Here, we review the biology underlying the activity of PD-1/programmed cell death-ligand 1 (PD-L1) antagonists, and data describing their clinical activity in breast and ovarian cancer. Recent findings Several antagonists of PD-1 and PD-L1 have been tested in breast and ovarian cancer. These drugs are generally well tolerated, with some immune-related adverse events that are typically easily managed. Objective response rates generally range from about 10 to 20% in both breast cancer and ovarian cancer, with durable responses noted in multiple trials. Selecting patients with PD-L1 expression by cells within the tumor microenvironment appears to enrich for responses. These agents are under accelerated development based on these promising early data. Summary Monoclonal antibody-based blockade of the PD-1 pathway results in objective and durable clinical responses in a subset of patients with breast or ovarian cancers, particularly those with PD-L1-positive cells within the tumor microenvironment. Current priorities are to refine biomarkers of therapeutic response, and to develop combination immunotherapy strategies that integrate PD-1/PD-L1 antagonists with both standard and immune-based cancer therapies to increase efficacy.

Additional value of the 70-gene signature and levels of ER and PR for the prediction of outcome in tamoxifen-treated ER-positive breast cancer

BACKGROUND Breast cancer patients with node positive disease can have an excellent outcome with tamoxifen only. It is unclear whether analysing both the 70-gene signature and hormone receptors provides superior prediction of outcome in tamoxifen-treated patients than either alone. METHODS Three series were evaluated: 121 patients (81% node positive) received adjuvant tamoxifen, 151 patients did not receive tamoxifen (10% node positive) and 92 patients received tamoxifen for metastatic disease. The 70-gene signature was analysed using MammaPrint. Oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry was evaluated following St. Gallen Consensus (Highly Endocrine Responsive: ER and PR ≥ 50%, Incompletely Endocrine Responsive: ER and/or PR low or either one absent). RESULTS In patients treated with adjuvant tamoxifen, both the 70-gene signature (adjusted for Endocrine Response Categories HR 2.17, 95%CI 1.01-4.66) as well as the Endocrine Response Categories (adjusted for 70-gene signature HR 6.35, 95%CI 1.90-21.3) were associated with breast-cancer-specific-survival (BCSS). Also in patients treated with tamoxifen for metastatic disease, combined analysis of the 70-gene signature and ER/PR revealed additional value (multivariate Cox regression, p = 0.013). In patients who did not receive tamoxifen, only the 70-gene signature was associated with outcome. CONCLUSION In the series analysed, the 70-gene signature was mainly a prognostic factor, while ER and PR levels were mainly associated with outcome after tamoxifen. Combination of these three factors may improve outcome prediction in tamoxifen-treated patients.

Neoadjuvant Therapy for Breast Cancer: Established Concepts and Emerging Strategies

In the last decade, the systemic treatment approach for patients with early breast cancer has partly shifted from adjuvant treatment to neoadjuvant treatment. Systemic treatment administration started as a ‘one size fits all’ approach but is currently customized according to each breast cancer subtype. Systemic treatment in a neoadjuvant setting is at least as effective as in an adjuvant setting and has several additional advantages. First, it enables response monitoring and provides prognostic information; second, it downstages the tumor, allowing for less extensive surgery, improved cosmetic outcomes, and reduced postoperative complications such as lymphedema; and third, it enables early development of new treatment strategies by using pathological complete remission as a surrogate outcome of event-free and overall survival. In this review we give an overview of the current standard of neoadjuvant systemic treatment strategies for the three main subtypes of breast cancer: hormone receptor-positive, triple-negative, and human epidermal growth factor receptor 2-positive. Additionally, we summarize drugs that are under investigation for use in the neoadjuvant setting.

Decreased expression of ABAT and STC2 hallmarks ER‐positive inflammatory breast cancer and endocrine therapy resistance in advanced disease

Patients with Estrogen Receptor α‐positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non‐IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer.