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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., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Background: N6-methyladenosine (m 6 A) is the most common internal RNA modification and is involved in regulation of RNA and protein expression. AlkB family member 5 (ALKBH5) is a m 6 A demethylase. Given the important role of m 6 A in biological mechanisms, m 6 A 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 m 6 A methylation in BC progression., (© 2024. The Author(s).)

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 mm 2 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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: None., (AJCR Copyright © 2023.)

Background: Increasing data indicate that HER2-positive (HER2 + ) breast cancer (BC) subtypes exhibit differential responses to targeted anti-HER2 therapy. This study aims to investigate these differences and the potential underlying molecular mechanisms., Methods: A large cohort of BC patients (n = 7390) was utilised. The clinicopathological characteristics and differential gene expression (DGE) of HER2+ immunohistochemical (IHC) subtypes, specifically HER2 IHC 3+ and IHC 2 + /Amplified, were assessed and correlated with pathological complete response (pCR) and survival in the neoadjuvant and adjuvant settings, respectively. The role of oestrogen receptor (ER) status was also investigated., Results: Compared to HER2 IHC 3+ tumours, BC patients with IHC 2 + /Amplified showed a significantly lower pCR rate (22% versus 57%, P < 0.001), shorter survival regardless of HER2 gene copy number, were less classified as HER2 enriched, and enriched for trastuzumab resistance and ER signalling pathway genes. ER positivity significantly decreased response to anti-HER2 therapy in IHC 2 + /Amplified, but not in IHC 3 + BC patients., Conclusion: In HER2 + BC, overexpression of HER2 protein is the driver of the oncogenic pathway, and it is the main predictor of response to anti-HER2 therapy. ER signalling pathways are more dominant in BC with equivocal HER2 expression. personalised anti-HER2 therapy based on IHC classes should be considered., (© 2023. The Author(s).)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

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., (© 2023 John Wiley & Sons Ltd.)

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.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Metzler, de Brot, Haigh, Woodcock, Lothion-Roy, Harris, Nilsson, Ntekim, Persson, Robinson, Khani, Laursen, Gudas, Toss, Madhusudan, Rakha, Heery, Rutland, Mongan and Jeyapalan.)

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., (© 2023 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

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., (© 2022. The Author(s).)

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., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

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., (© 2023. The Author(s).)

Background: Mitotic count in breast cancer is an important prognostic marker. Unfortunately, substantial inter- and intraobserver variation exists when pathologists manually count mitotic figures. To alleviate this problem, we developed a new technique incorporating both haematoxylin and eosin (H&E) and phosphorylated histone H3 (PHH3), a marker highly specific to mitotic figures, and compared it to visual scoring of mitotic figures using H&E only., Methods: Two full-face sections from 97 cases were cut, one stained with H&E only, and the other was stained with PHH3 and counterstained with H&E (PHH3-H&E). Counting mitoses using PHH3-H&E was compared to traditional mitoses scoring using H&E in terms of reproducibility, scoring time, and the ability to detect mitosis hotspots. We assessed the agreement between manual and image analysis-assisted scoring of mitotic figures using H&E and PHH3-H&E-stained cells. The diagnostic performance of PHH3 in detecting mitotic figures in terms of sensitivity and specificity was measured. Finally, PHH3 replaced the mitosis score in a multivariate analysis to assess its significance., Results: Pathologists detected significantly higher mitotic figures using the PHH3-H&E (median ± SD, 20 ± 33) compared with H&E alone (median ± SD, 16 ± 25), P < 0.001. The concordance between pathologists in identifying mitotic figures was highest when using the dual PHH3-H&E technique; in addition, it highlighted mitotic figures at low power, allowing better agreement on choosing the hotspot area (k = 0.842) in comparison with standard H&E (k = 0.625). A better agreement between image analysis-assisted software and the human eye was observed for PHH3-stained mitotic figures. When the mitosis score was replaced with PHH3 in a Cox regression model with other grade components, PHH3 was an independent predictor of survival (hazard ratio [HR] 5.66, 95% confidence interval [CI] 1.92-16.69; P = 0.002), and even showed a more significant association with breast cancer-specific survival (BCSS) than mitosis (HR 3.63, 95% CI 1.49-8.86; P = 0.005) and Ki67 (P = 0.27)., Conclusion: Using PHH3-H&E-stained slides can reliably be used in routine scoring of mitotic figures and integrating both techniques will compensate for each other's limitations and improve diagnostic accuracy, quality, and precision., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

N 6 -methyladenosine (m 6 A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m 6 A modification. These proteins, and the m 6 A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m 6 A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m 6 A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m 6 A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m 6 A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m 6 A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m 6 A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m 6 A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lothion-Roy, Haigh, Harris, Metzler, Alsaleem, Toss, Kariri, Ntekim, Robinson, Khani, Gudas, Allegrucci, James, Madhusudan, Mather, Emes, Archer, Fray, Rakha, Jeyapalan, Rutland, Mongan and Woodcock.)

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., Competing Interests: HL, TB, BM, NI, IM, AB, SM, SC, MR, MS, MT, JM, WT, DC, ER No competing interests declared, VB, HB received funding from Nimbus Therapeutics for an unrelated project, (© 2023, Luan, Bielecki, Mohapatra et al.)

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 &lt; 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., (© 2023 S. Karger AG, Basel.)

Background: Emerging evidence indicates that breast cancer (BC) patients whose tumours express HER2 protein without HER2 gene amplification (HER2-low), can benefit from antibody-drug conjugates (ADC). However, the current definition of HER2-low BC remains incomplete with low rates of concordance. This study aims to refine HER2-low definition with emphasis on distinguishing HER2 score 0 from score 1+ to identify patients who are eligible for ADC., Methods: A BC cohort (n = 363) with HER2 IHC scores 0, 1+ and 2+ (without HER2 gene amplification) and available HER2 mRNA was included. HER2 staining intensity, pattern and subcellular localisation were reassessed. Artificial neural network analysis was applied to cluster the cohort and to distinguish HER2 score 0 from 1+. Reproducibility and reliability of the refined criteria were tested., Results: HER2 IHC score 1+ was refined as membranous staining in invasive cells as either: (1) faint intensity in ≥ 20% of cells regardless the circumferential completeness, (2) weak complete staining in ≤ 10%, (3) weak incomplete staining in > 10% and (4) moderate incomplete staining in ≤ 10%. Based on this, 63% of the HER2-negative cases were reclassified as positive (HER2-low). The refined score showed perfect observer agreement compared to the moderate agreement in the original clinical scores. Similar results were generated when the refined score was applied on the independent BC cohorts. A proposal to refine the definition of other HER2 classes is presented., Conclusion: This study refined the definition of HER2-low BC based on correlation with HER2 mRNA and distinguished between HER2 IHC score 1+ and score 0 tumours., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Ki67 reflects the proliferation activity in breast cancer (BC). However, an optimal method for its assessment in clinical settings has yet to be robustly defined. In this study we compared several methods to score Ki67 to identify a reliable and reproducible method for routine practice., Methods: Sections from luminal BC cohort (n = 1662) were immunohistochemically stained with Ki67 and were assessed for the percentage, pattern, and intensity of expression. Ki67 positivity was evaluated using three methods: (i) quantification of Ki67-positive cells among 1000 invasive tumour cells within hotspot, (ii) average estimation of Ki67 within a defined hotspot, and (iii) average estimation of Ki67 positivity within the whole section. Time required for scoring, interobserver agreement and association with outcome were determined., Results: The mean percentage of Ki67 expression per 1000 cells method was 16%, while the mean value of Ki67 scores using the average estimation within hotspot and whole slide were 14% and 12%, respectively. Quantification of Ki67-positive cells within 1000 cells had the highest degree of consistency between observers, and the highest hazard ratio predicting patient outcome when compared to using different common Ki67 cutoffs, which was independent of the other two methods. Granular pattern of Ki67 expression was associated with poorer outcome as compared to the other patterns., Conclusion: Assessment of Ki67 expression using quantification positive cells among 1000 tumour cells is an optimal method to achieve high reliability and reproducibility. Comment on the predominant Ki67 expression pattern would add prognostic and predictive value in luminal BC., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Cell Division Cycle Associated 5 ( CDCA5 ) plays a role in the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling pathway involving cell division, cancer cell migration and apoptosis. This study aims to assess the prognostic and biological value of CDCA5 in breast cancer (BC)., Methods: The biological and prognostic value of CDCA5 were evaluated at mRNA ( n = 5109) and protein levels ( n = 614) utilizing multiple well-characterized early stage BC cohorts. The effects of CDCA5 knockdown (KD) on multiple oncogenic assays were assessed in vitro using a panel of BC cell lines., Results: this study examined cohorts showed that high CDCA5 expression was correlated with features characteristic of aggressive behavior and poor prognosis, including the presence of high grade, large tumor size, lymphovascular invasion (LVI), hormone receptor negativity and HER2 positivity. High CDCA5 expression, at both mRNA and protein levels, was associated with shorter BC-specific survival independent of other variables ( p = 0.034, Hazard ratio (HR) = 1.6, 95% CI; 1.1-2.3). In line with the clinical data, in vitro models indicated that CDCA5 depletion results in a marked decrease in BC cell invasion and migration abilities and a significant accumulation of the BC cells in the G2/M-phase., Conclusions: These results provide evidence that CDCA5 plays an important role in BC development and metastasis and could be used as a potential biomarker to predict disease progression in BC.

Extensive intratumoral heterogeneity (ITH) is believed to contribute to therapeutic failure and tumor recurrence, as treatment-resistant cell clones can survive and expand. However, little is known about ITH in triple-negative breast cancer (TNBC) because of the limited number of single-cell sequencing studies on TNBC. In this study, we explored ITH in TNBC by evaluating gene expression-derived and imaging-derived multi-region differences within the same tumor. We obtained tissue specimens from 10 TNBC patients and conducted RNA sequencing analysis of 2-4 regions per tumor. We developed a novel analysis framework to dissect and characterize different types of variability: between-patients (inter-tumoral heterogeneity), between-patients across regions (inter-tumoral and region heterogeneity), and within-patient, between-regions (regional intratumoral heterogeneity). We performed a Bayesian changepoint analysis to assess and classify regional variability as low (convergent) versus high (divergent) within each patient feature (TNBC and PAM50 subtypes, immune, stroma, tumor counts and tumor infiltrating lymphocytes). Gene expression signatures were categorized into three types of variability: between-patients (108 genes), between-patients across regions (183 genes), and within-patients, between-regions (778 genes). Based on the between-patient gene signature, we identified two distinct patient clusters that differed in menopausal status. Significant intratumoral divergence was observed for PAM50 classification, tumor cell counts, and tumor-infiltrating T cell abundance. Other features examined showed a representation of both divergent and convergent results. Lymph node stage was significantly associated with divergent tumors. Our results show extensive intertumoral heterogeneity and regional ITH in gene expression and image-derived features in TNBC. Our findings also raise concerns regarding gene expression based TNBC subtyping. Future studies are warranted to elucidate the role of regional heterogeneity in TNBC as a driver of treatment resistance.

Prostate cancer (PCa) is a leading cause of cancer-related deaths and is driven by aberrant androgen receptor (AR) signalling. For this reason, androgen deprivation therapies (ADTs) that suppress androgen-induced PCa progression either by preventing androgen biosynthesis or via AR signalling inhibition (ARSi) are common treatments. The N 6-methyladenosine (m6A) RNA modification is involved in regulating mRNA expression, translation, and alternative splicing, and through these mechanisms has been implicated in cancer development and progression. RNA-m6A is dynamically regulated by the METTL3 RNA methyltransferase complex and the FTO and ALKBH5 demethylases. While there is evidence supporting a role for aberrant METTL3 in many cancer types, including localised PCa, the wider contribution of METTL3, and by inference m6A, in androgen signalling in PCa remains poorly understood. Therefore, the aim of this study was to investigate the expression of METTL3 in PCa patients and study the clinical and functional relevance of METTL3 in PCa. It was found that METTL3 is aberrantly expressed in PCa patient samples and that siRNA-mediated METTL3 knockdown or METTL3-pharmacological inhibition significantly alters the basal and androgen-regulated transcriptome in PCa, which supports targeting m6A as a novel approach to modulate androgen signalling in PCa.

Purpose: The EndoPredict prognostic assay is validated to predict distant recurrence and response to chemotherapy primarily in post-menopausal women with estrogen receptor-positive (ER+), HER2- breast cancer. This study evaluated the performance of EndoPredict in pre-menopausal women., Experimental Design: Tumor samples from 385 pre-menopausal women with ER+, HER2- primary breast cancer (pT1-3, pN0-1) who did not receive chemotherapy in addition to endocrine therapy were tested with EndoPredict to produce a 12-gene EP molecular score and an integrated EPclin score that includes pathologic tumor size and nodal status. Associations of molecular and EPclin scores with 10-year distant recurrence-free survival (DRFS) were evaluated by Cox proportional hazards models and Kaplan-Meier analysis., Results: After a median follow-up of 9.7 years, both the EP molecular score and the molecular-clinicopathologic EPclin score were associated with increased risk of distant recurrence [HR, 1.33; 95% confidence interval (CI), 1.18-1.50; P = 7.2 × 10-6; HR, 3.58; 95% CI, 2.26-5.66; P = 9.8 × 10-8, respectively]. Both scores remained significant after adjusting for clinical factors in multivariate analysis. Patients with low-risk EPclin scores (64.7%) had significantly improved DRFS compared with high-risk patients (HR, 4.61; 95% CI, 1.40-15.17; P = 4.2 × 10-3). At 10 years, patients with low-risk and high-risk EPclin scores had a DRFS of 97% (95% CI, 93%-99%) and 76% (95% CI, 67%-82%), respectively., Conclusions: The EPclin score is strongly associated with DRFS in pre-menopausal women who received adjuvant endocrine therapy alone. On the basis of these data, pre-menopausal women with EPclin low-risk breast cancer may be treated with endocrine therapy only and safely forgo adjuvant chemotherapy., (©2022 The Authors; Published by the American Association for Cancer Research.)

Androgen deprivation therapies (ADTs) are important treatments which inhibit androgen-induced prostate cancer (PCa) progression by either preventing androgen biosynthesis (e.g. abiraterone) or by antagonizing androgen receptor (AR) function (e.g. bicalutamide, enzalutamide, darolutamide). A major limitation of current ADTs is they often remain effective for limited durations after which patients commonly progress to a lethal and incurable form of PCa, called castration-resistant prostate cancer (CRPC) where the AR continues to orchestrate pro-oncogenic signalling. Indeed, the increasing numbers of ADT-related treatment-emergent neuroendocrine-like prostate cancers (NePC), which lack AR and are thus insensitive to ADT, represents a major therapeutic challenge. There is therefore an urgent need to better understand the mechanisms of AR action in hormone dependent disease and the progression to CRPC, to enable the development of new approaches to prevent, reverse or delay ADT-resistance. Interestingly the AR regulates distinct transcriptional networks in hormone dependent and CRPC, and this appears to be related to the aberrant function of key AR-epigenetic coregulator enzymes including the lysine demethylase 1 (LSD1/KDM1A). In this review we summarize the current best status of anti-androgen clinical trials, the potential for novel combination therapies and we explore recent advances in the development of novel epigenetic targeted therapies that may be relevant to prevent or reverse disease progression in patients with advanced CRPC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Harris, Metzler, Lothion-Roy, Varun, Woodcock, Haigh, Endeley, Haque, Toss, Alsaleem, Persson, Gudas, Rakha, Robinson, Khani, Martin, Moyer, Brownlie, Madhusudan, Allegrucci, James, Rutland, Fray, Ntekim, de Brot, Mongan and Jeyapalan.)

Background and Aims: Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score., Methods and Results: A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5-< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007)., Conclusion: The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Atypical mitosis is considered a feature of malignancy, however, its significance in breast cancer (BC) remains elusive. Here, we aimed to assess the clinical value of atypical mitoses in BC and to explore their underlying molecular features. Atypical and typical mitotic figures were quantified and correlated with clinicopathological variables in a large cohort of primary BC tissue sections (n = 846) using digitalized hematoxylin and eosin whole-slide images (WSIs). In addition, atypical mitoses were assessed in The Cancer Genome Atlas (TCGA) BC dataset (n = 1032) and were linked to the genetic alterations and pathways. In this study, the median of typical mitoses was 17 per 3 mm 2 (range 0-120 mitoses), while the median of atypical mitoses was 4 (range 0-103 mitoses). High atypical mitoses were significantly associated with parameters characteristic of aggressive tumor behavior. The total number of mitoses, and a high atypical-to-typical mitoses ratio (>0.27) were associated with poor BC specific survival (BCSS), (p = 0.04 and 0.01, respectively). The atypical-to-typical mitoses ratio dichotomized triple negative-BC (TNBC) patients into two distinct groups in terms of the association with the outcome, while the overall number of mitoses was not. Moreover, TNBC patients with high atypical-to-typical mitoses ratio treated with adjuvant chemotherapy were associated with shorter survival (p = 0.003). Transcriptomic analysis of the TCGA-BRCA cohort dichotomized based on atypical mitoses identified 2494 differentially expressed genes. These included genes linked to pathways involved in chromosomal localization and segregation, centrosome assembly, spindle and microtubule formation, regulation of cell cycle and DNA repair. To conclude, the atypical-to-typical mitoses ratio has prognostic value independent of the overall mitotic count in BC patients and could predict the response to chemotherapy in TNBC., (© 2022. The Author(s).)

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer., (© 2022. The Author(s).)

Using digitalized whole slide images (WSI) in routine histopathology practice is a revolutionary technology. This study aims to assess the clinical impacts of WSI quality and representation of the corresponding glass slides. 40,160 breast WSIs were examined and compared with their corresponding glass slides. The presence, frequency, location, tissue type, and the clinical impacts of missing tissue were assessed. Scanning time, type of the specimens, time to WSIs implementation, and quality control (QC) measures were also considered. The frequency of missing tissue ranged from 2% to 19%. The area size of the missed tissue ranged from 1-70%. In most cases (>75%), the missing tissue area size was <10% and peripherally located. In all cases the missed tissue was fat with or without small entrapped normal breast parenchyma. No missing tissue was identified in WSIs of the core biopsy specimens. QC measures improved images quality and reduced WSI failure rates by seven-fold. A negative linear correlation between the frequency of missing tissue and both the scanning time and the image file size was observed (p < 0.05). None of the WSI with missing tissues resulted in a change in the final diagnosis. Missing tissue on breast WSI is observed but with variable frequency and little diagnostic consequence. Balancing between WSI quality and scanning time/image file size should be considered and pathology laboratories should undertake their own assessments of risk and provide the relevant mitigations with the appropriate level of caution., (© 2021. The Author(s).)

Although counting mitoses is part of breast cancer grading, concordance studies showed low agreement. Refining the criteria for mitotic counting can improve concordance, particularly when using whole slide images (WSIs). This study aims to refine the methodology for optimal mitoses counting on WSI. Digital images of 595 hematoxylin and eosin stained sections were evaluated. Several morphological criteria were investigated and applied to define mitotic hotspots. Reproducibility, representativeness, time, and association with outcome were the criteria used to evaluate the best area size for mitoses counting. Three approaches for scoring mitoses on WSIs (single and multiple annotated rectangles and multiple digital high-power (×40) screen fields (HPSFs)) were evaluated. The relative increase in tumor cell density was the most significant and easiest parameter for identifying hotspots. Counting mitoses in 3 mm 2 area was the most representative regarding saturation and concordance levels. Counting in area <2 mm 2 resulted in a significant reduction in mitotic count (P = 0.02), whereas counting in area ≥4 mm 2 was time-consuming and did not add a significant rise in overall mitotic count (P = 0.08). Using multiple HPSF, following calibration, provided the most reliable, timesaving, and practical method for mitoses counting on WSI. This study provides evidence-based methodology for defining the area and methodology of visual mitoses counting using WSI. Visual mitoses scoring on WSI can be performed reliably by adjusting the number of monitor screens., (© 2021. The Author(s).)

Background: The Ubiquitin-conjugating enzyme 2C (UBE2C) is essential for the ubiquitin-proteasome system and is involved in cancer cell migration and apoptosis. This study aimed to determine the prognostic value of UBE2C in invasive breast cancer (BC)., Methods: UBE2C was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium (n = 1980), The Cancer Genome Atlas (n = 854) and Kaplan-Meier Plotter (n = 3951) cohorts. UBE2C protein expression was assessed using immunohistochemistry in the BC cohort (n = 619). The correlation between UBE2C, clinicopathological parameters and patient outcome was assessed., Results: High UBE2C mRNA and protein expressions were correlated with features of poor prognosis, including high tumour grade, large size, the presence of lymphovascular invasion, hormone receptor negativity and HER2 positivity. High UBE2C mRNA expression showed a negative association with E-cadherin, and a positive association with adhesion molecule N-cadherin, matrix metalloproteinases and cyclin-related genes. There was a positive correlation between high UBE2C protein expression and cell cycle-associated biomarkers, p53, Ki67, EGFR and PI3K. High UBE2C protein expression was an independent predictor of poor outcome (p = 0.011, HR = 1.45, 95% CI; 1.10-1.93)., Conclusion: This study indicates that UBE2C is an independent prognostic biomarker in BC. These results warrant further functional validation for UBE2C as a potential therapeutic target in BC., (© 2022. The Author(s).)

Centrosome amplification (CA) has been implicated in the progression of various cancer types. Although studies have shown that overexpression of PLK4 promotes CA, the effect of tumor microenvironment on polo-like kinase 4 (PLK4) regulation is understudied. The aim of this study was to examine the role of hypoxia in promoting CA via PLK4. We found that hypoxia induced CA via hypoxia-inducible factor-1α (HIF1α). We quantified the prevalence of CA in tumor cell lines and tissue sections from breast cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, and prostate cancer and found that CA was prevalent in cells with increased HIF1α levels under normoxic conditions. HIF1α levels were correlated with the extent of CA and PLK4 expression in clinical samples. We analyzed the correlation between PLK4 and HIF1A mRNA levels in The Cancer Genome Atlas (TCGA) datasets to evaluate the role of PLK4 and HIF1α in breast cancer and PDAC prognosis. High HIF1A and PLK4 levels in patients with breast cancer and PDAC were associated with poor overall survival. We confirmed PLK4 as a transcriptional target of HIF1α and demonstrated that in PLK4 knockdown cells, hypoxia-mimicking agents did not affect CA and expression of CA-associated proteins, underscoring the necessity of PLK4 in HIF1α-related CA. To further dissect the HIF1α-PLK4 interplay, we used HIF1α-deficient cells overexpressing PLK4 and showed a significant increase in CA compared with HIF1α-deficient cells harboring wild-type PLK4. These findings suggest that HIF1α induces CA by directly upregulating PLK4 and could help us risk-stratify patients and design new therapies for CA-rich cancers., Implications: Hypoxia drives CA in cancer cells by regulating expression of PLK4, uncovering a novel HIF1α/PLK4 axis., (©2021 The Authors; Published by the American Association for Cancer Research.)

Lymphovascular invasion (LVI) is a key step in breast cancer (BC) metastasis. Targeting the molecular drivers of LVI can improve BC patients' management. However, the underlying molecular mechanisms of LVI are complex and interconnected with various carcinogenesis pathways. This study aimed to identify the key regulatory gene associated with LVI and to investigate its mechanisms of action and prognostic significance. Artificial neural network (ANN) was applied to two large transcriptomic datasets of BC with well-characterised LVI status. Cyclin B2 ( CCNB2 ) was identified in the top genes associated with LVI positivity. In vitro functional assays were carried out to assess the role of CCNB2 in tumour cell behaviour and their interactions with endothelial cells using a panel of BC cell lines. Large annotated BC cohorts were used to assess the clinical and prognostic role of CCNB2 at the transcriptomic and protein levels. Knockdown (KD) of CCNB2 mRNA reduced BC cell migration, inhibited proliferation, blocked the G2/M transition during the cell cycle and increased the number of apoptotic cells. Importantly, KD of CCNB2 reduced BC cell lines adherence and transmigration across endothelial cell lines. High CCNB2 protein expression was independently associated with LVI positivity in addition to other features of aggressive behaviour, including larger tumour size, higher histological grade, hormonal receptor-negativity, and HER2-positivity, and with shorter survival. We conclude that CCNB2 plays a crucial role in LVI development in BC, implying that CCNB2 could confer a promising therapeutic target to inhibit LVI and reduce metastatic events., Competing Interests: None., (AJCR Copyright © 2022.)

Despite the importance of atypia in diagnosing and classifying breast lesions, the definition of atypia varies depending on the context, with a lack of consistent and objective criteria for assessment. Atypia in breast pathology may be cytonuclear and/or architectural with different applications and implications. Cytonuclear atypia is used to assist the distinction of various intraductal epithelial proliferative lesions including usual ductal hyperplasia (UDH) versus atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS), and to grade DCIS. In invasive carcinoma, nuclear atypia (i.e., nuclear pleomorphism) is a component of the histological grading system. Stromal cell cytonuclear atypia is one of the key features used to distinguish fibroadenoma from phyllodes tumour (PT) and to classify PT as benign, borderline or malignant. Similarly, cytonuclear atypia is used in the evaluation of myoepithelial cell alterations in the breast. Architectural atypia is used to differentiate flat epithelial atypia (FEA) from ADH or DCIS. In addition to the inherent subjectivity in the interpretation of atypia, which presents as a morphological continuum reflecting a biological spectrum, the lack of standardisation in defining atypia augments diagnostic discordance in breast pathology, with potential implications for patient management. Evidence to date suggests that the traditional criteria used to assess atypia may require modification in the era of digital pathology primary diagnosis. This review aims to provide a comprehensive review of atypia in breast pathology with reference to inconsistencies, challenges and limitations., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Aims: Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions., Methods and Results: ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified., Conclusion: This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology., (© 2021 John Wiley & Sons Ltd.)

Introduction: Protein arginine deiminases (PADIs) are a family of enzymes that catalyse the post-translational modification of proteins. Association between PADI expression and clinicopathology, protein expression, and outcome was determined., Methods: PADI2 and PADI4 expression was assessed immunohistochemically in a cohort of colorectal cancer (CRC) patients., Results: CRC tissues expressed variable levels of PADI2 which was mainly localised in the cytoplasm and correlated with patient survival (p = 0.005); high expression increased survival time from 43.5 to 67.6 months. Expression of cytoplasmic PADI2 correlated with the expression of nuclear β catenin, PADI4, and alpha-enolase. In contrast, expression of nuclear PADI2 correlated with a decrease in survival (p = 0.010), with high expression decreasing survival from 76.4 to 42.9 months. CRC tissues expressed variable levels of PADI4 in both the nucleus and cytoplasm. Expression of cytoplasmic PADI4 correlated with survival (p = 0.001) with high expression increasing survival time from 48.1 to 71.8 months. Expression of cytoplasmic PADI4 correlated with expression of nuclear β catenin, alpha-enolase (p ≤ 0.0001, p = 0.002), and the apoptotic related protein, Bcl-2. Expression of nuclear PADI4 also correlated with survival (p = 0.011), with high expression of nuclear PADI4 increasing survival time from 55.4 to 74 months. Expression of nuclear PADI4 correlated with p53, alpha-enolase, and Bcl-2. Multivariate analysis showed that TNM stage, cytoplasmic PADI2, and PADI4 remained independent prognostic factors in CRC. Both PADI2 and PADI4 are good prognostic factors in CRC., Conclusion: High expression of cytoplasmic PADI2, PADI4, and nuclear PADI4 were associated with an increase in overall survival., (© 2021 S. Karger AG, Basel.)

Introduction: Aurora Kinase A (AURKA/STK15) has a role in centrosome duplication and is a regulator of mitotic cell proliferation. It is over-expressed in breast cancer and other cancers, however; its role in ductal carcinoma in situ (DCIS) remains to be defined. This study aims to characterize AURKA protein expression in DCIS and evaluate its prognostic significance., Methods: AURKA was assessed immunohistochemically in a large well-characterized cohort of DCIS (n = 776 pure DCIS and 239 DCIS associated with invasive breast cancer [DCIS-mixed]) with long-term follow-up data (median = 105 months) and basic molecular characterization., Results: High AURKA expression was observed in 15% of DCIS cases and was associated with features of aggressiveness including larger tumour size, high nuclear grade, hormone receptor negativity, HER2 positivity, and high Ki67 proliferation index. AURKA expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher AURKA expression than the DCIS component (p < 0.0001). Outcome analysis revealed that AURKA was a predictor of invasive recurrence (p = 0.002)., Conclusion: High AURKA expression is associated with poor prognosis in DCIS and might be a potential marker to predict DCIS progression to invasive disease., (© 2022 S. Karger AG, Basel.)

Background and Aims: Proliferation is an important indicator of breast cancer (BC) prognosis, but is assessed using different approaches. Not all cells in the cell cycle are committed to division. This study aimed to characterise quantitative differences between BC cells in the cell cycle and those in mitosis and assess their relationship with other pathological parameters., Methods and Results: A cohort of BC sections (n = 621) was stained with haematoxylin and eosin and immunohistochemistry for Ki-67. The proportion of mitotic cells and Ki-67-positive cells was assessed in the same areas. The Cancer Genome Atlas (TCGA) BC cohort was used to assess MKI-67 transcriptome level and its association with the mitotic counts. The mean proportion of BC cells in the cell cycle was 24% (range = 1-90%), while the mean proportion of BC cells in mitosis was 5% (range = 0-73%). A low proportion of mitoses to whole cycling cells was associated with low histological grade tumours and the luminal A molecular subtype, while tumours with a high proportion of mitoses to the overall cycling cells were associated with triple-negative subtype, larger tumour size, grade 3 tumours and lymph node metastasis. The high mitosis/low Ki-67-positive cells tumours showed a significant association with variables of poor prognosis, including high-grade and triple-negative subtypes., Conclusion: The proportion of BC cells in the cell cycle and mitosis is variable. We show that not only the number of cells in the cell cycle or mitosis, but also the difference between them, provides valuable information on tumour aggressiveness., (© 2021 John Wiley & Sons Ltd.)