Phenotypic and molecular characterisation of breast ductal carcinoma in situ

Background Ductal carcinoma in situ (DCIS) comprises more than 20% of screen-detected breast cancer cases. The current understanding of the biology and clinical behaviour of DCIS and prediction of its progression to invasive disease remains incomplete. Treatment modalities vary; however, in most cases it involves surgical excision with or without adjuvant radiotherapy and endocrine therapy. To date, predictors of DCIS progression are not well defined; therefore, the aim of the current study was to characterise a large cohort of DCIS with long term follow-up data and identify possible predictors of DCIS outcome. Patients and Methods In this study, 1,249 pure DCIS cases and a control group of 239 DCIS cases mixed with invasion were identified between 1987–2017. Collection of the clinicopathological, management and outcome data was performed. Cases were reviewed and representative tumour blocks were selected and retrieved. Tissue microarrays (TMAs) were constructed (776 pure DCIS and 239 DCIS mixed with invasion), followed by molecular characterisation of DCIS for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and proliferation marker (Ki67), using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH). Mining for biomarkers with potential prognostic and predictive values in DCIS was performed with the identification of thioredoxin interacting protein (TXNIP), aurora kinase A (AURKA), a targeting protein for Xenopus kinesin-like protein 2 (TPX2), and a non-receptor tyrosine kinase (SRC) and their expression was assessed in the TMA using IHC. In addition, whole tissue sections of DCIS were used to assess tumour infiltrating lymphocytes (TILs) as a component of the tumours’ microenvironment. Results Analysis of the clinicopathological data of the DCIS patients revealed a significant increase in incidence of screen-detected DCIS during the period of the study. This was associated with a significant increase in the rate of breast conserving surgery (BCS) and the use of adjuvant radiotherapy (RT), along with a marked decline in the rate of re-excisions and recurrence. Screen-detected DCIS, localised DCIS lesions, small tumour size and negative surgical margins were the contributing factors to the reduced rate of re-excisions. Large tumour size, high tumour grade, and no adjuvant radiotherapy were independent predictors of the development of ipsilateral local recurrence. Molecular characterisation of DCIS using ER, PR, HER2 and Ki67 revealed that the highest proportion (60%) of cases were of Luminal A subtype, however the lowest attribution (11%) was for HER2 positive (+) tumours. Tumours of HER2+, Luminal B/HER2+ and Triple Negative subtypes were of larger tumour size (>40mm), higher tumour grade, and more frequently treated with mastectomy. When the molecular classes were investigated against the development of local recurrence, HER2 positivity was associated with ipsilateral DCIS local recurrence. The assessment of the selected tissue marker panel with potential role in predicting DCIS progression revealed a significant difference in their protein expression between pure DCIS tumours and those associated with invasion. An association between low expression of TXNIP, and high expression of AURKA, TPX2 and SRC with the development of local recurrence was detected and that AURKA, TPX2 and SRC were predictors of invasive recurrence independent of other predictors of recurrence, including tumour size, grade, and radiotherapy. Analysis of tumour infiltrating lymphocytes (TILs) showed that the most reproducible and prognostically significant method of TILs evaluation in DCIS is the assessment of touching TILs (defined as lymphocytes touching or within one lymphocyte cell thickness from the DCIS basement membrane), and that dense TILs predicted the development of local recurrence. Conclusions The outcome of DCIS improved significantly during the period of the study. This study identified several predictors of DCIS progression. In addition to some clinical and pathological variables including tumour size, grade and radiotherapy use, expression of TXNIP, AURKA, TPX2 and SRC, together with TILs density were independent predictors of DCIS outcome. These variables can potentially be used in combination to refine the prognostic stratification of DCIS patients in routine practice

Diagnostic concordance and discordance in digital pathology : a systematic review and meta-analysis

Background – Digital pathology (DP) has the potential to fundamentally change the way that histopathology is practiced, by streamlining the workflow, increasing efficiency, improving diagnostic accuracy and facilitating the platform for implementation of artificial intelligence-based computerassisted diagnostics. Although the barriers to wider adoption of digital pathology have been multifactorial, limited evidence of reliability has been a significant contributor. A meta-analysis to demonstrate the combined accuracy and reliability of DP is still lacking in the literature. Objectives – We aimed to review the published literature on the diagnostic use of DP and to synthesise a statistically pooled evidence on safety and reliability of DP for routine diagnosis (primary and secondary) in the context of validation process. Methods – A comprehensive literature search was conducted through PubMed, Medline, EMBASE, Cochrane Library and Google Scholar for studies published between 2013 and August 2019. The search protocol identified all studies comparing DP with light microscopy (LM) reporting for diagnostic purposes, predominantly including H&E stained slides. Random-effects meta-analysis was used to pool evidence from the studies. Results – Twenty five studies were deemed eligible to be included in the review which examined a total of 10,410 histology samples (average sample size 176). For overall concordance (clinical concordance) the agreement percentage was 98.3% (95% Confidence interval: 97.4 – 98.9) across 24 studies. A total of 546 major discordances were reported across 25 studies. Over half (57%) of these were related to assessment of nuclear atypia, grading of dysplasia and malignancy. These were followed by challenging diagnoses (26%) and identification of small objects (16%). Conclusion - The results of this meta-analysis indicate equivalent performance of DP in comparison to LM for routine diagnosis. Furthermore, the results provide valuable information concerning the areas of diagnostic discrepancy which may warrant particular attention in the transition to DP

Myxovirus resistance 1 (MX1) is an independent predictor of poor outcome in invasive breast cancer

Background: Breast cancer (BC) is a disease with variable morphology, clinical behaviour and response to therapy. Identifying factors associated with the progression of early stage BC can help understand the risk of metastasis and guide treatment decisions. Myxovirus resistance 1 (MX1), which is involved in the cellular antiviral mechanism, plays a role in some solid tumours; however, its role in invasive BC remains unknown. In this study, we aimed to explore the clinicopathological and prognostic significance of MX1 in BC.Methods: MX1 was assessed at the protein level using tissue microarrays from a large well-annotated BC cohort (n=845). The expression of MX1 mRNA was assessed at the transcriptomic level using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1980) and validated using three publicly available cohorts on Breast Cancer Gene-Expression Miner (bc-GenExMiner version 4.4). The associations between MX1 expression and clinicopathological factors, and outcome were evaluated.Results: High MX1 protein expression was associated with features of aggressiveness, including large tumour size, high tumour grade, high Nottingham prognostic index scores, hormone receptor negativity and high Ki67 expression. High MX1 expression showed an association with poor patient outcome and it was an independent predictor of short BC- specific survival (p=0.028; HR=1.5; 95%CI=1.0–2.2). Consistent with the protein results, high MX1 mRNA levels showed an association with features of aggressive behaviour and with shorter survival.Conclusion: This study identified MX1 as an independent predictor of poor outcome in patients with BC. Further functional studies are needed to investigate the biological role of MX1 in BC and its potential value as a therapeutic target

Surgical management of ductal carcinoma in situ of the breast: A large retrospective study from a single institution

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 emphasise on re-operation rates and outcome. Methods: DCIS cases diagnosed at the Nottingham Breast Institute between 1987 and 2017 were identified (n=1,249). Clinicopathological data was collected. Cases were histologically reviewed, and different factors associated with primary operation selection, re-excision, presence of residual tumour in the re-excision specimens, use of radiotherapy and ipsilateral recurrences were analysed. Results: 34% of DCIS patients were initially treated by mastectomy and were more frequently symptomatic, of high nuclear tumour grade, size >40mm, and associated with comedo necrosis and Paget’s disease of the nipple. Further surgery was due to involved or narrow surgical margins. Residual tumour 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

The clinical and biological significance of HER2 over-expression in breast ductal carcinoma in situ: a large study from a single institution

© 2019, Cancer Research UK. Background: Previous studies have reported up to 50% of ductal carcinoma in situ (DCIS), is HER2 positive, but the frequency of HER2-positive invasive breast cancer (IBC) is lower. The aim of this study is to characterise HER2 status in DCIS and assess its prognostic value. Methods: HER2 status was evaluated in a large series of DCIS (n = 868), including pure DCIS and DCIS associated with IBC, prepared as tissue microarrays (TMAs). HER2 status was assessed using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH). Results: In pure DCIS, HER2 protein was over-expressed in 9% of DCIS (3+), whereas 15% were HER2 equivocal (2+). Using CISH, the final HER2 status was positive in 20%. In mixed DCIS, HER2 amplification of the DCIS component was detected in 15% with amplification in the invasive component of only 12%. HER2-positive DCIS was associated with features of aggressiveness (p < 0.0001) and more frequent local recurrence (p = 0.03). On multivariate analysis, combined HER2+/Ki67+ profile was an independent predictor of local recurrence (p = 0.006). Conclusions: The frequency of HER2 positivity in DCIS is comparable to IBC- and HER2-positive DCIS is associated with features of poor prognosis. The majority of HER2 over-expression in DCIS is driven by gene amplification

Legumain is an independent predictor for invasive recurrence in breast ductal carcinoma in situ

© 2018, United States & Canadian Academy of Pathology. Legumain is a proteolytic enzyme that plays a role in the regulation of cell proliferation in invasive breast cancer. Studies evaluating its role in ductal carcinoma in situ (DCIS) are lacking. Here, we aimed to characterize legumain protein expression in DCIS and evaluate its prognostic significance. Legumain was assessed immunohistochemically in a tissue microarray of a well-characterized cohort of DCIS (n = 776 pure DCIS and n = 239 DCIS associated with invasive breast cancer (DCIS-mixed)). Legumain immunoreactivity was scored in tumor cells and surrounding stroma and related to clinicopathological parameters and patient outcome. High legumain expression was observed in 23% of pure DCIS and was associated with features of high-risk DCIS including higher nuclear grade, comedo necrosis, hormone receptor negativity, HER2 positivity, and higher proliferation index. Legumain 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 legumain expression than the DCIS component (p < 0.0001). Legumain was an independent predictor of shorter local recurrencefree interval for all recurrences (p = 0.0003) and for invasive recurrences (p = 0.002). When incorporated with other risk factors, legumain provided better patient risk stratification. High legumain expression is associated with poor prognosis in DCIS and could be a potential marker to predict DCIS progression to invasive disease

The prognostic significance of lysosomal protective protein (Cathepsin A) in breast ductal carcinoma in situ

Background: 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: A large cohort of DCIS (n=776 for pure DCIS and n=239 for DCIS associated with IBC (DCIS/IBC)) prepared as tissue microarray was immunohistochemically stained for CTSA. Results: 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 (less than 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 DCIS component (p less than 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

The prognostic significance of immune microenvironment in breast ductal carcinoma in situ

BackgroundThe role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B and T lymphocytes and immune checkpoint proteins expression in DCIS.MethodsA well characterised DCIS cohort (n = 700) with long-term follow-up comprising pure DCIS (n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases (n = 58).ResultsCD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma (Z = 5.8, p

Aurora Kinase A Is an Independent Predictor of Invasive Recurrence in Breast Ductal Carcinoma in situ

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

ATM Regulated PTEN Degradation Is XIAP E3 Ubiquitin Ligase Mediated in p85α Deficient Cancer Cells and Influence Platinum Sensitivity

Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85α are key tumour suppressors. Whether ATM regulates PTEN expression and influence platinum sensitivity is unknown. We generated ATM knockdowns (KD) and CRISPR knock outs (KO) in glioblastoma (LN18, LN229) and ovarian cancer cells (OVCAR3, OVCAR4). Doxycycline inducible PTEN expression was generated in LN18 and LN229 cells. Transient KD of p85α, CK2, and XIAP was accomplished using siRNAs. Stable p85α knock-in was isolated in LN18 cells. Molecular biology assays included proteasome activity assays, PCR, flow cytometry analysis (cell cycle, double strand break accumulation, apoptosis), immunofluorescence, co-immunoprecipitation, clonogenic, invasion, migration, and 3D neurosphere assays. The clinicopathological significance of ATM, PTEN, p85α, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85α dependant. ATM also controls CK2α level which in turn phosphorylates and stabilizes PTEN. In addition, p85α physically interacts with CK2α and protects CK2α from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2α thereby directing them to degradation. ATM depletion in the context of p85α deficiency impaired cancer cell migration and invasion reduced 3D-neurosphere formation and increased toxicity to cisplatin chemotherapy. Increased sensitivity to platinum was associated with DNA double strand breaks accumulation, cell cycle arrest, and induction of autophagy. In ovarian cancer patients, ATM, PTEN, p85α, and XIAP protein levels predicted better progression free survival after platinum therapy. We unravel a previously unknown function of ATM in the regulation of PTEN throμgh XIAP mediated proteasome degradation