The role of the ALKBH5 RNA demethylase in invasive breast cancer.

BACKGROUND: N6-methyladenosine (m6A) is the most common internal RNA modification and is involved in regulation of RNA and protein expression. AlkB family member 5 (ALKBH5) is a m6A demethylase. Given the important role of m6A in biological mechanisms, m6A and its regulators, have been implicated in many disease processes, including cancer. However, the contribution of ALKBH5 to invasive breast cancer (BC) remains poorly understood. The aim of this study was to evaluate the clinicopathological value of ALKBH5 in BC. METHODS: Publicly available data were used to investigate ALKBH5 mRNA alterations, prognostic significance, and association with clinical parameters at the genomic and transcriptomic level. Differentially expressed genes (DEGs) and enriched pathways with low or high ALKBH5 expression were investigated. Immunohistochemistry (IHC) was used to assess ALKBH5 protein expression in a large well-characterised BC series (n = 1327) to determine the clinical significance and association of ALKBH5 expression. RESULTS: Reduced ALKBH5 mRNA expression was significantly associated with poor prognosis and unfavourable clinical parameters. ALKBH5 gene harboured few mutations and/or copy number alternations, but low ALKBH5 mRNA expression was seen. Patients with low ALKBH5 mRNA expression had a number of differentially expressed genes and enriched pathways, including the cytokine-cytokine receptor interaction pathway. Low ALKBH5 protein expression was significantly associated with unfavourable clinical parameters associated with tumour progression including larger tumour size and worse Nottingham Prognostic Index group. CONCLUSION: This study implicates ALKBH5 in BC and highlights the need for further functional studies to decipher the role of ALKBH5 and RNA m6A methylation in BC progression.

Prognostic and Clinical Significance of the Proliferation Marker MCM7 in Breast Cancer.

INTRODUCTION: Minichromosome maintenance complex component 7 (MCM7) plays an essential role in proliferation and DNA replication of cancer cells. However, the expression and prognostic significance of MCM7 in breast cancer (BC) remain to be defined. In this study, we aimed to evaluate the role of MCM7 in BC. METHODS: We conducted immunohistochemistry staining of MCM7 in 1,156 operable early-stage BC samples and assessed MCM7 at the transcriptomic levels using publicly available cohorts (n = 13,430). MCM7 expression was evaluated and correlated with clinicopathological parameters including Ki67 labelling index and patient outcome. RESULTS: At the transcriptomic level, there was a significant association between high MCM7 mRNA levels and shorter patient survival in the whole cohort and in luminal BC class but not in the basal-like molecular subtype. High MCM7 protein expression was detected in 43% of patients and was significantly associated with parameters characteristic of aggressive tumour behaviour. MCM7 was independently associated with shorter survival, particularly in oestrogen receptor-positive (luminal) BC. MCM7 stratified luminal tumours with aggressive clinicopathological features into distinct prognostic groups. In endocrine therapy-treated BC patients, high MCM7 was associated with poor outcome, but such association disappeared with administration of adjuvant chemotherapy. Patients with high expression of Ki67 and MCM7 showed worst survival, while patients with double low expression BC showed the best outcome compared with single expression groups. CONCLUSION: The current findings indicate that MCM7 expression has a prognostic value in BC and can be used to identify luminal BC patients who can benefit from adjuvant chemotherapy.

Novel 2 Gene Signatures Associated With Breast Cancer Proliferation: Insights From Predictive Differential Gene Expression Analysis.

The use of proliferation markers provides valuable information about the rate of tumor growth, which can guide treatment decisions. However, there is still a lack of consensus regarding the optimal molecular markers or tests to use in clinical practice. Integrating gene expression data with clinical and histopathologic parameters enhances our understanding of disease processes, facilitates the identification of precise prognostic predictors, and supports the development of effective therapeutic strategies. The purpose of this study was to apply an integrated approach that combines morphologic, clinical, and bioinformatic data to reveal effective regulators of proliferation. Whole-slide images generated from hematoxylin-and-eosin-stained sections of The Cancer Genome Atlas (TCGA) breast cancer (BC) database (n = 1053) alongside their transcriptomic and clinical data were used to identify genes differentially expressed between tumors with high and low mitotic scores. Genes enriched in the cell-cycle pathway were used to predict the protein-protein interaction (PPI) network. Ten hub genes (ORC6, SKP2, SMC1B, CDKN2A, CDC25B, E2F1, E2F2, ORC1, PTTG1, and CDC25A) were identified using CytoHubba a Cytoscape plugin. In a multivariate Cox regression model, ORC6 and SKP2 were predictors of survival independent of existing methods of proliferation assessment including mitotic score and Ki67. The prognostic ability of these genes was validated using the Molecular Taxonomy of Breast Cancer International Consortium, Nottingham cohort, Uppsala cohort, and a combined multicentric cohort. The protein expression of these 2 genes was investigated on a large cohort of BC cases, and they were significantly associated with poor prognosis and patient outcome. A positive correlation between ORC6 and SKP2 mRNA and protein expression was observed. Our study has identified 2 gene signatures, ORC6 and SKP2, which play a significant role in BC proliferation. These genes surpassed both mitotic scores and Ki67 in multivariate analysis. Their identification provides potential opportunities for the development of targeted treatments for patients with BC.

Artificial Intelligence-Based Mitosis Scoring in Breast Cancer: Clinical Application.

In recent years, artificial intelligence (AI) has demonstrated exceptional performance in mitosis identification and quantification. However, the implementation of AI in clinical practice needs to be evaluated against the existing methods. This study is aimed at assessing the optimal method of using AI-based mitotic figure scoring in breast cancer (BC). We utilized whole slide images from a large cohort of BC with extended follow-up comprising a discovery (n = 1715) and a validation (n = 859) set (Nottingham cohort). The Cancer Genome Atlas of breast invasive carcinoma (TCGA-BRCA) cohort (n = 757) was used as an external test set. Employing automated mitosis detection, the mitotic count was assessed using 3 different methods, the mitotic count per tumor area (MCT; calculated by dividing the number of mitotic figures by the total tumor area), the mitotic index (MI; defined as the average number of mitotic figures per 1000 malignant cells), and the mitotic activity index (MAI; defined as the number of mitotic figures in 3 mm2 area within the mitotic hotspot). These automated metrics were evaluated and compared based on their correlation with the well-established visual scoring method of the Nottingham grading system and Ki67 score, clinicopathologic parameters, and patient outcomes. AI-based mitotic scores derived from the 3 methods (MCT, MI, and MAI) were significantly correlated with the clinicopathologic characteristics and patient survival (P < .001). However, the mitotic counts and the derived cutoffs varied significantly between the 3 methods. Only MAI and MCT were positively correlated with the gold standard visual scoring method used in Nottingham grading system (r = 0.8 and r = 0.7, respectively) and Ki67 scores (r = 0.69 and r = 0.55, respectively), and MAI was the only independent predictor of survival (P < .05) in multivariate Cox regression analysis. For clinical applications, the optimum method of scoring mitosis using AI needs to be considered. MAI can provide reliable and reproducible results and can accurately quantify mitotic figures in BC.

The expression of high mobility group protein 3 (HMGB3) in breast cancer with emphasis on its role in lymphovascular invasion.

Lymphovascular invasion (LVI) is a common phenomenon in breast cancer (BC), and it is correlated to poor outcome. However, the biomarkers that influence the development of LVI remain to be defined. Through rigorous bioinformatics analyses, high mobility group protein 3 (HMGB3) was revealed as a driver gene that is associated with the presence of LVI. The purpose of this study was to further investigate the role of HMGB3 in the pathogenesis of LVI in BC. In vitro functional assays were performed to investigate the effect of HMGB3 silencing on cell proliferation, migration, adherence and transmigration of BC cell lines with dermal lymphatic endothelial cells (DLECs) and human vascular endothelial cells (HUVECs). The correlation of HMGB3 expression with clinicopathological parameters was also assessed at the transcriptomic and the proteomic levels using large BC cohorts with well-characterised LVI status. Silencing HMGB3 reduced cell proliferation, migration, adherence and transmigration across endothelial cell lines. At the mRNA and protein levels, high HMGB3 expression was significantly correlated with LVI-positivity, higher tumour grade, lymph nodal stage, hormone receptor negativity, HER2 positivity and poor outcome. Moreover, high HMGB3 expression was an independent predictor of shorter breast cancer-specific survival. HMGB3 plays an oncogenic function and contributes to the development of LVI in BC. Results warrant further investigation as a potential target to inhibit LVI in BC.

Characterisation of luminal and triple-negative breast cancer with HER2 Low protein expression.

BACKGROUND: Breast cancer (BC) expressing low levels of human epidermal growth factor receptor 2 (HER2 Low) is an emerging category that needs further refining. This study aims to provide a comprehensive clinico-pathological and molecular profile of HER2 Low BC including response to therapy and patient outcome in the adjuvant and neoadjuvant settings. METHODS: Two different independent and well-characterised BC cohorts were included. Nottingham cohort (A) (n = 5744) and The Cancer Genome Atlas (TCGA) BC cohort (B) (n = 854). The clinical, molecular, biological and immunological profile of HER2 Low BC was investigated. Transcriptomic and pathway enrichment analyses were performed on the TCGA BC cohort and validated through next-generation sequencing in a subset of Nottingham cases. RESULTS: Ninety percent of HER2 Low tumours were hormone receptor (HR) positive (HR+), enriched with luminal intrinsic molecular subtype, lacking significant expression of HER2 oncogenic signalling genes and of favourable clinical behaviour compared to HER2 negative (HER2-) BC. In HR+ BC, no significant prognostic differences were detected between HER2 Low and HER2- tumours. However, in HR- BC, HER2 Low tumours were less aggressive with longer patient survival. Transcriptomic data showed that the majority of HR- /HER2 Low tumours were of luminal androgen receptor (LAR) intrinsic subtype, enriched with T-helper lymphocytes, activated dendritic cells and tumour associated neutrophils, while most HR-/HER2- tumours were basal-like, enriched with tumour associated macrophages. CONCLUSION: HER2 Low BC is mainly driven by HR signalling in HR+ tumours. HR-/HER2 Low tumours tend to be enriched with LAR genes with a unique immune profile.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Preoperative diagnosis of ductal carcinoma in situ of the breast over a 24-year period.

AIMS: The method of diagnosis of ductal carcinoma in situ (DCIS) has changed since the 1980s. The aim of this audit was to assess changes in the preoperative diagnosis of DCIS since the introduction of needle core biopsy, particularly the proportion with a preoperative biopsy diagnosis of DCIS. METHODS AND RESULTS: The preoperative diagnoses of patients with a final diagnosis of DCIS in the surgical specimen were reviewed (i) in 809 patients who presented through breast screening from 1997 to 2021, and (ii) in all patients in 5 individual years at 5-year intervals from 2000 to 2020 (254 in total). For screening-detected DCIS the proportion with a preoperative diagnosis of DCIS increased from 75% to 98% over the study period. In a detailed analysis of all cases of DCIS in 5 separate years the proportion with a preoperative diagnosis of DCIS increased from 68% in 2000 to 96% in 2020. For high-grade DCIS the proportion increased from 87% to 97%, and for low- or intermediate-grade DCIS from 48% to 93%. The proportion of women who had vacuum-assisted biopsy increased from 7% in 2000 to 58% in 2015. There was a small increase in the number of biopsies that had basal cytokeratin and oestrogen receptor immunohistochemistry to aid diagnosis. CONCLUSION: There has been an increase in the preoperative diagnosis of DCIS, particularly of low- or intermediate-grade, over the last two decades. The increasing use of vacuum-assisted biopsy is likely to be a major contributory factor to this increase.

Deciphering the Morphology of Tumor-Stromal Features in Invasive Breast Cancer Using Artificial Intelligence.

Tumor-associated stroma in breast cancer (BC) is complex and exhibits a high degree of heterogeneity. To date, no standardized assessment method has been established. Artificial intelligence (AI) could provide an objective morphologic assessment of tumors and stroma, with the potential to identify new features not discernible by visual microscopy. In this study, we used AI to assess the clinical significance of (1) stroma-to-tumor ratio (S:TR) and (2) the spatial arrangement of stromal cells, tumor cell density, and tumor burden in BC. Whole-slide images of a large cohort (n = 1968) of well-characterized luminal BC cases were examined. Region and cell-level annotation was performed, and supervised deep learning models were applied for automated quantification of tumor and stromal features. S:TR was calculated in terms of surface area and cell count ratio, and the S:TR heterogeneity and spatial distribution were also assessed. Tumor cell density and tumor size were used to estimate tumor burden. Cases were divided into discovery (n = 1027) and test (n = 941) sets for validation of the findings. In the whole cohort, the stroma-to-tumor mean surface area ratio was 0.74, and stromal cell density heterogeneity score was high (0.7/1). BC with high S:TR showed features characteristic of good prognosis and longer patient survival in both the discovery and test sets. Heterogeneous spatial distribution of S:TR areas was predictive of worse outcome. Higher tumor burden was associated with aggressive tumor behavior and shorter survival and was an independent predictor of worse outcome (BC-specific survival; hazard ratio: 1.7, P = .03, 95% CI, 1.04-2.83 and distant metastasis-free survival; hazard ratio: 1.64, P = .04, 95% CI, 1.01-2.62) superior to absolute tumor size. The study concludes that AI provides a tool to assess major and subtle morphologic stromal features in BC with prognostic implications. Tumor burden is more prognostically informative than tumor size.

The KDM5B and KDM1A lysine demethylases cooperate in regulating androgen receptor expression and signalling in prostate cancer.

Histone H3 lysine 4 (H3K4) methylation is key epigenetic mark associated with active transcription and is a substrate for the KDM1A/LSD1 and KDM5B/JARID1B lysine demethylases. Increased expression of KDM1A and KDM5B is implicated in many cancer types, including prostate cancer (PCa). Both KDM1A and KDM5B interact with AR and promote androgen regulated gene expression. For this reason, there is great interested in the development of new therapies targeting KDM1A and KDM5B, particularly in the context of castrate resistant PCa (CRPC), where conventional androgen deprivation therapies and androgen receptor signalling inhibitors are no longer effective. As there is no curative therapy for CRPC, new approaches are urgently required to suppress androgen signalling that prevent, delay or reverse progression to the castrate resistant state. While the contribution of KDM1A to PCa is well established, the exact contribution of KDM5B to PCa is less well understood. However, there is evidence that KDM5B is implicated in numerous pro-oncogenic mechanisms in many different types of cancer, including the hypoxic response, immune evasion and PI3/AKT signalling. Here we elucidate the individual and cooperative functions of KDM1A and KDM5B in PCa. We show that KDM5B mRNA and protein expression is elevated in localised and advanced PCa. We show that the KDM5 inhibitor, CPI-455, impairs androgen regulated transcription and alternative splicing. Consistent with the established role of KDM1A and KDM5B as AR coregulators, we found that individual pharmacologic inhibition of KDM1A and KDM5 by namoline and CPI-455 respectively, impairs androgen regulated transcription. Notably, combined inhibition of KDM1A and KDM5 downregulates AR expression in CRPC cells. Furthermore, combined KDM1A and KDM5 inhibition impairs PCa cell proliferation and invasion more than individual inhibition of KDM1A and KDM5B. Collectively our study has identified individual and cooperative mechanisms involving KDM1A and KDM5 in androgen signalling in PCa. Our findings support the further development of KDM1A and KDM5B inhibitors to treat advanced PCa. Further work is now required to confirm the therapeutic feasibility of combined inhibition of KDM1A and KDM5B as a novel therapeutic strategy for targeting AR positive CRPC.

Characteristics and prognostic significance of polo-like kinase-1 (PLK1) expression in breast cancer.

AIM: Polo-like kinase-1 (PLK1) plays a crucial role in cell cycle progression, and it is considered a potential therapeutic target in many cancers. Although the role of PLK1 is well established in triple-negative breast cancer (TNBC) as an oncogene, its role in luminal BC is still controversial. In this study, we aimed to evaluate the prognostic and predictive role of PLK1 in BC and its molecular subtypes. METHODS: A large BC cohort (n = 1208) were immunohistochemically stained for PLK1. The association with clinicopathological, molecular subtypes, and survival data was analysed. PLK1 mRNA was evaluated in the publicly available datasets (n = 6774), including The Cancer Genome Atlas and the Kaplan-Meier Plotter tool. RESULTS: 20% of the study cohort showed high cytoplasmic PLK1 expression. High PLK1 expression was significantly associated with a better outcome in the whole cohort, luminal BC. In contrast, high PLK1 expression was associated with a poor outcome in TNBC. Multivariate analyses indicated that high PLK1 expression is independently associated with longer survival in luminal BC, and in poorer prognosis in TNBC. At the mRNA levels, PLK1 expression was associated with short survival in TNBC consistent with the protein expression. However, in luminal BC, its prognostic value significantly varies between cohorts. CONCLUSION: The prognostic role of PLK1 in BC is molecular subtype-dependent. As PLK1 inhibitors are introduced to clinical trials for several cancer types, our study supports evaluation of the pharmacological inhibition of PLK1 as an attractive therapeutic target in TNBC. However, in luminal BC, PLK1 prognostic role remains controversial.

High Inner Centromere Protein Expression Correlates with Aggressive Features and Predicts Poor Prognosis in Patients with Invasive Breast Cancer.

INTRODUCTION: Inner centromere protein (INCENP) is a member of the chromosomal passenger complex and plays a key role in mitosis and cell proliferation. This study aimed to evaluate the clinical and prognostic significance of INCENP in invasive breast cancer (BC). METHODS: INCENP expression was evaluated on a tissue microarray of a large BC cohort (n = 1,295) using immunohistochemistry. At the mRNA level, INCENP expression was assessed using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n = 1,980) and The Cancer Genome Atlas (TCGA) BC cohorts (n = 854). The correlations between INCENP expression, clinicopathological parameters, and patient outcome were investigated. RESULTS: INCENP expression was detected in the nucleus and cytoplasm of the tumour cells. Its expression was significantly associated with features characteristic of aggressive BC behaviour including high tumour grade, larger tumour size, and high Nottingham prognostic index scores. High INCENP nuclear expression was a predictor of shorter BC-specific survival in the whole cohort, as well as in the luminal subtype (p < 0.001). High INCENP nuclear expression was predictive of poor prognosis in BC patients who received hormone treatment or chemotherapy. CONCLUSION: High INCENP expression is a poor prognostic biomarker in BC with potential therapeutic benefits.

Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers.

Replication Protein A (RPA), a heterotrimeric complex consisting of RPA1, 2, and 3 subunits, is a single-stranded DNA (ssDNA)-binding protein that is critically involved in replication, checkpoint regulation and DNA repair. Here we have evaluated RPA in 776 pure ductal carcinomas in situ (DCIS), 239 DCIS that co-exist with invasive breast cancer (IBC), 50 normal breast tissue and 4221 IBC. Transcriptomic [METABRIC cohort (n = 1980)] and genomic [TCGA cohort (n = 1090)] evaluations were completed. Preclinically, RPA deficient cells were tested for cisplatin sensitivity and Olaparib induced synthetic lethality. Low RPA linked to aggressive DCIS, aggressive IBC, and shorter survival outcomes. At the transcriptomic level, low RPA tumours overexpress pseudogene/lncRNA as well as genes involved in chemical carcinogenesis, and drug metabolism. Low RPA remains linked with poor outcome. RPA deficient cells are sensitive to cisplatin and Olaparib induced synthetic lethality. We conclude that RPA directed precision oncology strategy is feasible in breast cancers.

Expression, assessment and significance of Ki67 expression in breast cancer: an update.

Ki67 expression is one of the most important and cost-effective surrogate markers to assess for tumour cell proliferation in breast cancer (BC). The Ki67 labelling index has prognostic and predictive value in patients with early-stage BC, particularly in the hormone receptor-positive, HER2 (human epidermal growth factor receptor 2)-negative (luminal) tumours. However, many challenges exist in using Ki67 in routine clinical practice and it is still not universally used in the clinical setting. Addressing these challenges can potentially improve the clinical utility of Ki67 in BC. In this article, we review the function, immunohistochemical (IHC) expression, methods for scoring and interpretation of results as well as address several challenges of Ki67 assessment in BC. The prodigious attention associated with use of Ki67 IHC as a prognostic marker in BC resulted in high expectation and overestimation of its performance. However, the realisation of some pitfalls and disadvantages, which are expected with any similar markers, resulted in an increasing criticism of its clinical use. It is time to consider a pragmatic approach and weigh the benefits against the weaknesses and identify factors to achieve the best clinical utility. Here we highlight the strengths of its performance and provide some insights to overcome the existing challenges.

Combined proliferation and apoptosis index provides better risk stratification in breast cancer.

AIMS: Breast cancer (BC) risk stratification is critical for predicting behaviour and guiding management decision-making. Despite the well-established prognostic value of cellular proliferation in BC, the interplay between proliferation and apoptosis remains to be defined. In this study, we hypothesised that the combined proliferation and apoptosis indices can provide a more accurate in-vivo growth rate measure and a precise prognostic predictor. METHODS AND RESULTS: Apoptotic and mitotic figures were counted in whole slide images (WSI) generated from haematoxylin and eosin-stained sections of 1545 BC cases derived from two well-defined BC cohorts. Counts were carried out visually within defined areas. There was a significant correlation between mitosis and apoptosis scores. High apoptotic counts were associated with features of aggressive behaviour, including high grade, high pleomorphism score and hormonal receptor negativity. Although the mitotic index (MI) and apoptotic index (AI) were independent prognostic indicators, the prognostic value was synergistically higher when combined. BC patients with a high combined AI and MI had the shortest survival. Replacing the mitosis score with the mitosis-apoptosis index in the Nottingham grading system revealed that the modified grade with the new score had a higher significant association with BC-specific survival with a higher hazard ratio. CONCLUSION: Apoptotic figures count provides additional prognostic value in BC when combined with MI; such a combination can be implemented to assess the behaviour of BC and provides an accurate prognostic indicator. This can be considered when using artificial intelligence algorithms to assess proliferation in BC.

The clinical value of progesterone receptor expression in luminal breast cancer: A study of a large cohort with long-term follow-up.

BACKGROUND: The routine assessment of progesterone receptor (PR) expression in breast cancer (BC) remains controversial. This study aimed to evaluate the role of PR expression in luminal BC, with emphasis on the definition of positivity and its prognostic significance as compared to Ki67 expression. METHODS: A large cohort (n = 1924) of estrogen receptor (ER)-positive/HER2-negative BC was included. PR was immunohistochemically (IHC) stained on full face sections and core needle biopsies (CNB) where the optimal scoring cutoff was evaluated. In addition, the association of PR with other clinicopathological factors, cellular proliferation, disease outcome, and response to adjuvant therapy were analyzed. RESULTS: Although several cutoffs showed prognostic significance, the optimal cutoff to categorize PR expression into two clinically distinct prognostic groups on CNB was 10%. PR negativity showed a significant association with features of aggressive tumor behavior and poor outcome. Multivariate analyses indicated that the association between PR negativity and poor outcome was independent of tumor grade, size, node stage, and Ki67. PR negativity showed independent association with shorter survival in patients who received endocrine therapy whereas Ki67did not. CONCLUSION: PR IHC expression provides independent prognostic value superior to Ki67. Routine assessment of PR expression in BC using 10% cutoff in the clinical setting is recommended. PLAIN LANGUAGE SUMMARY: In this study, we have established an optimal approach to determine the prognostic value of progesterone receptor expression in estrogen receptor-positive breast cancer patients. To do this, the levels of progesterone receptor were measured in a large cohort of estrogen receptor-positive breast cancer patients. We have refined the definition of progesterone receptor positivity in estrogen receptor-positive breast cancer. We show that progesterone receptor expression adds prognostic and predictive value of endocrine therapy in estrogen receptor-positive breast cancer patients, and our results show that the absence of progesterone receptor is associated with poorer outcomes independent of tumor grade, size, node stage, and Ki67 expression.

Evaluation oncotype DX® 21-gene recurrence score and clinicopathological parameters: a single institutional experience.

AIMS: Oncotype DX recurrence score (RS) is a clinically validated assay, which predicts the likelihood of disease recurrence in oestrogen receptor-positive/HER2-negative (ER+/HER2-) breast cancer (BC). In this study we aimed to compare the performance of Oncotype DX against the conventional clinicopathological parameters using a large BC cohort diagnosed in a single institution. METHODS AND RESULTS: A cohort (n = 430) of ER+/HER2- BC patients who were diagnosed at the Nottingham University Hospitals NHS Trust and had Oncotype DX testing was included. Correlation with the clinicopathological and other biomarkers, including the proliferation index, was analysed. The median Oncotype DX RS was 17.5 (range = 0-69). There was a significant association between high RS and grade 3 tumours. No grade 1 BC or grade 2 tumours with mitosis score 1 showed high RS. Low RS was significantly associated with special tumour types where none of the patients with classical lobular or tubular carcinomas had a high RS. There was an inverse association between RS and levels of ER and progesterone receptor (PR) expression and a positive linear correlation with Ki67 labelling index. Notably, six patients who developed recurrence had an intermediate RS; however, four of these six cases (67%) were identified as high-risk disease when the conventional clinical and molecular parameters were considered. CONCLUSION: Oncotype DX RS is correlated strongly with the conventional clinicopathological parameters in BC. Some tumour features such as tumour grade, type, PR status and Ki67 index can be used as surrogate markers in certain scenarios.

EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry.

With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2- and CAV1/2-overexpressing BC.

The clinical significance of cyclin B1 (CCNB1) in invasive breast cancer with emphasis on its contribution to lymphovascular invasion development.

BACKGROUND: Lymphovascular invasion (LVI) is regulated through complex molecular mechanisms. Cyclin B1 (CCNB1) was previously determined as being associated with LVI using large cohorts of breast cancer (BC) and artificial neural network (ANN) technique. In this study, we aimed to assess the association between CCNB1 and LVI, other clinicopathological and other LVI-related biomarkers at the molecular (RNA transcriptomic) and proteomic levels in BC. METHODS: Two transcriptomic BC cohorts (n = 2834) were used to assess the association between the expression of CCNB1 at the mRNA level and clinicopathological characteristics and patient outcome. Tissue microarrays (TMAs) from a well-characterised BC cohort (n = 2480) with long-term outcome were also used to assess the clinical significance of CCNB1 protein expression using immunohistochemistry. RESULTS: High CCNB1 mRNA expression was associated with aggressive tumour behaviour, including LVI, larger size, higher tumour grade, high lymph nodal stage, hormonal receptor negativity, HER2 positivity and poor clinical outcome (all p < 0.0001). Similarly, high CCNB1 protein expression was associated with higher tumour grade, hormonal receptor negativity and HER2 positivity (all p < 0.0001). Additionally, there was a significant association between CCNB1- and LVI-related biomarkers including N-cadherin, P-cadherin and TWIST2 at the transcriptomic and proteomic level. Multivariate analysis revealed that CCNB1 was an independent predictor of shorter BC-specific survival (HR = 1.3; 95% CI 1.2-1.5; p = 0.010). CONCLUSION: CCNB1 is a key gene associated with LVI in BC and has prognostic value. More functional studies are warranted to unravel the mechanistic role of CCNB1 in the development of LVI.