Background Breast cancer prognosis changes over time in complex ways depending on individual risk factors. This study aimed to analyze how breast cancer outcomes in New Zealand women change over time and identify features associated with breast cancer specific survival and locoregional recurrence across different receptor subtypes. Methods A retrospective cohort study was conducted using data from Te Rēhita Mate Ūtaetae (Breast Cancer Foundation National Register) on 21,574 women diagnosed with invasive breast cancer between 2000-2019. We applied k-medians survival clustering, landmark analysis, and piecewise Cox regression to identify time-specific risk patterns and prognostic features. Results Survival improved significantly for women diagnosed more recently. Triple-negative breast cancer had the poorest 5-year breast cancer specific survival but demonstrated better outcomes for women surviving beyond this period. In contrast, ER+/HER2- tumors, associated with favorable short-term outcomes, showed the highest risk of late recurrence and breast cancer mortality beyond 10 years. Younger age at diagnosis (≤44 years) was associated with increased recurrence risks, especially for ER-/HER2+ tumors. Radiation therapy reduced early LRR across subtypes. Tumor grade was inversely associated with late recurrence, while stage 2 disease in ER+ tumors markedly elevated late recurrence odds compared to stage 1. Conclusions This study demonstrates the dynamic nature of breast cancer prognosis, with key findings emphasizing the time-dependent shifts in risk across receptor subtypes. These findings underscore the importance of personalized, receptor-specific follow-up strategies, including extended monitoring for subgroups at heightened long-term risk.

Accurate prediction of breast cancer-specific survival is crucial for guiding personalized treatment decisions and improving patient outcomes. This study evaluated the performance of machine learning approaches (Random Survival Forest, RSF and Generalized Boosted Model, GBM) alongside traditional Cox proportional hazards models for predicting survival in 21,574 women diagnosed with stage I-IV breast cancer in New Zealand between 2000-2019. Performance comparisons using time-dependent Area Under the Curve and Brier score metrics demonstrated that RSF consistently outperformed both Cox regression variants and GBM across all time points. Distinct differences emerged in survival predictions between modelling approaches: RSF captured a sharper initial decline in survival for most tumour receptor subtypes and better differentiated the favourable prognosis of ER+/HER2- tumours compared to other subtypes. Notably, variable importance analysis revealed fundamentally different prognostic emphases between modelling approaches—disease stage dominated Cox model predictions while tumour receptor subtype most strongly influenced RSF predictions. These findings highlight how machine learning approaches can capture complex, nonlinear relationships between clinical variables and survival outcomes that may be missed by traditional statistical models. The complementary insights provided by different modelling approaches suggest potential value in their combined use for enhanced risk stratification and more tailored treatment planning in breast cancer management, particularly when accounting for tumour biological characteristics alongside conventional staging factors.

Objectives: To perform the first national analysis of demographic and clinicopathological features associated with the HER2 positive, HER2-low, and HER2-zero invasive breast cancers in New Zealand. The study will reveal the proportion of women who may benefit from new HER2-targeted antibody drug conjugate (ADC) therapies. Methods: Utilising data from Te Rēhita Mate Ūtaetae (Breast Cancer Foundation NZ National Register), the study analysed data from women diagnosed with invasive breast cancer over a 21-year period. The HER2 status of tumours was classified into three categories—HER2-zero, HER2-low, HER2-positive. Results: From 2009–2021, 94% of women underwent HER2 testing, with 14% diagnosed with HER2-positive breast cancer. For advanced-stage disease, 38% of those formerly classified as HER2-negative were reclassified as HER2-low. Including HER2-positive breast cancers, this indicates that 60% of women with advanced breast cancer may potentially benefit from the new HER2-directed ADCs (approximately 120 women per year). Conclusions: The findings suggest a significant proportion of women with invasive breast cancer in New Zealand could benefit from new HER2-targeted treatments. There is a need to standardise HER2 testing to enhance personalised treatment and improve outcomes.

The clinical importance of assessing and combining data on TP53 mutations and isoforms is discussed in this article. It gives a succinct overview of the structural makeup and key biological roles of the isoforms. It then provides a comprehensive summary of the roles that p53 isoforms play in cancer development, therapy response and resistance. The review provides a summary of studies demonstrating the role of p53 isoforms as potential prognostic indicators. It further provides evidence on how the presence of TP53 mutations may affect one or more of these activities and the association of p53 isoforms with clinicopathological data in various tumour types. The review gives insight into the present diagnostic hurdles for identifying TP53 isoforms and makes recommendations to improve their evaluation. In conclusion, this review offers suggestions for enhancing the identification and integration of TP53 isoforms in conjunction with mutation data within the clinical context.

Supplementary Table S1: Summary of the TCGA tumour sets for which RNAseq V2 RSEM normalised data was downloaded on 20th October 2015 and used for TP53 transcript expression analyses. Supplementary Table S2: Summary of QC for RNAseq and RNA expression using RNAseq for the nine human cell lines. Supplementary Table S3: UCSC and Locus Reference Genomic (LRG) IDs for canonical TP53 transcript variants. Supplementary Table S4: Primer sequences used for ddPCR and RT-qPCR. Supplementary Table S5: Read data for RNAseq RSEM Expected count, RNAseq Tuxedo-FPKM, RNAseq RSEM-FPKM, ddPCR and RT-qPCR. Supplementary Table S6: RNAseq reads containing both PCR primers Supplementary Table S7: RNAseq reads in 125nt specific regions in exon 4 and AluJB upstream and exon5/6 downstream of the 125nt unique to the 5'-UTR of delta133TP53 transcript subclass Supplementary Table S8: Mutations and sequencing errors do not significantly affect alignment of sequence reads to the TP53 locus Supplementary Figure S1: Shows examples of fluorescence amplitude by ddPCR Supplementary Figure S2: Dissociation curves for TP53 primer pairs by RT-qPCR Supplementary Figure S3: Sanger Sequencing of the amplicons for FL/delta40TP53_T1, FL/delta40TP53_T2, delta133TP53, TP53alpha and TP53beta Supplementary Figure S4: Relative abundance of TP53 transcript subclasses relative to FL/delta40TP53_T1.

Nine of the ten papers published in this Special Issue explore various aspects of the multifunctional protein Y-box binding protein-1 (YB-1) and its role in cancer [...].

Circulating tumour DNA (ctDNA) analysis promises to improve the care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 New Zealand (NZ) unresectable advanced-stage cutaneous melanoma patients through multiple cycles of immunotherapy, to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. During the course of treatment, a high level of dynamic mutational complexity was identified in blood plasma of these patients, including: multiple BRAF mutations in the same patient, clinically-relevant BRAF mutations emerging through therapy, and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis-reanalysis concordance as well as by concordance between three ctDNA measurement technologies: droplet digital polymerase chain reaction (ddPCR), a custom melanoma-specific amplicon next-generation sequencing (NGS) panel and mass spectrometry. In addition, we observed >90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared to standard EDTA blood collection protocols with rapid processing. We also found that undetectability of ctDNA at a proportion of treatment cycles was associated with both clinical benefit (best RECIST response) and prognosis (disease-specific survival). In summary, we found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically-relevant mutations, adding support for expanded implementation of this technology to guide in-treatment tailored cancer therapy.

The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34–5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53β transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53’s alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5’ and 3’ ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.

To assist clinicians with treatment decisions for patients with breast cancer, we investigated whether the preoperative plasma levels of RNAs and the protein cancer antigen 15-3 were associated with prognosis. We identified a novel biomarker, microRNA-923, and combined this with cancer antigen 15-3 and clinicopathological features to build a multivariable model predictive of prognosis, irrespective of treatment, in 253 patients with breast cancer. Introduction: Circulating biomarkers have been increasingly used in the clinical management of breast cancer. The present study evaluated whether RNAs and a protein present in the plasma of patients with breast cancer might have utility as prognostic biomarkers complementary to existing clinical tests. Patients and Methods: We performed microarray profiling of small noncoding RNAs in plasma samples from 30 patients with breast cancer and 10 control individuals. Two small noncoding RNAs, including microRNA (miR)-923, were selected and quantified in plasma samples from an evaluation cohort of 253 patients with breast cancer, using droplet digital polymerase chain reaction. We also measured cancer antigen (CA) 15-3 protein levels in these samples. Cox regression survival analysis was used to determine which markers were associated with patient prognosis. Results: As independent markers of prognosis, the plasma levels of miR-923 and CA 15-3 at the time of surgery for breast cancer were significantly associated with prognosis, irrespective of treatment (Cox proportional hazards, P = 3.9 x 10(-3) and 1.9 x 10(-9), respectively). After building a multivariable model with standard clinical and pathological features, the addition of miR923 and CA 15-3 information into the model resulted in a significantly better predictor of disease recurrence in patients, irrespective of treatment, compared with the use of clinicopathological data alone (area under the curve at 3 years, 0.858 vs. 0.770 with clinicopathological markers only; P = .017). Conclusion: We propose that the plasma levels of miR-923 and CA 15-3, combined with standard clinicopathological predictors, could be used as a preoperative, noninvasive estimate of patient prognosis to identify which women might need more aggressive treatment or closer surveillance after surgery for breast cancer. (C) 2019 Elsevier Inc. All rights reserved.

We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the E2F1 promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

Abstract A sea-change is imminent for cancer medicine, due to the use of non-invasive genomic biomarkers in blood to inform screening, diagnosis and the selection of treatment. This technology may be used routinely in oncology within five years. Although numerous studies, including work in our laboratory, have shown that genomic analysis of blood can detect the presence and even the type of cancer, researchers have only scratched the surface of what this technology can do. In our laboratory, we are generating new methods to improve the sensitivity and accuracy of such tests. Cell-free DNA from a metastatic colorectal cancer patient has been used to directly compare the sensitivity of mutation detection across three platforms - a custom QiaSeq Targeted amplicon sequencing panel, droplet digital PCR (ddPCR) and UltraSEEK lung panel. UltraSEEK and ddPCR platforms show better sensitivity for detection of the KRAS G12A mutation, compared to the QiaSeq Targeted DNA sequencing panel. This pilot study shows that when looking to specifically detect a small number of known mutations, UltraSEEK or ddPCR gives the highest sensitivity, but when mutations are not limited to a few or are completely unknown, the QiaSeq Targeted DNA panel allows identification of the entire repertoire of mutations in specific cancer-associated genes. Citation Format: Sandra Fitzgerald, Annette Lasham, Cherie Blenkiron, Paula Shields, Ben Lawrence, Cristin Print. Technical advances in plasma genomic biomarkers for mutation detection and monitoring in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3667.

Malignant pleural mesothelioma (MPM) is a devastating disease characterized by aggressive growth and local invasion, poor outcome and limited therapeutic options. YB-1 is a multifunctional oncoprotein, which is often up-regulated in cancer and associated with aggressiveness and poor patient outcome. Also, YB-1 is actively secreted by cells upon various stresses. Besides numerous other functions, YB-1 has been described to stimulate cancer cell migration and invasion via regulation of EMT-related factors such as Snail and Twist. Cells were treated with LPS (20 ng/ml) or grown under hypoxic conditions (1% O2, N2 balanced) for 24 hours. YB-1 levels in supernatants were quantified via western blot. Changes in EMT markers were measured by qPCR. Cell migration and cell cycle was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. YB-1 overexpression was achieved by stable transfection with an expression plasmid and confirmed via qPCR and western blot. YB-1 is secreted by MPM cells as well as the immortalised mesothelial cell line Met-5A after exposure to LPS or under hypoxic conditions. When MPM cells as well as primary mesothelial cells derived from pericardial fluid were exposed to soluble YB-1, we observed an upregulation of EMT markers such as SNAIL and TWIST as well as significantly increased migratory capacity. Similar effects were observed in cell lines which overexpress YB-1. YB-1 overexpressing cells migrated at significantly higher speed and covered a larger area. Also, when grown at low density, EMT-like changes in cell morphology as well as scattering was observed. Additionally, while cell divisions occurred at higher frequencies, the duration of the M phase was significantly prolonged. Our data highlight a crucial role of both intracellular and soluble YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, YB-1 also interferes with the cell cycle of MPM cells. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy and current therapy is essentially palliative. Novel therapy targets are urgently needed. YB-1 is a multifunctional oncoprotein associated with poor patient outcome and is related to increased chemoresistance in tumours including NSCLC. It is widely accepted that YB-1 plays a role in the cell growth of many cancers, and we recently confirmed this in MPM cells. Here, we begin to evaluate YB-1 as a therapeutic target in this disease. YB-1 expression was determined by Western blot in MPM cell lines and their drug resistant sublines. Growth and colony formation assays were conducted after transfection with YB-1- or control-siRNA. These were also carried out in combination with cisplatin, gemcitabine or vinorelbine treatment. Apoptosis was assessed by PI and annexin V staining in YB-1 knockdown MPM cells. Migration of MPM cells was measured using videomicroscopy and manual cell tracking. Luciferase-expressing MPM cells transfected with YB-1- or control-siRNA were injected into female SCID mice (n=10, intra-peritoneal injection). Tumour growth was monitored via luminescence by injecting luciferin (intra-peritoneal injection) and measuring bioluminescence on an In Vitro Imaging System (IVIS) once a week for 4 weeks. Tumour weight determined after humane euthanasia of animals at the termination of the experiment. YB-1-siRNA significantly inhibited the growth of MPM cell lines in vitro and was overexpressed in MPM cells compared to the immortalised mesothelial cell line MeT-5A. Growth of MeT-5A and primary mesothelial cell lines was not affected significantly by YB-1 knockdown. Mice injected with YB-1 knockdown cells displayed significantly lower tumour burden, evidenced by bioluminescence in live mice using IVIS and lower tumour weight after harvest. TALI assays showed an increase in apoptotic cells after YB-1 siRNA transfection in vitro, and cells transfected with siRNA showed sensitisation to cisplatin and vinorelbine. YB-1 was expressed at higher levels, and higher migratory capacity was observed in drug resistant MPM cell lines compared to parental cell lines. These results highlight the importance of YB-1 in MPM biology both in vitro and in vivo. YB-1 knockdown inhibits growth via apoptosis, sensitises MPM cells to commonly prescribed drugs and contributes to a change in behaviour of drug resistant MPM cells. This project serves as a basis for the further investigation of YB-1 as a novel therapeutic target.

The TP53 family consists of three sets of transcription factor genes, TP53, TP63 and TP73, each of which expresses multiple RNA variants and protein isoforms. Of these, TP53 is mutated in 25-30% of breast cancers. How TP53 mutations affect the interaction of TP53 family members and their isoforms in breast cancer is unknown. To investigate this, 3 independent breast cancer cohorts were stratified into 4 groups based on oestrogen receptor (ER) and TP53 mutation status. Using bioinformatic methodologies, principal signalling pathways associated with the expression of TP53 family members were identified. Results show an enrichment of IFN-γ signalling associated with TP63 RNA in wild type TP53 (wtTP53), ER negative (ER-) tumours and with Δ133TP53 RNA in mutant TP53 (mTP53) ER positive (ER+) tumours. Moreover, tumours with low IFN-γ signalling were associated with significantly poorer patient outcome. The predicted changes in expression of a subset of RNAs involved in IFN-γ signalling were confirmed in vitro. Our data show that different members of the TP53 family can drive transcription of genes involved in IFN-γ signalling in different breast cancer subgroups.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy and current therapy is essentially palliative. YB-1 is a multifunctional oncoprotein associated with poor patient outcome in tumors including NSCLC and is related to increased chemoresistance. It is widely accepted that YB-1 plays a role in the cell growth of many tumors. YB-1 has been implicated in suppressing apoptotic pathways such as the mTOR/STAT3 pathway and disrupting the cell cycle via transcriptionally regulating cyclins A, B1 and D1 in multiple cancers. We recently found YB-1 to be overexpressed in MPM cells and that siRNA-mediated knockdown inhibited growth. Here we investigate the mechanisms behind YB-1's role in MPM cell growth and subsequent effects on drug resistance. YB-1 expression and YBX1 mRNA was determined by Western blot and RT-qPCR, respectively, in MPM cell lines and their drug resistant sublines. Growth assays and colony formation assays with or without siRNA transfection elucidated the role of YB-1 in MPM growth. These were also conducted in combination with cisplatin, gemcitabine and vinorelbine treatment. TALI apoptosis assays were conducted to investigate the effect of YB-1 silencing in MPM cells. YB-1 siRNA significantly inhibited the growth of MSTO, VMC23 and MM05 cells (P<0.05) and was overexpressed compared to the immortalized mesothelial cell line MeT-5A in MSTO and VMC23. TALI apoptosis assays revealed that growth inhibition was due to apoptosis and necrosis in MSTO cells but not in VMC23, suggesting cell cycle arrest to be the cause of growth inhibition in this cell line. Interestingly, YB-1 knockdown in MSTO cells resulted in a sensitization to cisplatin, gemcitabine and vinorelbine, but increased resistance to these drugs in VMC23 and MM05, suggesting a link between the mode of growth regulation YB-1 plays and the effect of its silencing on innate drug resistance in MPM cells. Additionally, YB-1 levels were upregulated in MSTO and MM05 cells with acquired drug resistance, compared to parental cells. YB-1 plays different roles in MPM cell growth which are cell type dependent. When acting upon apoptotic pathways, YB-1 knockdown sensitized MPM cells to chemotherapy. In other cases, YB-1-mediated cell cycle arrest resulted in heightened resistance. Finally, YB-1 is upregulated in cells with acquired drug resistance, indicating that it plays an important role in the acquired resistance to cisplatin, gemcitabine and vinorelbine in MPM.

Introduction: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure. On a genomic level, MPM is characterized by frequent chromosomal deletions of tumor suppressors, including microRNAs. MiR-137 plays a tumor suppressor role in other cancers, so the aim of this study was to characterize it and its target Y-box binding protein 1 (YBX1) in MPM. Methods: Expression, methylation, and copy number status of miR-137 and its host gene MIR137HG were assessed by polymerase chain reaction. Luciferase reporter assays confirmed a direct interaction between miR-137 and Y-box binding protein 1 gene (YBX1). Cells were transfected with a miR-137 inhibitor, miR-137 mimic, and/or YBX1 small interfering RNA, and growth, colony formation, migration and invasion assays were conducted. Results: MiR-137 expression varied among MPM cell lines and tissue specimens, which was associated with copy number variation and promoter hypermethylation. High miR-137 expression was linked to poor patient survival. The miR-137 inhibitor did not affect target levels or growth, but interestingly, it increased miR-137 levels by means of mimic transfection suppressed growth, migration, and invasion, which was linked to direct YBX1 downregulation. YBX1 was overexpressed in MPM cell lines and inversely correlated with miR-137. RNA interference-mediated YBX1 knockdown significantly reduced cell growth, migration, and invasion. Conclusions: MiR-137 can exhibit a tumor-suppressive function in MPM by targeting YBX1. YBX1 knockdown significantly reduces tumor growth, migration, and invasion of MPM cells. Therefore, YBX1 represents a potential target for novel MPM treatment strategies. (C) 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

We investigated the transcriptional pathways activated by estrogen receptor-cc and their relationship with patient survival after treatment with endocrine therapy. From this analysis, we propose that multimodal assessment Of breast tumors, using a combination of estrogen receptor-alpha status, estrogen receptor-alpha mRNA expression, and genomic indicators of estrogen pathway activity, could be useful for both research and treatment stratification.Background: Molecular markers have transformed our understanding of the heterogeneity of breast cancer and have allowed the identification of genomic profiles of estrogen receptor (ER)-alpha signaling. However, our understanding of the transcriptional profiles of ER signaling remains inadequate. Therefore, we sought to identify the genomic indicators of ER pathway activity that could supplement traditional immunohistochemical (IHC) assessments of ER status to better understand ER signaling in the breast tumors of individual patients. Materials and Methods: We reduced ESR1 (gene encoding the ER-alpha protein) mRNA levels using small interfering RNA in ER+ MCF7 breast cancer cells and assayed for transcriptional changes using Affymetrix HG U133 Plus 2.0 arrays. We also compared 1034 ER+ and ER breast tumors from publicly available microarray data. The principal components of ER activity generated from these analyses and from other published estrogen signatures were compared with ESR1 expression, ER-alpha IHC, and patient survival. Results: Genes differentially expressed in both analyses were associated with ER-alpha IHC and ESR1 mRNA expression. They were also significantly enriched for estrogen-driven molecular pathways associated with ESR1, cyclin D1 (CCND1), MYC (v-myc avian myelocytomatosis viral oncogene homolog), and NFKB (nuclear factor kappa B). Despite their differing constituent genes, the principal components generated from these new analyses and from previously published ER-associated gene lists were all associated with each other and with the survival of patients with breast cancer treated with endocrine therapies. Conclusion: A biomarker of ER-alpha pathway activity, generated using ESR1-responsive mRNAs in MCF7 cells, when used alongside ER-alpha IHC and ESR1 mRNA expression, could provide a method for further stratification of patients and add insight into ER pathway activity in these patients. (C) 2016 The Author(s). Published by Elsevier Inc.