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Background: It remains clinically important to identify ER positive breast cancers likely to respond to tamoxifen (TAM) and so we aimed to select a group of biomarkers able to predict response. We also assessed whether data from different sample types [tumor microarrays (TMAs) and core biopsies] or tumor sites could be combined for biomarker studies. Methods: A total of 123 endocrine treatment naïve patients with known ER and HER2 status treated with TAM had paraffin-embedded tumor tissue available either as TMAs (n=102) or core biopsies (n=21). TMA cores were collected from three different tumor sites, two central and one peripheral. Ten biomarkers were evaluated by immunohistochemistry, for % positivity and/or H-Score, comprising: ER, HER2, Ki67, phosphorylated forms of ER (Ser118), IGF1R, PRAS40, Akt & MAPK (ERK1/2), and PTEN & androgen receptor expression (AR). Each tumor was analysed for Akt1 E17K somatic mutation using BEAMing technology. Patient outcome was assessed by clinical benefit (CB) rate & survival analyses [time to progression (TTP) and time to death (TTD)]. Results: There was no significant difference in % positivity or H-Score between central & peripheral tumor sites for all biomarkers examined. After False Discovery Rate (FDR) correction differences (P

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

Purpose Identification of effective biomarkers for the benefit of endocrine treatment and understanding the molecular pathways that contribute to the development of resistance are of crucial importance to the management of luminal breast cancer. The amino acid transporter SLC1A5 has emerging importance as a prognostic marker and potential therapeutic target in various types of cancer. This study aims to investigate its role in luminal breast cancer as a potential predictive marker for endocrine treatment. Methods SLC1A5 expression was assessed at the transcriptomic and proteomic levels in large, well-characterized cohorts of luminal breast cancer. The sensitivity to endocrine therapy after SLC1A5 knockdown was investigated in vitro, using MCF7 and MDA-MB-175 cell lines. Bioinformatic analyses were performed to study the interacting networks of SLC1A5 and to identify a key co-expressed gene with SLC1A5. Results Here, we showed that patients with tumors that highly expressed SLC1A5 associated with a high risk of relapse after endocrine treatment. In vitro, depletion of SLC1A5 increases the sensitivity of luminal breast cancer cells to tamoxifen. TALDO1 was identified as key co-expressed gene with SLC1A5, and in vitro knockdown of SLC1A5 showed reduction in TALDO1 expression. Indeed, TALDO1 was associated with poor clinical outcomes in patients who were subject to endocrine therapy. Conclusion These findings suggest that metabolic alterations, particularly the interaction between the key amino acid transporter SLC1A5 and metabolic enzyme TALDO1, could affect the sensitivity of endocrine therapy. This study demonstrated the prognostic value of both SLC1A5 and TALDO1 as biomarkers in luminal breast cancer.

Purpose Prediction of response to primary endocrine therapy (PET) in older women is based on measurement of oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor (HER)-2. This study uses a unique method for construction of core needle biopsy (CNB) tissue microarray (TMA), to correlate expression of a panel of 17 biomarkers with clinical outcome, in patients receiving PET. Methods Over 37 years (1973–2010), 1758 older (≥ 70 years) women with operable primary breast cancer were managed in a single institution. Of these, 693 had sufficient good-quality CNB to construct TMA, of which 334 had ER-positive tumours treated by PET with a minimum of 6-month follow-up. A panel of biomarkers was measured by immunohistochemistry (ER, PgR, HER2, Ki-67, p53, CK5/6, CK 7/8, EGFR, BCL-2, MUC1, VEGF, LKB1, BRCA1, HER3, HER4, PTEN and AIB1). Expression of each biomarker was dichotomised into ‘low’ or ‘high’ based on breast cancer-specific survival (BCSS). Results From the panel of biomarkers, multivariate analysis showed: High ER (p = 0.003) and PgR (p = 0.002) were associated with clinical benefit of PET at 6 months, as opposed to progressive disease. High ER (p = 0.0023), PgR (p p = 0.043) and low LKB1 (p = 0.022) were associated with longer time to progression. High PgR (p p = 0.021) were associated with better BCSS. Expression of other biomarkers did not show any significant correlation. Conclusions In addition to ER and PgR; MUC1, BCL-2 and LKB1 are important in determining the outcome of PET in this cohort.

Summary Background The appropriate age range for breast cancer screening remains a matter of debate. We aimed to estimate the effect of mammographic screening at ages 40–48 years on breast cancer mortality. Methods We did a randomised, controlled trial involving 23 breast screening units across Great Britain. We randomly assigned women aged 39–41 years, using individual randomisation, stratified by general practice, in a 1:2 ratio, to yearly mammographic screening from the year of inclusion in the trial up to and including the calendar year that they reached age 48 years (intervention group), or to standard care of no screening until the invitation to their first National Health Service Breast Screening Programme (NHSBSP) screen at approximately age 50 years (control group). Women in the intervention group were recruited by postal invitation. Women in the control group were unaware of the study. The primary endpoint was mortality from breast cancers (with breast cancer coded as the underlying cause of death) diagnosed during the intervention period, before the participant's first NHSBSP screen. To study the timing of the mortality effect, we analysed the results in different follow-up periods. Women were included in the primary comparison regardless of compliance with randomisation status (intention-to-treat analysis). This Article reports on long-term follow-up analysis. The trial is registered with the ISRCTN registry, ISRCTN24647151. Findings 160 921 women were recruited between Oct 14, 1990, and Sept 24, 1997. 53 883 women (33·5%) were randomly assigned to the intervention group and 106 953 (66·5%) to the control group. Between randomisation and Feb 28, 2017, women were followed up for a median of 22·8 years (IQR 21·8–24·0). We observed a significant reduction in breast cancer mortality at 10 years of follow-up, with 83 breast cancer deaths in the intervention group versus 219 in the control group (relative rate [RR] 0·75 [95% CI 0·58–0·97]; p=0·029). No significant reduction was observed thereafter, with 126 deaths versus 255 deaths occurring after more than 10 years of follow-up (RR 0·98 [0·79–1·22]; p=0·86). Interpretation Yearly mammography before age 50 years, commencing at age 40 or 41 years, was associated with a relative reduction in breast cancer mortality, which was attenuated after 10 years, although the absolute reduction remained constant. Reducing the lower age limit for screening from 50 to 40 years could potentially reduce breast cancer mortality. Funding National Institute for Health Research Health Technology Assessment programme.

BackgroundInvasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancers and is typically estrogen receptor alpha positive (ER+) and ERBB2 non-amplified. Somatic mutations in ERBB2/3 are emerging as a tractable mechanism underlying enhanced human epidermal growth factor 2 (HER2) activity. We tested the hypothesis that therapeutically targetable ERBB2/3 mutations in primary ILC of the breast associate with poor survival outcome in large public datasets.MethodsWe performed in silico comparison of ERBB2 non-amplified cases of ER+ stage I-III primary ILC (N=279) and invasive ductal carcinoma (IDC, N=1,301) using METABRIC, TCGA and MSK-IMPACT information. Activating mutations amenable to HER2-directed therapy with neratinib were identified using existing functional data from in vitro cell line and xenograft experiments. Multivariate analysis of 10-year overall survival (OS) with tumor size, grade and lymph node status was performed using a Cox regression model. Differential gene expression analyses by ERBB2 mutation and amplification status was performed using weighted average differences and an in-silico model of response to neratinib derived from breast cancer cell lines.ResultsILC tumors comprised 17.7% of all cases in the dataset but accounted for 47.1% of ERBB2-mutated cases. Mutations in ERBB2 were enriched in ILC versus IDC cases (5.7%, N=16 vs. 1.4%, N=18, pERBB3 mutations were not enriched in ILC (1.1%, N=3 vs. 1.8%, N=23; p=0.604). Median OS for patients with ERBB2-mutant ILC tumors was 66 months versus 211 months for ERBB2 wild-type (p=0.0001), and 159 vs. 166 months (p=0.733) for IDC tumors. Targetable ERBB2 mutational status was an independent prognostic marker of 10-year OS – but only in ILC (hazard ratio, HR=3.7, 95% CI 1.2–11.0; p=0.021). Findings were validated using a novel ERBB2 mutation gene enrichment score (HR for 10-year OS in ILC=2.3, 95% CI 1.04–5.05; p=0.040).ConclusionsTargetable ERBB2 mutations are enriched in primary ILC and their detection represents an actionable strategy with the potential to improve patient outcomes. Biomarker-led clinical trials of adjuvant HER-targeted therapy are warranted for patients with ERBB2-mutated primary ILC.

SCOPUS: no.j, info:eu-repo/semantics/published

The majority of biological profiling studies use surgical excision (SE) samples, excluding patients receiving nonsurgical and neoadjuvant therapy. We propose using core needle biopsy (CNB) for biological profiling in older women. Over 37 years (1973–2010), 1 758 older (≥70 years) women with operable primary breast cancer attended a dedicated clinic. Of these, 693 had sufficient quality CNB to construct tissue microarray (TMA). The pattern of biomarkers was analysed in oestrogen receptor (ER)-positive cases, using immunohistochemistry and partitional clustering analysis. The biomarkers measured were: progesterone receptor (PgR), Ki67, Epidermal Growth Factor Receptor (EGFR), Human Epidermal Growth Factor Receptor (HER)-2, HER3, HER4, p53, cytokeratins CK5/6 and CK7/8, Mucin (MUC)1, liver kinase B1 (LKB1), Breast Cancer Associated gene (BRCA) 1, B-Cell Lymphoma (BCL)-2, phosphate and tensin homolog (PTEN), vascular endothelial growth factor (VEGF), and Amplified in breast cancer 1 (AIB1). CNB TMA construction was possible in 536 ER-positive cases. Multivariate analysis showed progesterone receptor (PgR) (p = 0.015), Ki67 (p = 0.001), and mucin (MUC)1 (p = 0.033) as independent predictors for breast-cancer-specific survival (BCSS). Cluster analysis revealed three biological clusters, which were consistent with luminal A, luminal B, and low-ER luminal. The low-ER luminal cluster had lower BCSS compared to luminal A and B. The presence of the low-ER luminal cluster unique to older women, identified in a previous study in SE TMAs in the same cohort, is confirmed. This present study is novel in its use of core needle biopsy tissue microarrays to profile the biology of breast cancer in older women.

Endocrine therapy is the mainstay of adjuvant treatment for patients with luminal breast cancer. Despite ongoing advances in endocrine therapy to date, a proportion of patients ultimately develop endocrine resistance, resulting in failure of therapy and poor prognosis. Therefore, as part of the growing concept of personalised medicine, the need for identification of predictive markers of endocrine therapy response at an early stage, is recognised. The METABRIC series was used to identify differentially expressed genes (DEGs) in term of response to adjuvant endocrine therapy. Drebrin 1 (DBN1) was identified as a key DEG associated with response to hormone treatment. Next, large, well-characterised cohorts of primary luminal breast cancer with long-term follow-up were assessed at the mRNA and protein levels for the value of DBN1 as a prognostic marker in luminal breast cancer, as well as its potential for predicting the benefit of endocrine therapy. DBN1 positivity was associated with aggressive clinicopathological variables and poor patient outcomes. Importantly, high DBN1 expression predicted relapse patients who were subject to adjuvant endocrine treatment. Our results further demonstrate that DBN1 is an independent prognostic marker in luminal breast cancer. Its association with the response to endocrine therapy and outcome provides evidence for DBN1 as a potential biomarker in luminal breast cancer, particularly for the benefit of endocrine treatment. Further functional investigations into the mechanisms underlying sensitivity to endocrine therapy is required.

There is strong interest in targeting the androgen receptor (AR) in estrogen receptor (ER) positive breast cancer, but widespread confusion exits as to what therapeutic strategy - agonism or antagonism - is appropriate. Current understanding of AR predominantly stems from the field of prostate cancer, where AR is the key oncogenic driver and therapeutic target. An ensuing assumption is that AR promotes malignancy in breast cancer and should be therapeutically antagonised. However, compelling pre-clinical data to support this assumption is lacking. Since estrogen stimulates and androgen inhibits the development of normal breast tissue, we hypothesized that AR acts as a tumour suppressor in the breast and that AR agonism is the appropriate therapeutic strategy for ER-driven breast cancer. We tested this hypothesis using a large suite of cell line and patient-derived explant (PDE) and xenograft (PDX) models of breast cancer, including those that were resistant to current therapies and those harbouring genomic anomalies of ESR1 associated with treatment-resistant disease. Across the diverse models we found compelling evidence that AR agonism, but not antagonism, potently and durably inhibited tumour growth. A signature of AR activity derived from the xenograft models positively predicted disease survival in multiple large clinical cohorts of ER+ breast cancer, out-performing other breast cancer-specific prognostic signatures. We also show that an AR agonist can be combined with current ER target therapies such as Tamoxifen or a CDK4/6 inhibitor to maximize growth inhibition. Mechanistically, agonist-bound AR opposed ER signalling by repositioning ER and the co-activator p300 in the chromatin landscape, resulting in down-regulation of cell cycle genes. Introduction of an AR DNA binding mutant had no effect on ER signalling or estrogen-stimulated growth in breast cancer cells. As part of this study, we have generated consensus AR cistromes representing ER+ breast cancer cell lines and ER+ tumours that provide a new understanding of AR activity and clearly show differences to those associated with prostate cancer cell lines and tumours. In conclusion, our data provides a compelling biological rationale for AR agonism as a therapeutic strategy in multiple, clinically relevant contexts of ER-positive breast cancer. These findings should dispel widespread confusion over the role of AR in ER-driven breast cancer, an issue that currently hinders progress in leveraging modern AR-targeted therapies (e.g. selective androgen receptor modulators) that lack the undesirable side-effects of androgens for clinical benefit.

Multi-cohort analysis demonstrated that cytoplasmic cyclin E expression in primary breast tumors predicts aggressive disease. However, compared to their younger counterparts, older patients have favorable tumor biology and are less likely to die of breast cancer. Biomarkers therefore require interpretation in this specific context. Here, we assess data on cytoplasmic cyclin E from a UK cohort of older women alongside a panel of &gt, 20 biomarkers. Between 1973 and 2010, 813 women &ge, 70 years of age underwent initial surgery for early breast cancer, from which a tissue microarray was constructed (n = 517). Biomarker expression was assessed by immunohistochemistry. Multivariate analysis of breast cancer-specific survival was performed using Cox&rsquo, s proportional hazards. We found that cytoplasmic cyclin E was the only biological factor independently predictive of breast cancer-specific survival in this cohort of older women (hazard ratio (HR) = 6.23, 95% confidence interval (CI) = 1.93&ndash, 20.14, p = 0.002). At ten years, 42% of older patients with cytoplasmic cyclin E-positive tumors had died of breast cancer versus 8% of negative cases (p &lt, 0.0005). We conclude that cytoplasmic cyclin E is an exquisite marker of aggressive tumor biology in older women. Patients with cytoplasmic cyclin E-negative tumors are unlikely to die of breast cancer. These data have the potential to influence treatment strategy in older patients.

Pathology in the UK is on the verge of transformation from analogue to digital practice through the development of digital pathology (DP). Advances in technology has allowed for this change to occur through the use of high-throughput slide scanners to obtain whole histopathology glass slides onto computer workstations rather than the use of a conventional light microscope (LM). Previous studies have shown that the use of digital imaging to view histopathology slides has proven to be of benefit to pathology departments. It allows pathologists to analyse samples remote from the laboratory, making sharing of the slides between pathologists more straight-forward, and also enables expert review out of hours. With the ability to electronically transfer slides from the laboratory to the reporting pathologist, it may provide solutions for local shortages of pathologists across NHS trusts in the UK. However, a number of researchers argue that the costs of implementing digital pathology may outweigh its advantages. Moreover, images produced by DP systems are often of inferior resolution when compared to conventional light microscopy. The lack of literature on this subject limits the adoption of this new technology by laboratories across the country. This multi-centre study aims to analyse how the study pathologists examine DP images of different pathology modalities by using eye-tracking technology, thus using data on their reading and interpretation technique to improve performance and contribute to the adoption of DP across the UK.

Cyclin‐dependent kinase 5 (Cdk5) is an atypical member of the cyclin‐dependent kinase family and functions as a serine/threonine kinase that can be activated by non‐cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early‐stage invasive breast cancer tumours (n = 1110) with long‐term follow‐up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer‐specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418‐0.896, P = .011 and HR = 0.507, 95% CI = 0.318‐0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.

The majority of breast cancers are oestrogen-receptor-positive (ER+) and are subject to endocrine therapy, however, an unpredictable subgroup of patients will develop resistance to endocrine therapy. The SLC7A5/SLC3A2 complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Alterations in the expression and function of those amino-acid transporters lead to metabolic reprogramming, which contributes to the tumorigenesis and drug resistance. This study aims to assess the effects and roles of SLC7A5/SLC3A2 co-expression in predicting responses to endocrine therapy in patients with ER+ breast cancer. The biological and clinical impact of SLC7A5/SLC3A2 co-expression was assessed in large annotated cohorts of ER+/HER2&minus, breast cancer with long-term follow-up at the mRNA and protein levels. In vitro experiments were conducted to investigate the effect of SLC7A5/SLC3A2 knockdown in the proliferation of cancer cells and to the sensitivity to tamoxifen. We found that proliferation-related genes are highly expressed in a subgroup of patients with high SLC7A5/SLC3A2, and knockdown of SLC7A5/SLC3A2 decreased proliferation of ER+ breast cancer cells. In patients treated with endocrine therapy, high SLC7A5/SLC3A2 co-expression was associated with poor patient outcome, and depletion of SLC7A5/SLC3A2 using siRNA increased the sensitivity of breast cancer cells to tamoxifen. On the basis of our findings, SLC7A5/SLC3A2 co-expression has the potential of identifying a subgroup of ER+/HER2&minus, breast cancer patients who fail to benefit from endocrine therapy and could guide the choice of other alternative therapies.

Key Points Question Are sperm-associated antigen 5 (SPAG5) transcript or protein expressions associated with treatment response in patients with estrogen receptor–positive breast cancer? Findings In this cohort study including 12 720 patients with estrogen receptor–positive breast cancer, SPAG5 transcript and SPAG5 protein overexpressions were associated with worse outcomes in patients who received endocrine therapy alone. Overexpressions of SPAG5 transcript or SPAG5 protein were associated with resistance to endocrine therapy but sensitivity to anthracycline-based combination chemotherapy, and downregulation of SPAG5 during the course of preoperative systemic therapies was associated with clinical benefit. Meaning These findings suggest that SPAG5 transcript or SPAG5 protein expression could be used as a clinical tool for selecting and monitoring response to neoadjuvant therapies and guide adjuvant therapy in estrogen receptor–positive breast cancer., This cohort study examines the association of sperm-associated antigen 5 (SPAG5) transcript and protein expression with treatment response in patients with estrogen receptor–positive breast cancer., Importance There is no proven test that can guide the optimal treatment, either endocrine therapy or chemotherapy, for estrogen receptor–positive breast cancer. Objective To investigate the associations of sperm-associated antigen 5 (SPAG5) transcript and SPAG5 protein expressions with treatment response in systemic therapy for estrogen receptor–positive breast cancer. Design, Settings, and Participants This retrospective cohort study included patients with estrogen receptor–positive breast cancer who received 5 years of adjuvant endocrine therapy with or without neoadjuvant anthracycline-based combination chemotherapy (NACT) derived from 11 cohorts from December 1, 1986, to November 28, 2019. The associations of SPAG5 transcript and SPAG5 protein expression with pathological complete response to NACT were evaluated, as was the association of SPAG5 mRNA expression with response to neoadjuvant endocrine therapy. The associations of distal relapse–free survival with SPAG5 transcript or SPAG5 protein expressions were analyzed. Data were analyzed from September 9, 2015, to November 28, 2019. Main Outcomes and Measures The primary outcomes were breast cancer–specific survival, distal relapse–free survival, pathological complete response, and clinical response. Outcomes were examined using Kaplan-Meier, multivariable logistic, and Cox regression models. Results This study included 12 720 women aged 24 to 78 years (mean [SD] age, 58.46 [12.45] years) with estrogen receptor–positive breast cancer, including 1073 women with SPAG5 transcript expression and 361 women with SPAG5 protein expression of locally advanced disease stage IIA through IIIC. Women with SPAG5 transcript and SPAG5 protein expressions achieved higher pathological complete response compared with those without SPAG5 transcript or SPAG5 protein expressions (transcript: odds ratio, 2.45 [95% CI, 1.71-3.51]; P

Deep learning approaches are widely applied to histopathological image analysis due to the impressive levels of performance achieved. However, when dealing with high-resolution histopathological images, utilizing the original image as input to the deep learning model is computationally expensive, while resizing the original image to achieve low resolution incurs information loss. Some hard-attention based approaches have emerged to select possible lesion regions from images to avoid processing the original image. However, these hard-attention based approaches usually take a long time to converge with weak guidance, and valueless patches may be trained by the classifier. To overcome this problem, we propose a deep selective attention approach that aims to select valuable regions in the original images for classification. In our approach, a decision network is developed to decide where to crop and whether the cropped patch is necessary for classification. These selected patches are then trained by the classification network, which then provides feedback to the decision network to update its selection policy. With such a co-evolution training strategy, we show that our approach can achieve a fast convergence rate and high classification accuracy. Our approach is evaluated on a public breast cancer histopathological image database, where it demonstrates superior performance compared to state-of-the-art deep learning approaches, achieving approximately 98% classification accuracy while only taking 50% of the training time of the previous hard-attention approach.

Granular cell tumors (GCTs) are rare tumors that can arise in multiple anatomical locations, and are characterized by abundant intracytoplasmic granules. The genetic drivers of GCTs are currently unknown. Here, we apply whole-exome sequencing and targeted sequencing analysis to reveal mutually exclusive, clonal, inactivating somatic mutations in the endosomal pH regulators ATP6AP1 or ATP6AP2 in 72% of GCTs. Silencing of these genes in vitro results in impaired vesicle acidification, redistribution of endosomal compartments, and accumulation of intracytoplasmic granules, recapitulating the cardinal phenotypic characteristics of GCTs and providing a novel genotypic–phenotypic correlation. In addition, depletion of ATP6AP1 or ATP6AP2 results in the acquisition of oncogenic properties. Our results demonstrate that inactivating mutations of ATP6AP1 and ATP6AP2 are likely oncogenic drivers of GCTs and underpin the genesis of the intracytoplasmic granules that characterize them, providing a genetic link between endosomal pH regulation and tumorigenesis., Granular cell tumors (GCTs) are rare tumors that arise in multiple anatomical locations. Here, the authors investigate the genomics of GCTs, finding inactivating somatic mutations in ATP6AP1 or ATP6AP2 in 72% of the 82 GCTs analyzed. In vitro manipulation of these genes recapitulated GCT phenotypes in cellular models.

The absence of a robust risk stratification tool for triple negative breast cancer (TNBC) underlies imprecise and nonselective treatment of these patients with cytotoxic chemotherapy. This study aimed to interrogate transcriptomes of TNBC resected samples using next generation sequencing to identify novel biomarkers associated with disease outcomes. A subset of cases (n = 112) from a large, well-characterized cohort of primary TNBC (n = 333) were subjected to RNA-sequencing. Reads were aligned to the human reference genome (GRCH38.83) using the STAR aligner and gene expression quantified using HTSEQ. We identified genes associated with distant metastasis-free survival and breast cancer-specific survival by applying supervised artificial neural network analysis with gene selection to the RNA-sequencing data. The prognostic ability of these genes was validated using the Breast Cancer Gene-Expression Miner v4. 0 and Genotype 2 outcome datasets. Multivariate Cox regression analysis identified a prognostic gene signature that was independently associated with poor prognosis. Finally, we corroborated our results from the two-gene prognostic signature by their protein expression using immunohistochemistry. Artificial neural network identified two gene panels that strongly predicted distant metastasis-free survival and breast cancer-specific survival. Univariate Cox regression analysis of 21 genes common to both panels revealed that the expression level of eight genes was independently associated with poor prognosis (p

Dopamine and cAMP regulated phosphoprotein 32 kDa (DARPP-32) also known as phosphoprotein phosphatase-1 regulatory subunit 1B and encoded by the PPP1R1B gene is an inhibitor of protein phosphatase-1 and protein kinase A. DARPP-32 is expressed in a wide range of epithelial cells and some solid tumours; however, its role in breast cancer is only partially defined. DARPP-32 expression was determined using immunohistochemistry in two independent cohorts of early stage invasive breast cancer patients (discovery n = 1352; validation n = 1655), and 112 HER2 positive breast cancer patients treated with trastuzumab and adjuvant chemotherapy. PPP1R1B mRNA expression was assessed in the METABRIC cohort (n = 1980), using artificial neural network analysis to identify associated genes. In the discovery cohort, low nuclear expression of DARPP-32 was significantly associated with shorter survival (P = 0.041), which was independent of other prognostic variables (P = 0.019). In the validation cohort, low cytoplasmic and nuclear expression was significantly associated with shorter survival (both P = 0.002), with cytoplasmic expression independent of other prognostic variables (P = 0.023). Stronger associations with survival in oestrogen receptor (ER) positive disease were observed. In patients treated with trastuzumab, low nuclear expression was significantly associated with adverse progression-free survival (P = 0.031). In the METABRIC cohort, low PPP1R1B expression was associated with shortened survival of ER positive patients. Expression of CDC42 and GRB7, amongst others, were associated with PPP1R1B expression. This data suggests a role for DARPP-32 as a prognostic marker with clinical utility in breast cancer.

Item does not contain fulltext CHD8 has been reported as an autism susceptibility/intellectual disability gene but emerging evidence suggests that it additionally causes an overgrowth phenotype. This study reports 27 unrelated patients with pathogenic or likely pathogenic CHD8 variants (25 null variants, two missense variants) and a male:female ratio of 21:6 (3.5:1, p < .01). All patients presented with intellectual disability, with 85% in the mild or moderate range, and 85% had a height and/or head circumference >/=2 standard deviations above the mean, meeting our clinical criteria for overgrowth. Behavioral problems were reported in the majority of patients (78%), with over half (56%) either formally diagnosed with an autistic spectrum disorder or described as having autistic traits. Additional clinical features included neonatal hypotonia (33%), and less frequently seizures, pes planus, scoliosis, fifth finger clinodactyly, umbilical hernia, and glabellar hemangioma (

BACKGROUND: Ras association and pleckstrin homology domains 1 (RAPH1) is involved in cytoskeleton regulation and re-epithelialisation in invasive carcinoma and therefore may play a key role in carcinogenesis and metastasis. We herein investigated the biological and clinical significance of RAPH1 in breast cancer using large annotated cohorts. METHODS: The clinicopathological and prognostic significance of RAPH1 was assessed at the genomic and transcriptomic levels using The Cancer Genome Atlas (TCGA) dataset (n=1039) and the results were validated using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n=1980). RAPH1 protein expression was evaluated by immunohistochemistry in a large, well-characterised cohort of early-stage breast cancer (n=1040). RESULTS: In both the TCGA-BRCA and METABRIC cohorts, RAPH1 mRNA expression and RAPH1 copy number alteration were strongly correlated. RAPH1 mRNA overexpression was significantly correlated with high expression of adhesion and EMT markers including CDH1, TGFbeta1 and CD44. RAPH1 mRNA overexpression was a significant predictor of a poor prognosis (Hazard ratio: 3.88; p = 0.049). High RAPH1 protein expression was associated with higher grade tumours with high proliferation index, triple negative phenotype and high E-cadherin expression. High RAPH1 protein expression was an independent predictor of shorter survival (Hazard ratio: 4.37; p = 0.037). CONCLUSIONS: High RAPH1 expression is correlated with aggressive breast cancer phenotypes and provides independent prognostic value in invasive breast cancer.

Background: Breast cancer (BC) is a heterogeneous disease characterised by variant biology, metabolic activity and patient outcome. This study aimed to evaluate the biological and prognostic value of the membrane solute carrier, SLC3A2 in BC with emphasis on the intrinsic molecular subtypes.\ud \ud Methods: SLC3A2 was assessed at the genomic level, using METABRIC data (n=1,980), and proteomic level, using immunohistochemistry on TMA sections constructed from a large well-characterised primary BC cohort (n=2,500). SLC3A2 expression was correlated with clinicopathological parameters, molecular subtypes, and patient outcome.\ud \ud Results: SLC3A2 mRNA and protein expression were strongly correlated with higher tumour grade and poor Nottingham prognostic index (NPI). High expression of SLC3A2 was observed in triple negative (TN), HER2+, and ER+ high proliferation subtypes. SLC3A2 mRNA and protein expression were significantly associated with the expression of c-MYC in all BC subtypes (p

Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors. Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays. Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition. Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells. Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-13.

Breast cancer (BC) displays striking clinical, morphological, and behavioural diversity within a single tumour and between tumours. Currently, mounting evidence indicates that the morphological heterogeneity of BC reflects an underlying spectrum of genetic and epigenetic portraits that control BC behaviour. Further understanding of BC heterogeneity will have an impact, not only on the routine diagnostic practices but also on patients' management decisions. Phenomena like diagnostic inconsistencies and therapeutic resistance, both primary and acquired, could be attributed, at least in part, to tumour heterogeneity within the same cancer and between the primary disease and subsequent recurrences. From a practical standpoint, and to minimise the impact of BC intratumoral heterogeneity, pragmatic approaches for adequate tumour sampling have been suggested in translational biomarker discovery and validation research studies and in the clinical setting. Here, we provide a brief overview of BC heterogeneity, with an emphasis on the clinical consequences of intratumoral heterogeneity.

Macrophage-associated cytokines play an important role in cancer metastasis; however, the functions of interleukins (IL) 6 and 10 in breast cancer (BC) progression and metastasis are not clear. In this study the roles of IL-6/IL-10 in regulating vascular invasion and their prognostic significance in BC are investigated. MDA-MB-231 and MCF-7 migration (± IL-6 or IL-10) was assessed by scratch wound assay. Cancer cell adhesion to IL-6/IL-10 stimulated blood and lymphatic endothelial cells (EC) was investigated. Expression of IL-6 /IL-10 was assessed using immunohistochemistry in an annotated cohort of early stage BC (n = 1380) and associations with clinicopathological variables and clinical outcome evaluated. IL-6 did not alter BC cell migration however a dose-dependent inhibition in MDA-MB-231 migration with IL-10 treatment was observed (P = 0.03). BC cells were more adhesive to blood vs lymphatic EC, however, IL-6/IL-10 had no effect on adhesion patterns. High expression of IL-6/IL-10 was associated with clinicopathological criteria (e.g. hormone receptor status, all P

Cancer cells must alter their metabolism in order to satisfy the demands of necessary energy and cellular building blocks. These metabolic alterations are mediated by many oncogenic changes that affect cellular signalling pathways, which result in sustained cell growth and proliferation. Recently, metabolomics has received great attention in the field of cancer research, and as the essential metabolic pathways that drive tumour growth and progression are determined the possibilities of new targets for therapeutic intervention are opened. More specifically, as breast cancer is a heterogeneous disease, there is growing evidence that differences in metabolic changes exist between molecular subtypes. In this review, the most recent findings in breast cancer cell metabolism are discussed, with particular emphasis on glutamine and its transporters, which is considered one of the key amino acids fuelling cancer growth. Furthermore, the metabolic differences between the molecular subtypes of breast cancer are examined, highlighting the clinical utility for breast cancer diagnosis and treatment.

Triple-negative breast cancer is viewed clinically as an aggressive subgroup of breast cancer. In fact, most triple-negative breast cancers are poor-prognosis tumors with a complex genomic landscape. However, triple-negative disease is vastly heterogeneous, encompassing multiple entities with marked genetic, transcriptional, histologic, and clinical differences, with neoplasms in this group ranging from low to high grade. Among the less common low-grade triple-negative lesions, two large subgroups, both with a rather indolent behavior, can be distinguished: a low-grade triple-negative breast neoplasia family, which includes nonobligate precursors of triple-negative breast cancer, and, despite being low-grade, harbors the complex genomic landscape of usual triple-negative breast cancer, and the salivary gland-like tumors of the breast, lacking all the cardinal molecular features of conventional triple-negative breast cancer and underpinned by specific fusion genes or hotspot mutations, which may be of diagnostic and possibly therapeutic utility. Progression to high-grade triple-negative breast cancer likely occurs in both subgroups but at different rates. In this review, we describe the heterogeneity of triple-negative disease, focusing on the histologic and molecular features of the low-grade lesions. Recognition that triple-negative breast cancer is an operational term and that triple-negative disease is heterogeneous and includes low-grade forms driven by distinct sets of genetic alterations is germane to the successful implementation of precision medicine.

Background:\ud The prognostic value of lymphovascular invasion (LVI) in breast cancer (BC) has been demonstrated in several independent studies. However, identification of driver molecules for LVI remains a challenging task. Large-scale transcriptomic profiling of histologically validated LVI can potentially identify genes that regulate LVI.\ud \ud Methods:\ud Integrative bio-informatics analyses of the METABRIC study were performed utilising a subset of strictly defined LVI using histological and immunohistochemical (IHC) criteria. ARHGAP18 was among the top differentially expressed genes between LVI+ and LVI− BC with a 1.8-fold change. The prognostic impact of ARHGAP18 gene expression was assessed in the METABRIC data set (n=1980) and externally validated using the online BC gene expression data sets utilising bc-GenExMiner v4.0 (n=2016). Subsequently, ARHGAP18 protein expression was assessed on a large cohort of invasive BC (n=959) with long-term follow-up using IHC.\ud \ud Results:\ud Pooled analysis of ARHGAP18 mRNA expression showed that overexpression was associated with better outcome (P

Purpose: Low molecular weight cyclin E (LMW-E) detected by Western blot analysis predicts for reduced breast cancer survival; however, it is impractical for clinical use. LMW-E lacks a nuclear localization signal that leads to accumulation in the cytoplasm that can be detected by IHC. We tested the hypothesis that cytoplasmic staining of cyclin E can be used as a predictor of poor outcome in different subtypes of breast cancer using patient cohorts with distinct clinical and pathologic features.Experimental Design: We evaluated the subcellular localization of cyclin E in breast cancer specimens from 2,494 patients from 4 different cohorts: 303 from a prospective study and 2,191 from retrospective cohorts [NCI, MD Anderson Cancer Center (MDA), and the United Kingdom (UK)]. Median follow-up times were 8.0, 10.1, 13.5, and 5.7 years, respectively.Results: Subcellular localization of cyclin E on IHC was associated with full-length (nuclear) and low molecular weight isoforms (cytoplasmic) of cyclin E on Western blot analysis. In multivariable analysis, cytoplasmic cyclin E staining was associated with the greatest risk of recurrence compared with other prognostic factors across all subtypes in three (NCI, MDA, and UK) of the cohorts. In the MDA cohort, cytoplasmic cyclin E staining outperformed Ki67 and all other variables as prognostic factors.Conclusions: Cytoplasmic cyclin E identifies patients with the highest likelihood of recurrence consistently across different patient cohorts and subtypes. These patients may benefit from alternative therapies targeting the oncogenic isoforms of cyclin E. Clin Cancer Res; 23(12); 2991–3002. ©2016 AACR.

Some previous studies have reported that the chemokine (C-C motif) receptor 7 (CCR7) plays a role in breast cancer, is associated with lymph node metastasis and drives the site of distant metastasis. However, the impact of its expression on patient outcome and its association with tumour infiltrating inflammatory cells remain to be validated. We evaluated CCR7 protein expression by immunohistochemistry in a large well characterized cohort (n = 866) of early invasive primary breast cancers. CCR7 was expressed in the cytoplasm and membrane of tumour cells. We observed a weak positive association of high CCR7 expression when in either cellular component, but not both together, with axillary lymph node stage 3 tumours (p = 0.043). Logistic regression analysis of lymph node stage revealed no independent predictive value for CCR7 expression. CCR7 expression was higher in HER2 positive tumours (p = 0.03) and associated with positive CD68+ FOXP3+ tumour infiltrating cells. CCR7 staining was negatively associated with CD3+ cells. There was no significant association of CCR7 expression with breast cancer recurrence or survival. We conclude that while CCR7 is not a useful biomarker for predicting lymph node metastasis, it may reflect altered intra- and inter-cellular signalling related to the immune microenvironment. The subcellular localization of CCR7 appears to affect the nature of these interactions.

Backgound and Aim: The best test to guide the choice of systemic therapy for breast cancer (BC) has not yet been identified. We did this study to identify factors that drive proliferation features in BC and assess their association with clinical outcomes after systemic therapy. Methods: We applied an artificial neural network-based integrative data mining approach to three cohorts of patients with untreated lymph node (LN)-negative BC (Wang et al; n=286, Desmedt et al; n=198 and Schmidt et al; n=200). The results were validated in four cohorts of BC patients (the Nottingham discovery cohort (n=171), Uppsala cohort (n=249), The Cancer Genome Atlas-Breast Cancer project [TCGA-BRCA; n= 970] and Molecular Taxonomy of Breast Cancer International Consortium [METABRIC cohort; n=1980]. Genes that featured prominently in our interactome map of proliferation have been chosen to take them forward to investigate their clinicopathological relevance of their gene copy number aberrations (CNAs), mRNA transcript expression, and protein expression and their associations with breast cancer-specific survival (BCSS), distant relapse-free survival (DRFS) and pathological complete response (pCR) in ten international cohorts of BC (n>12000 patients). Findings: ESR1, SPAG5, EGFR, BCL2, and FOXA1 were among the 39 common gene probes that were predictive across most proliferation features and datasets. In TCGA-BRCA cohort, SPAG5 gene mutation, gain/amplification and loss at the Ch17q11.2 locus were detected in 43 (4.4%), 177 (18.2%) and 180 (18.8%) of 970 patients, respectively and 65 (31%) of 479 ER-positive /HER-positive patients showed gain/amplification of SPAG5 gene. In multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were associated with reduced BCSS compared with lower expression (METABRIC: HR 1·27, 95% CI 1·02–1·58, p=0·034; untreated LN-negative cohort: 2·34, 1·24–4·42, p=0·0090; and Nottingham-cohort: 1·73, 1·23–2·46, p=0·0020). In patients with ER-negative/HER2-negative or ER-positive/HER2-negative BC, high SPAG5 transcript expression was associated with an increased pCR compared with low SPAG5 transcript expression after receiving anthracycline neoadjuvant chemotherapy (AC-NeoACT) [(Multicentre phase 2 clinical trial cohort; n=136; OR 2·47, 95% CI 1·17–5·21, p=0.016) and (MD Anderson- taxane+AC-NeoACT cohort; n=287; OR 3·16, 95% CI 1·46–6·84, p=0.003); respectively]. In patients with ER-positive/HER2-negative BC who received taxane+AC-NeoACT followed by adjuvant tamoxifen (Adj-Tam) for 5 years (MD Anderson- taxane+AC-NeoACT cohort; n=287), high and low SPAG5 transcript expression had similar DRFS (HR 1·40, 95% CI 0.76–2·58, p=0.282). Whereas in ER-positive/HER2-negative BC patients who received only adj-Tam (n=298), high SPAG5 transcript expression was associated with reduced DRF at 5 years compared with lower expression (HR 1.98, 95% CI 1.19–3.27, p=0.008). Interpretation: The transcript and protein products of SPAG5 are independent prognostic and predictive biomarkers that might have clinical utility as biomarkers for combination cytotoxic chemotherapy sensitivity in ER-positive/HER-negative BC. Citation Format: Abdel-Fatah TMA, Agarwal D, Zafeiris D, Pongor L, Györffy B, Rueda OM, Moseley PM, Green AR, Liu D-X, Pockley AG, Rees RC, Caldas C, Ellis IO, Ball GR, Chan SYT. Identification of proliferation related derivers and their roles in precision medicine for breast cancers: A retrospective multidimensional comparative, integrated genomic, transcriptomic, and protein analysis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-16.

The aim of our study was to assess various predictors for local recurrence (LR) in patients undergoing breast conservation surgery (BCS) for ductal carcinoma in situ (DCIS).An audit was performed of 582 consecutive patients with DCIS between Jan 1975 to June 2008. In patients undergoing BCS, local guidelines reported a margin of ≥10 mm during the above period. Guideline with regard to margin of excision changes soon after this period. We retrospectively analysed clinical and pathological risk factors for local recurrence in patients undergoing BCS. Statistical analysis was carried out using SPSS version 19, and a cox regression model for multivariate analysis of local recurrence was used.Overall 239 women had BCS for DCIS during the above period. The actuarial 5-year recurrence rate was 9.6%. The overall LR rate was 17% (40/239. LR was more common in patients ≤50 years: (10/31 patients, 32%) compared to patients 50 years (30/208, 14%, P = 0.02). Forty three per cent of patients (6/14) with5 mm margin developed LR which was significantly higher compared to patients with 5-9 mm margin (12%, 3/25) and with ≥10 mm margin (14%, 27/188, P = 0.01). On multivariate analysis age ≤50 years,5 mm pathological margin were independent prognostic factors for local recurrence.Our study shows that younger age (≤50 years) and a margin5 mm are poor prognostic factors for LR in patients undergoing breast conservation surgery for DCIS.

Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair–related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation.

Background: NACT is a standard option for BC (T2-4, N0-3, M0) and certain BC phenotypes. The choice of AT based on the pre-NACT biomarkers status may not be optimum for individual patient because dynamic phenotypic changes induced by NACT may alter the response to treatment. Aim: The aim of this study is to determine the incidence of changes in the receptor status (ER, PR and HER2) and other proliferation biomarkers (Ki67 and SPAG5) before and after NACT and to assess the clinical significance of such changes. Methods Immunohistochemistry staining of ER, PR, HER2, Ki67 and SPAG5 in pre and post NACT tumors tissues from a consecutive series of 850 of BC (T2-4, N0-3, M0) treated at the Nottingham University Hospital (NUH) from 2000 to 2018, have been centrally evaluated according to ASCO guidelines. All cases with conversion in HER2 status had also been retested by HER2-FISH. The results were validated in an external cohort of 250 cases. Treatment options and patients characteristics are the same between the centres: (68%) received anthracycline plus Taxane (AC+T) NACT and 32% of patients have received NACT Anthracycline only (AC). Neoadjuvant HER2 targeting agents (Trastuzumab) or (Trastuzumab + Petruzumab) had been prescribed to 16% of patients in addition to AC+T followed by adjuvant Trastuzumab (total=18 cycles). All pre-NACT ER+ patients were given at least 5 year of adjuvant endocrine therapy (ET). In 2013 NUH started a prospective audit of retesting of receptor status in all post NACT surgical tumour samples. The results of the tests were presented to the weekly tumour board meeting and any change in the receptor status (ER and HER2) from negative (in the pre NACT core biopsies) to positive (in the post NACT surgical specimen) being considered for additional AT (ET for ER+ and Trastuzumab for HER2+ cases).The primary end points for this study are the % changes of biomarker changes and the disease free survival (DFS). The median follow up was 72 months. Results In pre NACT core biopsies 32% and 68% were HER2+ and HER2-; respectively. Twelve percent (12%) of the pre NACT HER2- tumours had a conversion to HER2+ in the post NACT surgical specimens. In this group of patients who subsequently received adjuvant Trastuzumab, 95% 3-year DFS was reported; which was similar to those patients who achieved pCR (3-year DFS; 90%) and was superior to cases which remained HER- in post NACT specimens (3-year DFS; 41%); (p Citation Format: Tarek M. a. Abdel-Fatah, Rebekah Webb, Jennifer Walker, Jun Lim, A. Graham Pockley, Graham Ball, Matthew Griffiths, Ian Ellis, Emad Rakhah, Zsolt Hodi, Andrew Lee, Arlene Chan, Stephen Chan. Clinically significant changes of receptors status (ER, PR and HER2) after receiving neoadjuvant chemotherapy (NACT) in breast cancer (BC) predicts the prognosis and guides the choice of the optimal adjuvant therapy (AT): Retesting of the receptor status should be mandatory [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-06.

The Androgen receptor (AR) is expressed in up to 90% of primary ER-positive (ER+) breast cancer and high expression of AR is an independent prognostic factor associated with good outcome. Its role in the context of ER+ breast cancer is very controversial and has constrained clinical implementation of new drugs that influence AR activity for treatment of women with this disease, resulting in concurrent clinical trials of opposite AR agonist and antagonist strategies. Herein, using RNA-seq and Chip-seq approaches, we demonstrate that therapeutic activation of AR for treatment of breast cancer leverages a natural regulatory mechanism that inhibits estrogen-stimulated proliferation and induction of cell cycle genes in patient-derived explants (PDE) of primary ER+ breast cancers, and in contemporary in vivo patient-derived xenograft (PDX) and cell line xenograft models of ER+ breast cancer resistant to standard-of-care ER targeting agents, including those harbouring genomic aberrations of ESR1. AR-mediated growth inhibition mechanistically involved loss of ER or co-activator p300 recruitment to chromatin at key cell cycle genes, leading to transcriptional down-regulation. A signature of AR activity derived from the xenograft models positively predicted disease survival in multiple large clinical cohorts of ER+ breast cancer, outperforming other breast cancer-specific prognostic signatures. Finally, the combination strategy of a new class of non virilising Selective Androgen Receptor Modulator (SARM) in combination with CDK4/6 inhibitors demonstrated additive effects in our preclinical models. Collectively, these findings provide compelling evidence that AR has a tumour suppressor role in ER+ breast cancer and advocate an AR agonist strategy for treatment, even in the context of therapy-resistant, ESR1 mutant disease, and should dispel widespread confusion over the role of AR in ER-driven breast cancer, an issue that currently hinders progress in leveraging modern AR-targeted therapies, including available SARMs that lack the undesirable side-effects of androgens, for clinical benefit. Citation Format: Elgene Lim, Theresa A Hickey, Luke A Selth, Kee Ming Chia, Heloisa H Milioli, Daniel Roden, Geraldine Laven-Law, Shalini Jindal, Mun Hui, Esmaeil Ebrahimie, Stephen N Birrell, Suzan Stelloo, C. Elizabeth Caldon, Jessica Finlay-Schultz, Tarek M Abdel-Fatah, Ian O Ellis, Willbert Zwart, Carlo Palmieri, Carol A Sartorius, Alex Swarbrick, Jason S Carroll, Wayne D Tilley. The androgen receptor is a tumour suppressor in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-03.

Multigene assay is currently recommended for prognostic stratification of the clinically indeterminate group of breast cancer (BC) patients defined as lymph node (LN)-negative, oestrogen receptor (ER)-positive HER2-negative (LN-/ER+/HER2-) to determine the use of chemotherapy. However this cohort, comprising approximately 40% of BC, is not a homogeneous group and shows variable outcome. This study aims to determine the prognostic value of routinely assessed variables, singly and in combination, in the LN-/ER+/HER2- BC. Methods 830 LN-/ER+/HER2- chemotherapy naive BCs were investigated. The prognostic value of histologic grade, tumour size, lymphovascular invasion (LVI), progesterone receptor (PgR) and Ki67LI was assessed. In this series, only 25% of patients received hormone therapy. Median follow-up was 172 months. Results In the whole cohort, tumour grade, size, LVI, PgR and Ki67LI were highly correlated with outcome in a time-dependent manner. The outcome of this group varied widely from 97% (20% of cases) to 50% survival rate after 10-year follow-up using combination of these markers. A prognostic index (Nottingham Px) incorporating grade, size, PgR and Ki67LI was developed. The index can robustly stratify the whole cohort as well as the higher risk subgroup (NPI score >3.4) into distinct prognostic classes. Conclusion Current routinely assessed variables can provide additional prognostic information in LN-/ER+/HER2- BC. The proposed (Nottingham Px) index can stratify BC clinically indeterminate group of patients into excellent and poor prognostic subgroups and can reliably be used to identify patients for systemic chemotherapy or further multigene prognostic testing. Performance of prognostic variables in these tumours is time-dependent and should be considered in future studies. This article is protected by copyright. All rights reserved.

The introduction of mammographic screening has resulted in a rise in the detection rate of ductal carcinoma in situ (DCIS), currently accounting for one-fifth of screen-detected breast cancers. Although 60-70% of DCIS are treated with breast-conserving surgery (BCS) with or without radiotherapy, the frequency of subsequent surgery to re-excise positive margins in order to reduce the probability of recurrences remains high. DCIS recurrence is associated not only with financial, health and psychological implications; approximately half these recurrences are invasive disease. An appropriate margin width for patients undergoing BCS for invasive breast cancer has been largely agreed. Although there is a perception that such recommendations may be applicable to DCIS, major differences exist which may affect this application. Importantly, DCIS patients often do not receive systemic adjuvant (endocrine) therapy and not all receive radiotherapy in routine practice. There is evidence that wide margins (i.e. >10 mm) confer better protection against recurrence than positive (i.e. 0 mm) margins; however, there remains a debate concerning the optimum margin width between 0 and 10 mm. Previous studies have demonstrated that radiation therapy may not compensate for lack of re-excision in those patients with positive or close margins, while wide margins will inevitably compromise cosmesis and patients' body image perception. This review aims to address the clinical question of the minimal margin width in DCIS treated with BCS that is associated with the lowest recurrence rate and when, therefore, further surgical intervention for re-excision can be safely avoided. A range of clinical circumstances that might affect this are considered.

Background and Aim Although oestrogen receptor (ER)-negative breast cancers (BC) do not respond to hormone therapy, the response of ER-positive BC is reported to be variable which may suggest a dose dependent effect. This study aimed to assess the pattern of ER-expression in BC at the protein (IHC) and transcriptome (microarray-based gene expression) levels. Materials and Methods ER IHC expression was assessed in a large series of BC including 3649 core biopsies and 1892 cases prepared as TMA stained using specific antibodies. ESR1 mRNA expression was assessed in the METABRIC study (1980 cases) using Linear Models for Microarray Data (LIMMA) and results were compared with protein levels. Results IHC data confirmed bimodality of ER expression with 92.2% and 89.2% of the cases showed completely negative (

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

Purpose Breast cancer (BC) is a heterogeneous disease consisting of various subtypes, with different prognostic and therapeutic outcomes. The amino acid transporter, SLC7A8, is overexpressed in oestrogen receptor-positive BC. However, the consequence of this overexpression, in terms of disease prognosis, is still obscure. This study aimed to evaluate the biological and prognostic value of SLC7A8 in BC with emphasis on the intrinsic molecular subtypes. Methods SLC7A8 was assessed at the genomic, using METABRIC data (n = 1980), and proteomic, using immunohistochemistry and TMA (n = 1562), levels in well-characterised primary BC cohorts. SLC7A8 expression was examined with clinicopathological parameters, molecular subtypes, and patient outcome. Results SLC7A8 mRNA and SLC7A8 protein expression were strongly associated with good prognostic features, including small tumour size, low tumour grade, and good Nottingham Prognostic Index (NPI) (all P SLC7A8 mRNA was higher in luminal tumours compared to other subtypes (P SLC7A8 mRNA and SLC7A8 protein was associated with good patient outcome (P ≤ 0.001) but only in the low proliferative ER+/luminal A tumours (P = 0.01). In multivariate analysis, SLC7A8 mRNA and SLC7A8 protein were independent factors for longer breast cancer specific survival (P = 0.01 and P = 0.03), respectively. Conclusion SLC7A8 appears to play a role in BC and is a marker for favourable prognosis in the most predominant, ER+ low proliferative/luminal A, BC subtype. Functional assessment is necessary to reveal the specific role played by SLC7A8 in ER+ BC.

Zinc transporter ZIP7 activates key downstream signalling pathways and is correlated with important clinicopathological parameters that are associated with endocrine resistance., ZIP7, a member of the ZIP family of zinc importers, resides on the endoplasmic reticulum membrane and transports zinc from intracellular stores to the cytoplasm after activation by CK2 phosphorylation on two serine residues (S275 and S276). ZIP7 is known to be required for the growth of anti-hormone resistant breast cancer models, especially those with acquired tamoxifen resistance developed from MCF-7. Using our new pS275S276ZIP7 antibody which only recognises activated ZIP7 (pZIP7), we have demonstrated that the hyperactivation of ZIP7 is prevalent in tamoxifen-resistant breast cancer cells. This evidence suggests that pZIP7 might have potential as a biomarker of acquired resistance to such anti-hormones in breast cancer, a current unmet clinical need. In this regard, we have also developed a new immunohistochemical assay for pZIP7 which allowed pZIP7 to be tested on a small clinical series of breast cancer tissues confirming its prevalence in such tumours and relationship to a variety of clinicopathological parameters and biomarkers previously associated with endocrine resistant phenotypes, notably increased activated MAPK signalling, expression of ErbB2, CD71 and the proto-oncogene c-Fos, as well as with increased tumour grade.

BackgroundRetinoid X Receptor Gamma (RXRG) is a member of the nuclear receptor superfamily and plays a role in tumour suppression. This study aims to explore the prognostic significance of RXRG in breast cancer.MethodsPrimary breast cancer tissue microarrays (n = 923) were immuno-stained for RXRG protein and correlated with clinicopathological features, and patient outcome.ResultsNuclear RXRG expression was significantly associated with smaller tumour size (p = 0.036), lower grade(p p = 0.016), lower Nottingham Prognostic Index (p = 0.04) and longer breast cancer-specific survival (p p = 0.002). RXRG expression showed positive association with oestrogen receptor (ER)-related biomarkers: GATA3, FOXA1, STAT3 and MED7 (allp RXRGexpression was associated with improved patients’ outcome (p = 0.025). In ER-positive tumours, high expression of RXRG was associated with better patient outcome regardless of adjuvant systemic therapy. ER signalling pathway was the top predicted master regulator of RXRG protein expression (p = 0.005).ConclusionThis study provides evidence for the prognostic value of RXRG in breast cancer particularly the ER-positive tumours.

Endocrine therapy is the standard treatment for oestrogen receptor positive (ER+) breast cancer. Despite its efficacy, around half of patients will develop resistance to this treatment and eventually relapse. Identification of effective and reliable biomarkers to predict the efficacy of endocrine therapy is of crucial importance in the management of ER+ breast cancer. Emerging evidence has revealed that the cell division regulator CDC20 exhibits an oncogenic function and plays important roles in tumourigenesis and progression of solid tumours. In this study, we investigated the prognostic and predictive role of CDC20 in early ER+ breast cancer patients. The biological and clinical impact of CDC20 expression was assessed in large clinical annotated cohort of ER+ breast cancer with long-term follow-up at the mRNA level, using METABRIC and KM-Plotter datasets, and the protein level using immunohistochemistry on patients presenting at Nottingham. CDC20 expression was correlated with clinico-pathological parameters, molecular subtypes, clinical outcome and efficacy of endocrine therapy. High CDC20 mRNA expression was associated with poor clinico-pathological parameters including large tumour size and high tumour grade (P

E-cadherin is a tumor suppressor gene in invasive lobular breast cancer. However, a proportion of high-grade ductal carcinoma shows reduced/loss of E-cadherin. In this study, we assessed the underlying mechanisms and molecular implications of E-cadherin loss in invasive ductal carcinoma. This study used large, well-characterized cohorts of early-stage breast cancer-evaluated E-cadherin expression via various platforms including immunohistochemistry, microarray analysis using Illumina HT-12 v3, copy number analysis using Affymetrix SNP 6.0 arrays, and next-generation sequencing for differential gene expression. Our results showed 27% of high-grade invasive ductal carcinoma showed reduced/loss of E-cadherin membranous expression. CDH1 copy number loss was in 21% of invasive ductal carcinoma, which also showed low CDH1 mRNA expression (p = 0.003). CDH1 copy number was associated with copy number loss of TP53, ATM, BRCA1, and BRCA2 (p

Background: The androgen receptor (AR) has emerged as a potential therapeutic target for AR-positive triple-negative breast cancer (TNBC). However, conflicting reports regarding AR’s prognostic role in TNBC are putting its usefulness in question. Some studies conclude that AR positivity indicates a good prognosis in TNBC, whereas others suggest the opposite, and some show that AR status has no significant bearing on the patients’ prognosis. Methods: We evaluated the prognostic value of AR in resected primary tumors from TNBC patients from six international cohorts {US (n = 420), UK (n = 239), Norway (n = 104), Ireland (n = 222), Nigeria (n = 180), and India (n = 242); total n = 1407}. All TNBC samples were stained with the same anti-AR antibody using the same immunohistochemistry protocol, and samples with ≥1% of AR-positive nuclei were deemed AR-positive TNBCs. Results: AR status shows population-specific patterns of association with patients’ overall survival after controlling for age, grade, population, and chemotherapy. We found AR-positive status to be a marker of good prognosis in US and Nigerian cohorts, a marker of poor prognosis in Norway, Ireland and Indian cohorts, and neutral in UK cohort. Conclusion: AR status, on its own, is not a reliable prognostic marker. More research to investigate molecular subtype composition among the different cohorts is warranted.

Collagen plays a key role in normal and malignant tissue homeostasis. While the prognostic significance of collagen fiber remodeling in invasive breast cancer has been studied, its role in ductal carcinoma in situ (DCIS) remains poorly defined. Using image analysis, we aimed to evaluate the prognostic significance of the geometric characteristics of collagen surrounding DCIS. A large well-characterized cohort of DCIS comprising pure DCIS (n = 610) and DCIS coexisting with invasive carcinoma (n = 180) were histochemically stained for collagen using picrosirius red. ImageJ software was used to assess collagen density, degree of collagen fiber dispersion and directionality in relation to DCIS ducts’ boundary. We developed a collagen prognostic index and evaluated its prognostic significance. A poor index was observed in 24% of the pure DCIS and was associated with determinants of high-risk DCIS including higher nuclear grade, comedo type necrosis, hormonal receptor negativity, HER2 positivity and high proliferation index. High collagen prognostic index was associated with the collagen remodeling protein prolyl-4-hydroxlase alpha subunit 2 and the hypoxia-related protein hypoxia inducible factor 1α. DCIS coexisting with invasive breast cancer had a higher collagen prognostic index than pure DCIS ( p

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1–6. It is therefore essential to identify patients who have a high risk of late relapse7–9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47–62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials. A statistical framework for breast-cancer recurrence uses long-term follow-up data and a knowledge of molecular subcategories to model distinct disease stages and to predict the risk of relapse.

Background SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). Methods SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα− early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. Results As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc− [HR (95% CI) = 0.52 (0.34–0.78), p = 0.002]. Meanwhile, in ERα− patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto− [HR (95% CI) = 0.50 (0.34–0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc− or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28–27.03), p = 0.012], or SHON-Cyto− [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18–25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc− [HR (95% CI) = 0.41 (0.19–0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto− patients [HR (95% CI) = 4.63 (1.05–20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13–44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. Conclusion SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.