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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. This study provides further evidence that assessment of progesterone receptor status in the luminal breast cancer can provide valuable prognostic and predictive information independent of other clinicopathological variables comparable to that provided by Ki67 and can outperform it in certain situations. Using a 10% rather than a 1% cutoff for progesterone receptor expression provides the optimal prognostic significance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and aims Methods Results Conclusions Lobular carcinoma (LC) of the breast exhibits diverse morphology and clinical behaviour. The pleomorphic variant (pLC) displays distinct cytonuclear features and aggressiveness compared to the classic variant (cLC). However, diagnosing pLC remains subjective. This study aims to refine LC's cytonuclear features, focusing on pLC.Whole slide images of 59 LCs, including both in situ (LCIS) and invasive (ILC) lesions, were analysed. Nuclear measurements, including nuclear size and variability, were scored using QuPath image analysis software. For comparison, selected features were scored in normal cells (n = 10) and pleomorphism score‐matched invasive breast carcinoma (IBC) of NST type (n = 33). Additional visual assessment of the pleomorphic ILC (pILC) cohort (n = 90) was conducted for cytomorphological features characterisation.pILC demonstrated larger nuclear area and higher nuclear variability with abundance of cytoplasm than cILC. Compared to lymphocytes, pILC demonstrated a median area ranging from 2.7 to 4.7 times larger. Cut‐off values for differentiating pILC from other ILC subtypes included median nuclear area > 48.2 μm2 and interquartile range (IQR) > 19.4, nuclear perimeter median > 25.2 μm and IQR > 5.3 and maximum diameter > 9.1 μm and IQR > 2.2. Multivariable logistic regression confirmed these parameters as independent predictors of pILC, with the maximum diameter being the most significant (P < 0.001). Visual assessment recognised two pILC subtypes: apocrine and non‐apocrine. Apocrine variant showed nuclear roundness, pale vesicular chromatin patterns and prominent nucleoli, while non‐apocrine variant exhibited greater nuclear size and shape variation.Objective nuclear measurements, combined with cytoplasmic and architectural features, provide a robust framework for diagnosing LC subtypes, improving diagnostic accuracy and reproducibility. [ABSTRACT FROM AUTHOR]

Aims: Tumour genotype and phenotype are related and can predict outcome. In this study, we hypothesised that the visual assessment of breast cancer (BC) morphological features can provide valuable insight into underlying molecular profiles. Methods and results: The Cancer Genome Atlas (TCGA) BC cohort was used (n = 743) and morphological features, including Nottingham grade and its components and nucleolar prominence, were assessed utilising whole‐slide images (WSIs). Two independent scores were assigned, and discordant cases were utilised to represent cases with intermediate morphological features. Differentially expressed genes (DEGs) were identified for each feature, compared among concordant/discordant cases and tested for specific pathways. Concordant grading was observed in 467 of 743 (63%) of cases. Among concordant case groups, eight common DEGs (UGT8, DDC, RGR, RLBP1, SPRR1B, CXorf49B, PSAPL1 and SPRR2G) were associated with overall tumour grade and its components. These genes are related mainly to cellular proliferation, differentiation and metabolism. The number of DEGs in cases with discordant grading was larger than those identified in concordant cases. The largest number of DEGs was observed in discordant grade 1:3 cases (n = 1185). DEGs were identified for each discordant component. Some DEGs were uniquely associated with well‐defined specific morphological features, whereas expression/co‐expression of other genes was identified across multiple features and underlined intermediate morphological features. Conclusion: Morphological features are probably related to distinct underlying molecular profiles that drive both morphology and behaviour. This study provides further evidence to support the use of image‐based analysis of WSIs, including artificial intelligence algorithms, to predict tumour molecular profiles and outcome. [ABSTRACT FROM AUTHOR]

Aims: Aldehyde dehydrogenase family 1 member A1 (ALDH1A1) is reportedly a key ALDH isozyme linked to the cancer stem cells (CSC) of many solid tumours, where it is involved in self‐renewal, differentiation and self‐protection. In this study, the prognostic significance of ALDH1A1 expression in early invasive breast cancer (BC) and its role as a BC stem cell (BCSC) were evaluated. Methods and results: ALDH1A1 expression was assessed, using immunohistochemistry and tissue microarrays, in a large well‐characterised BC cohort. ALDH1A1 mRNA expression was also assessed at transcriptomic levels, utilising data from the Molecular Taxonomy of Breast Cancer International Consortium. The associations of ALDH1A1 with clinicopathological parameters, other stem cell markers and patient outcomes were determined. ALDH1A1 was expressed in 71% of BC cases at both the protein and mRNA levels. High ALDH1A1 expression was associated with poor prognostic features, including high grade, poor Nottingham Prognostic Index (NPI), lymph node metastasis and highly proliferative ER+ (luminal B) and triple‐negative (TNBC) subtypes. ALDH1A1 expression was positively correlated with the expression of CD44, CD24, TWIST, SOX9, EPCAM and CD133. The high immunoexpression of ALDH1A1 was significantly associated with poor BC‐specific survival (P < 0.001), and specifically in the luminal B and TNBC subtypes (P = 0.042 and P = 0.003, respectively). The immunoexpression of ALDH1A1 was an independent predictor of poor prognosis (P = 0.015). Conclusions: ALDH1A1, as assessed using immunohistochemistry, seems to act as a BCSC marker associated not only with other BCSC markers but also with poor prognostic characteristics and poor outcomes, particularly in the luminal B and TNBC subtypes. [ABSTRACT FROM AUTHOR]

Aims: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)‐positive AMEs have mutations in phosphoinositide 3‐kinase (PI3K) pathway genes, whereas ER‐negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs. Methods and results: Twenty‐six AMEs (14 ER‐positive; 12 ER‐negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R‐specific antibody, in addition to detailed histopathological analysis. Nine ER‐negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2). Conclusions: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER‐negative AMEs. [ABSTRACT FROM AUTHOR]

Aims: Nucleolar morphometric features have a potential role in the assessment of the aggressiveness of many cancers. However, the role of nucleoli in invasive breast cancer (BC) is still unclear. The aims of this study were to investigate the optimal method for scoring nucleoli in IBC and their prognostic significance, and to refine the grading of breast cancer (BC) by incorporating nucleolar score. Methods and results: Digital images acquired from haematoxylin and eosin‐stained sections from a large BC cohort were divided into training (n = 400) and validation (n = 1200) sets for use in this study. Four different assessment methods were evaluated in the training set to identify the optimal method associated with the best performance and significant prognostic value. These were: (i) a modified Helpap method; (ii) counting prominent nucleoli (size ≥2.5 µm) in 10 field views (FVs); (iii) counting prominent nucleoli in five FVs; and (iv) counting prominent nucleoli in one FV. The optimal method was applied to the validation set and to an external validation set, i.e. data from The Cancer Genome Atlas (n = 743). Scoring prominent nucleoli in five FVs showed the highest interobserver concordance rate (intraclass correlation coefficient of 0.8) and a significant association with BC‐specific survival (P < 0.0001). A high nucleolar score was associated with younger age, larger tumour size, and higher grade. Incorporation of nucleolar score in the Nottingham grading system resulted in a higher significant association with survival than the conventional grade. Conclusions: Quantification of nucleolar prominence in five FVs is a cost‐efficient and reproducible morphological feature that can predict BC behaviour and can provide an alternative to pleomorphism to improve BC grading performance. [ABSTRACT FROM AUTHOR]

Background: Management of breast ductal carcinoma in situ (DCIS) has various approaches with distinct institutional specific practice. Here, we review DCIS management in a single institution with emphasize on re‐operation rates and outcome. Methods: Breast ductal carcinoma in situ cases diagnosed at the Nottingham Breast Institute between 1987 and 2017 were identified (n = 1249). Clinicopathological data were collected. Cases were histologically reviewed, and different factors associated with primary operation selection, re‐excision, presence of residual tumor in the re‐excision specimens, use of radiotherapy and ipsilateral recurrences were analyzed. Results: 34% of DCIS patients were initially treated by mastectomy and were more frequently symptomatic, of high nuclear tumor grade, size >40 mm, and associated with comedo necrosis and Paget's disease of the nipple. Further surgery was due to involved or narrow surgical margins. Residual tumor tissue was detected in 53% of the re‐excision specimens. Re‐excision rates of patients treated with breast‐conserving surgery (BCS) were reduced from approximately 70% to 23%, and the final mastectomy rates decreased from 60% to 20%. Changes in surgical practice with acceptance of smaller excision margins and more frequent use of local radiotherapy have led to a significant decrease not only in the re‐excision rate but also in the final mastectomy rate together with non‐significant reduction in 5‐ and 10‐year local recurrence rates. Conclusion: Although BCS is increasingly the preferred primary surgical option for DCIS management, a proportion of low‐risk DCIS patients continue to undergo re‐excision surgery or completion mastectomy. Despite acceptance of smaller margins, recurrence rate is decreasing. [ABSTRACT FROM AUTHOR]

Cten is an oncogene promoting EMT in many signaling pathways, namely through Snail. We investigated whether Cten function could be mediated through Src. Cten levels were modulated by forced expression in HCT116 and gene knockdown in SW620 CRC (colorectal cancer) cell lines. In all cell lines, Cten was a positive regulator of Src expression. The functional importance of Src was tested by simultaneous Cten overexpression and Src knockdown. This resulted in abrogation of Cten motility‐inducing activity and reduction of colony formation ability together with failure to induce Cten targets. In SW620ΔCten reduced Src expression increased following restoration of Cten, also leading to increased cell motility and colony formation, which were lost if Src was concomitantly knocked down. By qRT‐PCR we showed modulation of Cten had no effect on Src mRNA. However, a CHX pulse chase assay demonstrated stabilization of Src protein by Cten. Finally, expression of Cten and Src was tested in a series of 84 primary CRCs and there was a significant correlation between them (P = 0.001). We conclude that Src is a novel and functionally important target of the Cten signaling pathway and that Cten protein causes post‐transcriptional stabilization of Src in promoting EMT and possibly metastasis in CRC. [ABSTRACT FROM AUTHOR]

Aims: Cathepsin A (CTSA) is a key regulatory enzyme for galactoside metabolism. Additionally, it has a distinct proteolytic activity and plays a role in tumour progression. CTSA is differentially expressed at the mRNA level between breast ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). In this study, we aimed to characterise CTSA protein expression in DCIS and evaluate its prognostic significance. Methods and results: A large cohort of DCIS [n = 776 for pure DCIS and n = 239 for DCIS associated with IBC (DCIS/IBC)] prepared as a tissue microarray was immunohistochemically stained for CTSA. High CTSA expression was observed in 48% of pure DCIS. High expression was associated with features of poor DCIS prognosis, including younger age at diagnosis (<50 years), higher nuclear grade, hormone receptor negativity, HER2 positivity, high proliferative index and high hypoxia inducible factor 1 alpha expression. High CTSA expression was associated with shorter recurrence‐free interval (RFI) (P = 0.0001). In multivariate survival analysis for patients treated with breast conserving surgery, CTSA was an independent predictor of shorter RFI (P = 0.015). DCIS associated with IBC showed higher CTSA expression than pure DCIS (P = 0.04). In the DCIS/IBC cohort, CTSA expression was higher in the invasive component than the DCIS component (P < 0.0001). Conclusion: CTSA is not only associated with aggressive behaviour and poor outcome in DCIS but also a potential marker to predict co‐existing invasion in DCIS. [ABSTRACT FROM AUTHOR]

Aims: Histological grade is an independent prognostic variable in breast cancer (BC). Previous concordance studies of BC grade have reported moderate levels of agreement; a typical finding in morphological assessment of biological variables. This study aimed to investigate the impact of discordance on the prognostic value of grade and to identify the best reporting approach in borderline cases. Methods and results: A large (n = 1675) well‐characterised annotated cohort of BC, originally graded in routine practice using glass slides, was re‐graded twice by an expert breast pathologist using virtual microscopy with a 3‐month washout period. Outcome was assessed using breast cancer‐specific and distant metastasis‐free survival (median follow‐up = 135 months). Fifty‐eight per cent of the cases showed absolute agreement in the three separate grading sessions, whereas grades 1/2 and 2/3 discordance were observed in 21% and 21%, respectively. Absolute intra‐observer agreement using virtual microscopy was observed in 77% of the cases, whereas 13% and 10% showed grades 1/2 and 2/3 discordance, respectively. Despite the concordance, outcome analysis revealed significant associations between tumour grade and patients' outcome in the three grading sessions. Grades 1/2 and 2/3 discordant cases showed intermediate survival between grades 1 and 2 tumours and grades 2 and 3 tumours, respectively. Grades 1/2 discordant cases showed a worse outcome when compared with grade 1 tumours (P = 0.008) but no statistical difference was identified when compared with grade 2 tumours. Similarly, grades 2/3 discordant cases showed a significant difference from grade 2 tumours (P < 0.001), but no statistical difference was identified when compared with grade 3 tumours. Conclusions: Breast cancer grade discordance is probably a reflection of biologically, and hence morphologically, borderline tumours. Cases with borderline features for grade are more likely to behave similarly to the higher‐grade category. Repeating histological grade of borderline cases or double reporting may improve correlation with outcome. [ABSTRACT FROM AUTHOR]

Despite the significant biological, behavioural and management differences between ductal carcinoma in situ (DCIS) and invasive carcinoma of the breast, they share many morphological and molecular similarities. Differentiation of these two different lesions in breast pathological diagnosis is based typically on the presence of an intact barrier between the malignant epithelial cells and stroma; namely, the myoepithelial cell (MEC) layer and surrounding basement membrane (BM). Despite being robust diagnostic criteria, the identification of MECs and BM to differentiate in‐situ from invasive carcinoma is not always straightforward. The MEC layer around DCIS may be interrupted and/or show an altered immunoprofile. MECs may be absent in some benign locally infiltrative lesions such as microglandular adenosis and infiltrating epitheliosis, and occasionally in non‐infiltrative conditions such as apocrine lesions, and in these contexts this does not denote malignancy or invasive disease with metastatic potential. MECs may also be absent around some malignant lesions such as some forms of papillary carcinoma, yet these behave in an indolent fashion akin to some DCIS. In Paget's disease, malignant mammary epithelial cells extend anteriorly from the ducts to infiltrate the epidermis of the nipple but do not typically infiltrate through the BM into the dermis. Conversely, BM‐like material can be seen around invasive carcinoma cells and around metastatic tumour cell deposits. Here, we review the role of MECs and BM in breast pathology and highlight potential clinical implications. We advise caution in interpretation of MEC features in breast pathology and mindfulness of the substantive evidence base in the literature associated with behaviour and clinical outcome of lesions classified as benign on conventional morphological examination before changing classification to an invasive lesion on the sole basis of MEC characteristics. [ABSTRACT FROM AUTHOR]

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

Positive PR expression (>=10%) identified 94% of luminal BC patients with a low recurrence score.[2] These results provide further evidence of the prognostic value of PR in luminal BC and support the routine immunohistochemical assessment of PR in clinical settings. We thank Dr Lehrer and Dr Rheinstein for their interest in our study[1] and for confirming our findings by using the publicly available breast cancer (BC) data set from The Cancer Genome Atlas. [Extracted from the article]