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Background: Monoclonal antibodies against PD-1 or PD-L1 have been established in clinical practice for the treatment of both early and advanced/metastatic triple-negative breast cancer. Beyond the established immune checkpoints (ICPs) (PD-1 and CTLA-4), additional ICPs, such as lymphocyte activation gene-3 (LAG-3), are subject of current research. In the present retrospective gene-expression analysis, we evaluated the prognostic significance of LAG-3 in 461 patients with early breast cancer. In addition, we examined whether there was a correlation between the different ICP and CD8 expressions. Methods: Using microarray-based gene-expression analysis, we examined the prognostic significance of LAG-3 mRNA expression for metastasis-free survival (MFS) in the whole cohort of 461 breast cancer patients and among different molecular subtypes. Correlations were analyzed using Spearman's rho correlation coefficient. Results: In the whole cohort, LAG-3 expression had no significant impact on MFS (p = 0.712, log-rank). In the subgroup analyses, there was a trend that a higher LAG-3 expression was associated with a favorable outcome in the luminal B (p = 0.217), basal-like (p = 0.370) and HER2 (p = 0.089) subtypes, although significance was not reached. In contrast, in a multivariate Cox regression analysis, adjusted for age, tumor size, axillary nodal status, histological grade of differentiation and proliferation marker Ki-67, LAG-3 showed a significant influence on MFS (HR 0.574; 95% CI 0.369–0.894; p = 0.014). High LAG-3 significantly correlated with CD8 (ρ = 0.571; p < 0.001). Conclusions: LAG-3 expression had an independent impact on MFS. In addition to PD-1 and PD-L1, further immune checkpoints, such as LAG-3, could serve as therapeutic targets in breast cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Inhibitors targeting the cell cycle-regulated aurora kinase A (AURKA) are currently being developed. Here, we examine the prognostic impact of AURKA in node-negative breast cancer patients without adjuvant systemic therapy (n = 766). Methods AURKA was analyzed using microarray-based gene-expression data from three independent cohorts of node-negative breast cancer patients. In multivariate Cox analyses, the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER), and HER2 were considered. Results Patients with higher AURKA expression had a shorter metastasis-free survival (MFS) in the Mainz (HR 1.93; 95% CI 1.34 – 2.78; P < 0.001), Rotterdam (HR 1.95; 95% CI 1.45– 2.63; P Conclusions AURKA is associated with worse prognosis in estrogen receptor positive breast carcinomas. Patients with the highest AURKA expression (>75% percentile) have a particularly bad prognosis and may profit from therapy with AURKA inhibitors.

Background: In breast cancer, gene signatures that predict the risk of metastasis after surgical tumor resection are mainly indicative of early events. The purpose of this study was to identify genes linked to metastatic recurrence more than three years after surgery. Methods: Affymetrix HG U133A and Plus 2.0 array datasets with information on metastasis-free, disease-free or overall survival were accessed via public repositories. Time restricted Cox regression models were used to identify genes associated with metastasis during or after the first three years post-surgery (early- and late-type genes). A sequential validation study design, with two non-adjuvantly treated discovery cohorts (n = 409) and one validation cohort (n = 169) was applied and identified genes were further evaluated in tamoxifen-treated breast cancer patients (n = 923), as well as in patients with non-small cell lung (n = 1779), colon (n = 893) and ovarian (n = 922) cancer. Results: Ten late- and 243 early-type genes were identified in adjuvantly untreated breast cancer. Adjustment to clinicopathological factors and an established proliferation-related signature markedly reduced the number of early-type genes to 16, whereas nine late-type genes still remained significant. These nine genes were associated with metastasis-free survival (MFS) also in a non-time restricted model, but not in the early period alone, stressing that their prognostic impact was primarily based on MFS more than three years after surgery. Four of the ten late-type genes, the ribosome-related factors EIF4B, RPL5, RPL3, and the tumor angiogenesis modifier EPN3 were significantly associated with MFS in the late period also in a meta-analysis of tamoxifen-treated breast cancer cohorts. In contrast, only one late-type gene (EPN3) showed consistent survival associations in more than one cohort in the other cancer types, being associated with worse outcome in two non-small cell lung cancer cohorts. No late-type gene was validated in ovarian and colon cancer. Conclusions: Ribosome-related genes were associated with decreased risk of late metastasis in both adjuvantly untreated and tamoxifen-treated breast cancer patients. In contrast, high expression of epsin (EPN3) was associated with increased risk of late metastasis. This is of clinical relevance considering the well-understood role of epsins in tumor angiogenesis and the ongoing development of epsin antagonizing therapies. [ABSTRACT FROM AUTHOR]

Background Cyclooxygenases (COX) play a key role in prostaglandin metabolism and are important for tumor development and progression. The aim of this study was to analyze the prognostic impact of COX-2 expression in a cohort of lymph node-negative breast cancer patients not treated in the adjuvant setting. Methods COX-2 expression was determined by immunohistochemistry (IHC) in tumor tissue of 193 node-negative breast cancer patients. Additionally, mRNA expression was determined in corresponding tumor samples using microarray based gene-expression data. Univariate and multivariate Cox regression analyses adjusted for age at diagnosis, tumor size, histological grade, human epithelial growth factor receptor 2 (HER2), estrogen receptor (ER) and progesterone receptor (PR) were performed to evaluate the association of both COX-2 protein and mRNA expression with survival. Survival rates were determined by the Kaplan-Meier method. Correlations between COX-2 expression and established prognostic factors were analyzed using the Chi-square test. A potential correlation between COX-2 protein expression and COX-2 mRNA expression was assessed utilizing the Kruscal-Wallis-H-test. Results COX-2 protein expression was positive in 24.9% of the breast cancer samples. Univariate analysis showed that COX-2 protein expression was associated with shorter disease-free survival (DFS) (P = 0.0001), metastasis-free survival (MFS) (P = 0.002) as well as breast cancer specific overall survival (OS) (P = 0.043). In multivariate analysis COX-2 expression retained its significance independent of established prognostic factors for shorter DFS (P < 0.001, HR = 2.767, 95% CI = 1.563-4.901) and for inferior MFS (P = 0.002, HR = 2.7, 95% CI = 1.469-5.263) but not for OS (P = 0.096, HR = 1.929, 95% CI = 0.889-4.187). In contrast, COX-2 mRNA expression was not related to survival and failed to show a correlation with protein expression (P = 0.410). Conclusions The present findings support the hypothesis that COX-2 protein but not mRNA expression is associated with an unfavorable outcome in node-negative breast cancer. [ABSTRACT FROM AUTHOR]

The importance of inflammation is increasingly noticed in cancer. The aim of this study was to analyze the prognostic influence of pre-operative serum C-reactive protein (CRP) in a cohort of 148 lymph node-negative breast cancer patients. The prognostic significance of CRP level for disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS) was evaluated using univariate and multivariate Cox regression, also including information on age at diagnosis, tumor size, tumor grade, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, proliferation index (Ki67) and molecular subtype, as well as an assessment of the presence of necrosis and inflammation in the tumor tissue. Univariate analysis showed that CRP, as a continuous variable, was significantly associated with DFS (P = 0.002, hazard ratio [HR]  = 1.04, 95% confidence interval [CI]  = 1.02–1.07) and OS (P = 0.036, HR  = 1.03, 95% CI  = 1.00–1.06), whereas a trend was observed for MFS (P = 0.111). In the multivariate analysis, CRP retained its significance for DFS (P = 0.033, HR  = 1.01, 95% CI  = 1.00–1.07) as well as OS (P = 0.023, HR  = 1.03, 95% CI  = 1.00–1.06), independent of established prognostic factors. Furthermore, large-scale gene expression analysis by Affymetrix HG-U133A arrays was performed for 72 (48.6%) patients. The correlations between serum CRP and gene expression levels in the corresponding carcinoma of the breast were assessed using Spearman's rank correlation, controlled for false-discovery rate. No significant correlation was observed between CRP level and gene expression indicative of an ongoing local inflammatory process. In summary, pre-operatively elevated CRP levels at the time of diagnosis were associated with shorter DFS and OS independent of established prognostic factors in node-negative breast cancer, supporting a possible link between inflammation and prognosis in breast cancer. [ABSTRACT FROM AUTHOR]

In order to avoid over- as well as undertreatment, it is advisable to select the appropriate treatment strategy on the basis of a careful risk assessment for each individual patient. At present, we guide our therapeutic decisions using clinicopathological risk classifications such as the St Gallen risk category or Adjuvant!. The St Gallen risk classification, in particular, has a high sensitivity but a low specificity, potentially leading to overtreatment of a considerable number of patients. To spare patients unnecessary toxicities, we need additional prognostic factors, and several tumor markers such as urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor 1 (PAI-1) or multiparameter gene-expression analyses have shown promising results, especially in node-negative breast cancer. This should allow us to quantify the risk of progression in each individual patient and tailor treatment accordingly, leading to a more personalized treatment recommendation. [ABSTRACT FROM AUTHOR]

This article focuses on the recent findings by Marsh and colleagues, and also discusses recent findings with regards to breast cancer. Taxanes are amongst the most active agents in the treatment of breast cancer. However, many tumors are intrinsically resistant. Therefore, it would be an enormous progress, if factors could be identified that reliably differentiate between taxane-sensitive and -resistant patients. Marsh and colleagues analyzed the CYP1B1*3 (Val432Leu) polymorphism in patients with high-risk stage III and IV breast cancer, who received dose-intense paclitaxel in combination with doxorubicin and cyclophosphamide. They report for the first time that patients with two leucine alleles in codon 432 of CYP1B1 experience a longer progression-free survival compared with patients with the Val/Leu or Val/Val genotypes. If confirmed in independent cohorts CYP1B1*3 may prove to be an important factor that helps to differentiate between paclitaxel-sensitive and resistant breast cancer patients. However, the mechanism behind the association between CYP1B1*3 and prognosis of paclitaxel-treated patients remains unclear. Several studies provide strong evidence that CYP1B1 does not influence tumor progression independently from paclitaxel chemotherapy, and that CYP1B1 itself does not alter paclitaxel resistance. In addition, CYP1B1 mRNA expression does not correlate with paclitaxel sensitivity of primary tumor cells. Although still speculative, a possible explanation is an association between CYP1B1*3 with still unknown factors that, on their part, influence paclitaxel sensitivity. In the future, studies with SNP chips and studies on the transcriptome, proteome and metabolome level should be performed in order to identify signatures differentiating between paclitaxel-sensitive and -resistant patients. [ABSTRACT FROM AUTHOR]

Interferons are crucial for adaptive immunity and play an important role in the immune landscape of breast cancer. Using microarray-based gene expression analysis, we examined the subtype-specific prognostic significance of interferon-γ (IFN-γ) as a single gene as well as an IFN-γ signature covering the signaling pathway in 461 breast cancer patients. Prognostic significance of IFN-γ, as well as the IFN-γ signature for metastasis-free survival (MFS), were examined using Kaplan–Meier as well as univariate and multivariate Cox regression analyses in the whole cohort and in different molecular subtypes. The independent prognostic significance of IFN-γ as a single gene was limited to basal-like breast cancer (hazard ratio (HR) 2.779, 95% confidence interval (95% CI) 1.117–6.919, p = 0.028). In contrast, the IFN-γ-associated gene signature was an independent prognostic factor in the whole cohort (HR 2.287, 95% CI 1.410–3.633, p < 0.001) as well as in the basal-like (HR 3.458, 95% CI 1.154–10.359, p = 0.027) and luminal B (HR 2.690, 95% CI 1.416–5.112, p = 0.003) molecular subtypes. These results underline the subtype-dependent prognostic influence of the immune system in early breast cancer. [ABSTRACT FROM AUTHOR]

High resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly used for profiling of breast cancer tissue, delivering quantitative information for approximately 40 metabolites. One unique advantage of the method is that it can be used to analyse intact tissue, thereby requiring only minimal sample preparation. Importantly, since the method is non-destructive, it allows further investigations of the same specimen using for instance transcriptomics. Here, we discuss technical aspects critical for a successful analysis—including sample handling, measurement conditions, pulse sequences for one- and two dimensional analysis, and quantification methods—and summarize available studies, with a focus on significant associations of metabolite levels with clinically relevant parameters. [ABSTRACT FROM AUTHOR]

Abstract The interplay of ErbB receptor homo- and heterodimers plays a crucial role in the pathology of breast cancer since activated signal transduction cascades coordinate proliferation, survival and migration of cells. EGF and β-Heregulin are well characterised ligands known to induce ErbB homo- and heterodimerisation, which have been associated with disease progression. In the present study, we investigated the impact of both factors on the migration of MDA-NEO and MDA-HER2 human breast cancer cells. MDA-NEO cells are positive for EGFR and HER3, while MDA-HER2 cells express EGFR, HER2 and HER3. Cell migration analysis revealed that β-Heregulin potently impaired EGF induced migration in both cell lines. Western blot studies showed that both ErbB receptor and PLC-γ1 tyrosine phosphorylation levels were diminished in EGF and β-Heregulin co-treated MDA-NEO and MDA-HER2 cells, which was further correlated to a significantly impaired calcium influx. Our data indicate that EGF and HRG may interfere with each other for receptor binding and dimerisation, which ultimately has an impact on signalling outcome. Highlights • β-Heregulin inhibits the EGF induced migration of human breast cancer cells. • EGF induced PLC-γ1 activation concomitant with calcium influx is impaired by β-Heregulin. • EGF induced tyrosine phosphorylation of EGFR Y992 and HER2 Y1248 is impaired by β-Heregulin. • β-Heregulin induced tyrosine phosphorylation of HER3 Y1289 is impaired by EGF. • β-Heregulin signalling interferes with EGF signalling. [ABSTRACT FROM AUTHOR]

Abstract: Alterations in lipid metabolism and in the lipid composition of cellular membranes are linked to the pathology of numerous diseases including cancer. However, the influence of oncogene expression on cellular lipid profile is currently unknown. In this work we analyzed changes in lipid profiles that are induced in the course of ERBB2-expression mediated premature senescence. As a model system we used MCF-7 breast cancer cells with doxycycline-inducible expression of NeuT, an oncogenic ERBB2 variant. Affymetrix gene array data showed NeuT-induced alterations in the transcription of many enzymes involved in lipid metabolism, several of which (ACSL3, CHPT1, PLD1, LIPG, MGLL, LDL and NPC1) could be confirmed by quantitative realtime PCR. A study of the glycerophospholipid and lyso-glycerophospholipid profiles, obtained by high performance liquid chromatography coupled to Fourier-transform ion cyclotron resonance-mass spectrometry revealed senescence-associated changes in numerous lipid species, including mitochondrial lipids. The most prominent changes were found in PG(34:1), PG(36:1) (increased) and LPE(18:1), PG(40:7) and PI(36:1) (decreased). Statistical analysis revealed a general trend towards shortened phospholipid acyl chains in senescence and a significant trend to more saturated acyl chains in the class of phosphatidylglycerol. Additionally, the cellular cholesterol content was elevated and accumulated in vacuoles in senescent cells. These changes were accompanied by increased membrane fluidity. In mitochondria, loss of membrane potential along with altered intracellular distribution was observed. In conclusion, we present a comprehensive overview of altered cholesterol and glycerophospholipid patterns in senescence, showing that predominantly mitochondrial lipids are affected and lipid species less susceptible to peroxidation are increased. [Copyright &y& Elsevier]

Originally identified in cultured cells, oncogenic cellular senescence is a growth-arrest mechanism which may inhibit tumor development by limiting the ability of cells to divide. However, literature shows that these cells secrete tumor-inducing and tumor-suppressing proteins leading to poor prognosis. Understanding the progression of oncogenic cellular senescence and associated mechanisms provides important implications for improving tumorigenesis therapeutic treatments. Micro-Raman spectroscopic imaging has grown in popularity as an imaging technique compared to the standard imaging predecessors and can be attributed to its numerous benefits such as no sample perturbation and the provision of direct chemical information. Through the use of label-free micro-Raman spectroscopy, control and senescent cells were noninvasively imaged. Resulting spectral images were processed using chemometric techniques, and average nuclei spectra from each sample set were compared. In turn, changes in the -cis and -trans unsaturated lipid isomer content were found to differ among proliferating and senescent cells. This may lead to increased nuclear fluidity and may contribute to the inability of senescent cells to complete the cell cycle. In the tumor environment, this detected increase in nuclear envelope fluidity could lead to downstream gene expression modifications and increased nucleo-cytoplasmic RNA translocation. Understanding nuclear envelope fluidity could provide insight into secretory profiles of senescent cells and their role in carcinogenesis, meriting further investigation into novel therapeutic technique development for oncogenic cellular senesce [ABSTRACT FROM AUTHOR]