A.R. Green

Caveolin 1 and Caveolin 2 are associated with breast cancer basal-like and triple-negative immunophenotype

Caveolin-1 (CAV1) and caveolin 2 (CAV2) are the principal structural proteins of caveolae, sphingolipid and cholesterol-rich invaginations of the plasma membrane involved in vesicular trafficking and signal transduction. Over the recent years there has been controversy about their role in breast cancer and their suitability as markers of basal-like phenotype. Caveolin-1 and CAV2 protein expression was assessed on a tissue microarray containing 880 unselected invasive breast cancer cases, by means of immunohistochemistry. Caveolin-1 and CAV2 expression was observed in 13.4 and 5.9% of all breast cancer, respectively. Their expression was strongly associated with high histological grade, lack of steroid hormone receptor positivity (ER and PR), and expression of basal markers (basal cytokeratins, P63, P-cadherin). Furthermore, there was a significant association between CAV1 and CAV2 expression and basal-like phenotype. On univariate analysis only CAV2 had a prognostic impact on breast cancer-specific survival; however, this was not independent from other traditional markers on multivariate analysis. Our results demonstrate that both CAV1 and CAV2 are associated with basal-like phenotype. Further studies are warranted to determine whether they play an oncogenic role in basal-like/triple-negative breast cancer development or are just surrogate markers for this subgroup.

Tumour-infiltrating macrophages and clinical outcome in breast cancer

Background Macrophages constitute a major component of the leucocytic infiltrate of tumours. Human studies show an association between tumour-associated macrophages and tumours with poor prognostic features. In breast cancer, the presence of macrophages has been correlated with increased angiogenesis and poor prognosis but little information is available about the independent prognostic role of macrophages infiltrating breast carcinomas. Aims and methods This study used immunohistochemistry and tissue microarrays to assess the density and localisation of CD68 macrophages infiltrating 1322 breast tumours and to identify any relationship with clinicopathological factors and patient outcome. Results Tumour-infiltrating macrophages were present in the majority of tumours with a predominantly diffuse pattern. The density of distant stromal macrophages (infiltrating stroma away from the carcinoma, median count 14 cells) was higher than intratumoural (median zero cells) and adjacent stromal macrophages (median three cells). Higher total macrophage number was associated with higher tumour grade (rs=0.39, p<0.001), ER and PgR negativity, HER-2 positivity and basal phenotype (p<0.001). In univariate survival analysis, higher numbers of CD68 macrophages were significantly associated with worse breast cancer-specific survival (p<0.001) and shorter disease-free interval (p=0.004). However in multivariate model analysis, the CD68 macrophage count was not an independent prognostic marker. Conclusions Macrophages are heterogeneous with different subsets having different functions. The present study suggests that overall macrophage numbers are not related to prognosis in breast cancer. However, further studies are needed to investigate the potential role of different subsets of macrophages.

MicroRNA involvement in the pathogenesis and management of breast cancer

MicroRNAs (miRNAs) are a highly abundant class of endogenous small non-coding RNAs (18–25 nucleotides in length) that regulate gene expression by targeting protein-coding mRNAs post-transcriptionally. miRNAs have been implicated in cancer development and progression. As miRNAs and their regulatory functions are further revealed, the more the importance of miRNA-directed gene regulation is emphasised. In the human genome, 695 mature miRNAs have been identified, although computational calculation predicts that this may increase to >1000. Deregulation of miRNA expression profiles is thought to be implicated in the pathogenesis of many human cancers including breast tumours. Breast cancer subtypes are observed to have deranged miRNA expression signatures, which makes miRNAs important targets for developing a novel molecular classification of breast cancer and opening avenues for more individualised treatment strategies for patients with breast cancer.

Biology of primary breast cancer in older women treated by surgery: with correlation with long-term clinical outcome and comparison with their younger counterparts

Background:As age advances breast cancer appears to change its biological characteristics, however, very limited data are available to define the precise differences between older and younger patients.Methods:Over 36 years (1973–2009), 1758 older (⩾70 years) women with early operable primary breast cancer were managed in a dedicated clinic. In all, 813 underwent primary surgery and 575 good quality tumour samples were available for biological analysis. The pattern of biomarkers was analysed using indirect immunohistochemistry on tissue microarrays. Comparison was made with a previously characterised series of younger (<70 years) patients.Results:There was high expression of oestrogen receptor (ER), PgR, Bcl2, Muc1, BRCA1 and 2, E-cadherin, luminal cytokeratins, HER3, HER4, MDM2 and 4 and low expression of human epidermal growth factor receptor (HER)-2, Ki67, p53, EGFR and CK17. Oestrogen receptor and axillary stage appeared as independent prognostic factors. Unsupervised partitional clustering showed six biological clusters in older patients, five of which were common in the younger patients, whereas the low ER luminal cluster was distinct in the older series. The luminal phenotype showed better breast cancer-specific survival, whereas basal and HER2-overexpressing tumours were associated with poor outcome.Conclusion:Early operable primary breast cancer in older women appears as a distinct biological entity, with existence of a novel cluster. Overall older women showed less aggressive tumour biology and ER appeared as an independent prognostic factor alongside the time-dependent axillary stage. These biological characteristics may explain the differences in clinical outcome and should be considered in making therapeutic decisions.

Lymph-node metastases in invasive lobular carcinoma are different from those in ductal carcinoma of the breast

Aim Invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) of the breast are distinct morphological entities with different biological features and clinical behaviour. In the present study, the authors compare the axillary-lymph-node (ALN) status of patients with grade-matched ILC (no=426) and IDC (no=820). The pattern of nodal metastatic deposits (nodular, sinusoidal and diffuse) and the proportion of involved nodes were also analysed in a selected group of 246 tumours, which were associated with a single positive ALN. Results Compared with grade-matched IDC, ILC was associated with a higher nodal stage (13.1% vs 4.5% of ILC and IDC were stage 3), higher absolute number of positive nodes and higher ratio of positive nodes (0.46±0.30 and 0.33±0.23 in ILC and IDC respectively). These differences were maintained in the different size subgroups. The most common metastatic morphological pattern was nodular in both types of carcinomas. A sinusoidal pattern was more frequent in IDC, and the diffuse pattern was more frequent in ILC. Despite these differences, ILC and grade-matched IDC exhibited similar rates of regional recurrences (RR) and breast-cancer survival. Conclusion This study provides clinical evidence which further demonstrates that ILC and IDC are biologically distinct entities with different lymph-node involvement patterns and ILC having a tendency to metastasise to more nodes than IDC. However, this difference was not associated with a significant impact on patient outcome.

Long-term clinical outcome of oestrogen receptor-positive operable primary breast cancer in older women: a large series from a single centre

Introduction:A Cochrane review of seven randomised trials (N=1571) comparing surgery and primary endocrine therapy (PET) (oestrogen receptor (ER) unselected) shows no difference in overall survival (OS). We report outcome of a large series with ER-positive (ER+) early invasive primary breast cancer.Methods:Between 1973 and 2009, 1065 older (⩾70 years) women (median age 78 years (70–99)) had either surgery (N=449) or PET (N=616) as initial treatment.Results:At 49-month median follow-up (longest 230 months), the 5-year breast cancer-specific survival (BCSS) and OS were 90 and 62%, respectively. Majority (74.2%) died from causes other than breast cancer. The rates (per annum) of local/regional recurrence (<1%) (following surgery), contralateral tumour (<1%) and metastases (<3%) were low. For patients on PET, 97.9% achieved clinical benefit (CB) at 6 months, with median time to progression of 49 months (longest 132 months) and significantly longer BCSS when compared with those who progressed (P<0.001). All patients with strongly ER+ (H-score >250) tumours achieved CB and had better BCSS (P<0.01). Patients with tumours having an H-score >250 were found to have equivalent BCSS regardless of treatment (surgery or PET; P=0.175), whereas for those with H-score ⩽250, surgery produced better outcome (P<0.001).Conclusion:Older women with ER+ breast cancer appear to have excellent long-term outcome regardless of initial treatment. Majority also die from non-breast cancer causes. Although surgery remains the treatment of choice, patients with ER-rich (H-score >250) tumours tend to do equally well when treated by PET. This should be taken into account when therapies are considered.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

Background:It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC.Methods:A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry.Results:KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables.Conclusions:This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Three dimensional reconstruction of a human breast carcinoma using routine laboratory equipment and immunohistochemistry

Aims: To establish a three dimensional reconstruction of an invasive breast carcinoma using basic laboratory equipment to evaluate and characterise the spatial arrangement of the parenchymal cells of the breast. Methods: One hundred and twenty eight sequential 4 μm sections (20 μm apart) of the tumour were stained immunohistochemically with an epithelial specific marker (AE1/AE3) or tumour specific marker (c-erbB-2) to reconstruct two different three dimensional images of the normal and malignant parenchymal cells. Sections were digitally imaged using a microscope, scanner, and digital camera linked to a conventional personal computer. Accurate alignment of the digitalised images was carried out using a semiautomatic graphical method of manual interaction, using the cross correlation coefficient as a goodness of fit measure, and an automatic search algorithm using the Fibonacci search algorithm for automatic alignment. The volume was reconstructed using maximum, minimum point projection and “back to front” opacity blending. Results: The quality of the reconstructed images was distinct and perfect, providing a comprehensive and explicit view of the normal and malignant parenchymal tissues of the breast that is not possible by viewing two dimensional histological sections. Specifically, this approach showed the spatial arrangement of the tumour cells and their relation to the surrounding tissues at a high resolution. Conclusion: This simple and reproducible approach enables the spread and infiltration of invasive carcinoma to be understood and could also be used to analyse the spatial relation between atypical hyperplastic and malignant in situ lesions of the breast.

Markers of Progression in Early-Stage Invasive Breast Cancer: a Predictive Immunohistochemical Panel Algorithm for Distant Recurrence Risk Stratification

Accurate distant metastasis (DM) prediction is critical for risk stratification and effective treatment decisions in breast cancer (BC). Many prognostic markers/models based on tissue marker studies are continually emerging using conventional statistical approaches analysing complex/dimensional data association with DM/poor prognosis. However, few of them have fulfilled satisfactory evidences for clinical application. This study aimed at building DM risk assessment algorithm for BC patients. A well-characterised series of early invasive primary operable BC (nxa0=xa01902), with immunohistochemical expression of a panel of biomarkers (nxa0=xa031) formed the material of this study. Decision tree algorithm was computed using WEKA software, utilising quantitative biomarkers’ expression and the absence/presence of distant metastases. Fifteen biomarkers were significantly associated with DM, with six temporal subgroups characterised based on time to development of DM ranging from <1 to >15xa0years of follow-up. Of these 15 biomarkers, 10 had a significant expression pattern where Ki67LI, HER2, p53, N-cadherin, P-cadherin, PIK3CA and TOMM34 showed significantly higher expressions with earlier development of DM. In contrast, higher expressions of ER, PR and BCL2 were associated with delayed occurrence of DM. DM prediction algorithm was built utilising cases informative for the 15 significant markers. Four risk groups of patients were characterised. Three markers p53, HER2 and BCL2 predicted the probability of DM, based on software-generated cut-offs, with a precision rate of 81.1xa0% for positive predictive value and 77.3xa0%, for the negative predictive value. This algorithm reiterates the reported prognostic values of these three markers and underscores their central biological role in BC progression. Further independent validation of this pruned panel of biomarkers is therefore warranted.

Three-dimensional reconstruction of sentinel lymph nodes with metastatic breast cancer indicates three distinct patterns of tumour growth

Aim: A better understanding of the biology of nodal metastatic disease is of indisputable value. Three-dimensional (3D) serial section alignment and reconstruction techniques can be used for visualisation of nodal metastasis and could provide better understanding of disease growth patterns. Methods: 19 tumour-involved sentinel nodes (SLNs) from breast cancer patients were serially sectioned, immunohistochemically stained, and digitally scanned. Digital image alignment and voxel-based rendering was used to construct informative 3D visual representations of metastatic tumour distribution within involved nodes. Results: The 3D reconstruction technique was successful and informative. The reconstructions of all 19 SLNs enabled the metastatic tumour cells to be viewed infiltrating normal SLN tissue from all angles. Metastases were present at the afferent lymphatic pole in 17/19 cases, confined to the afferent pole only in 7 cases, located at the efferent pole in 12/19 cases, and efferent pole only in just 2 cases. Finally, this study made the novel observation that metastatic growth occurs in three distinct patterns: sinusoidal, nodular and diffuse. Conclusions: This methodology provides improved understanding of metastatic disease development and potentially could be used to develop strategies to improve techniques for its routine detection. Further studies are required in order to evaluate the prognostic and biological significance of the growth patterns identified.