Total loss of MHC class I is an independent indicator of good prognosis in breast cancer

Tumours can be recognised by CTL and NK cells. CTL recognition depends on expression of MHC Class I loaded with peptides from tumour antigens. In contrast, loss of MHC Class I results in NK activation. In our study a large set of samples from patients with primary operable invasive breast cancer was evaluated for the expression of MHC Class I heavy and light by immunohistochemical staining of 439 breast carcinomas in a tissue microarray. Forty-seven percent (206 of 439) of breast carcinomas were considered negative for HLA Class I heavy chain (HC10), whereas lack of anti-β2m-antibody staining was observed in 39% (167 of 424) of tumours, with only 3% of the β2m-negative tumours expressing detectable HLA Class I heavy chain. Correlation with patient outcome showed direct relationship between patient survival and HLA-negative phenotype (log rank = 0.004). A positive relationship was found between the intensity of expression of MHC Class I light and heavy chains expression and histological grade of invasive tumour (p < 0.001) and Nottingham Prognostic Index (p < 0.001). To investigate whether HLA Class I heavy and light chains expression had independent prognostic significance, Cox multivariate regression analysis, including the parameters of tumour size, lymph node stage, grade and intensity of HC10 and anti-β2m staining, was carried out. In our analysis, lymph node stage (p < 0.001), tumour grade (p = 0.005) and intensity of MHC Class I light and heavy chains expression were shown to be independent prognostic factors predictive of overall survival (p-values HC10 = 0.047 and β2m = 0.018)

A comparison of three different methods for classification of breast cancer data

The classification of breast cancer patients is of great importance in cancer diagnosis. During the last few years, many algorithms have been proposed for this task. In this paper, we review different supervised machine learning techniques for classification of a novel dataset and perform a methodological comparison of these. We used the C4.5 tree classifier, a Multilayer Perceptron and a naïve Bayes classifier over a large set of tumour markers. We found good performance of the Multilayer Perceptron even when we reduced the number of features to be classified. We found naive Bayes achieved a competitive performance even though the assumption of normality of the data is strongly violated

Lymphatic expression of CLEVER-1 in breast cancer and its relationship with lymph node metastasis

BACKGROUND Mechanisms regulating breast cancer lymph node metastasis are unclear. Staining of CLEVER-1 (common lymphatic endothelial and vascular endothelial receptor-1) in human breast tumors was used, along with in vitro techniques, to assess involvement in the metastatic process. METHODS 148 sections of primary invasive breast cancers, with 10 yr follow-up, were stained with anti-CLEVER-1. Leukocyte infiltration was assessed, along with involvement of specific subpopulations by staining with CD83 (mature dendritic cells, mDC), CD209 (immature DC, iDC) and CD68 (macrophage, Mϕ). In vitro expression of CLEVER-1 on lymphatic (LEC) and blood endothelial cells (BEC) was examined by flow cytometry. RESULTS In vitro results showed that although both endothelial cell types express CLEVER-1, surface expression was only evident on LEC. In tumour sections CLEVER-1 was expressed in blood vessels (BV, 61.4% of samples), lymphatic vessels (LV, 18.2% of samples) and in Mϕ/DCs (82.4% of samples). However, only CLEVER-1 expression in LV was associated with LN metastasis (p = 0.027) and with Mϕ indices (p = 0.021). Although LV CLEVER-1 was associated with LN positivity there was no significant correlation with recurrence or overall survival, BV CLEVER-1 expression was, however, associated with increased risk of recurrence (p = 0.049). The density of inflammatory infiltrate correlated with CLEVER-1 expression in BV (p &lt; 0.001) and LV (p = 0.004). CONCLUSIONS The associations between CLEVER-1 expression on endothelial vessels and macrophage/leukocyte infiltration is suggestive of its regulation by inflammatory conditions in breast cancer, most likely by macrophage-associated cytokines. Its upregulation on LV, related surface expression, and association with LN metastasis suggest that it may be an important mediator of tumor cell metastasis to LN

High-resolution array CGH clarifies events occurring on 8p in carcinogenesis.

BACKGROUND: Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb - 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. METHODS: We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. RESULTS: We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb - 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb - 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb - 23.4 Mb) in two lines and one of 98 tumours. CONCLUSION: The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene

Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer.

INTRODUCTION: Programmed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK). METHODS: We have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan-Meier analyses. RESULTS: siRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself. CONCLUSIONS: The combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Chemokine (C-C motif) receptor 7 (CCR7) associates with the tumour immune microenvironment but not progression in invasive breast carcinoma

Some previous studies have reported that the chemokine (C-C motif) receptor 7 (CCR7) plays a role in breast cancer, is associated with lymph node metastasis and drives the site of distant metastasis. However, the impact of its expression on patient outcome and its association with tumour infiltrating inflammatory cells remain to be validated. We evaluated CCR7 protein expression by immunohistochemistry in a large well characterized cohort (n = 866) of early invasive primary breast cancers. CCR7 was expressed in the cytoplasm and membrane of tumour cells. We observed a weak positive association of high CCR7 expression when in either cellular component, but not both together, with axillary lymph node stage 3 tumours (p = 0.043). Logistic regression analysis of lymph node stage revealed no independent predictive value for CCR7 expression. CCR7 expression was higher in HER2 positive tumours (p = 0.03) and associated with positive CD68+ FOXP3+ tumour infiltrating cells. CCR7 staining was negatively associated with CD3+ cells. There was no significant association of CCR7 expression with breast cancer recurrence or survival. We conclude that while CCR7 is not a useful biomarker for predicting lymph node metastasis, it may reflect altered intra- and inter-cellular signalling related to the immune microenvironment. The subcellular localization of CCR7 appears to affect the nature of these interactions

Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers

Background Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. Methods Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. Results MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER? subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER? and ER- cell lines. Conclusions MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathwa

Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections.

BACKGROUND: Although the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested. METHOD: A large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study. RESULTS: There was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes. CONCLUSION: Ki67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used