Integrated MicroRNA-mRNA Profiling Identifies Oncostatin M as a Marker of Mesenchymal-Like ER-Negative/HER2-Negative Breast Cancer

MicroRNAs (miRNAs) simultaneously modulate different oncogenic networks, establishing a dynamic system of gene expression and pathway regulation. In this study, we analyzed global miRNA and messenger RNA (mRNA) expression profiles of 17 cell lines representing different molecular breast cancer subtypes. Spearman's rank correlation test was used to evaluate the correlation between miRNA and mRNA expression. Hierarchical clustering and pathway analysis were also performed. Publicly available gene expression profiles (n = 699) and tumor tissues (n = 80) were analyzed to assess the relevance of key miRNA-regulated pathways in human breast cancer. We identified 39 significantly deregulated miRNAs, and the integration between miRNA and mRNA data revealed the importance of immune-related pathways, particularly the Oncostatin M (OSM) signaling, associated with mesenchymal-like breast cancer cells. OSM levels correlated with genes involved in the inflammatory response, epithelial-to-mesenchymal transition (EMT), and epidermal growth factor (EGF) signaling in human estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Our results suggest that the deregulation of specific miRNAs may cooperatively impair immune and EMT pathways. The identification of the OSM inflammatory pathway as an important mediator of EMT in triple-negative breast cancer (TNBC) may provide a novel potential opportunity to improve therapeutic strategies

miRpower: a web-tool to validate survival-associated miRNAs utilizing expression data from 2178 breast cancer patients

PURPOSE: The proper validation of prognostic biomarkers is an important clinical issue in breast cancer research. MicroRNAs (miRNAs) have emerged as a new class of promising breast cancer biomarkers. In the present work, we developed an integrated online bioinformatic tool to validate the prognostic relevance of miRNAs in breast cancer. METHODS: A database was set up by searching the GEO, EGA, TCGA, and PubMed repositories to identify datasets with published miRNA expression and clinical data. Kaplan-Meier survival analysis was performed to validate the prognostic value of a set of 41 previously published survival-associated miRNAs. RESULTS: All together 2178 samples from four independent datasets were integrated into the system including the expression of 1052 distinct human miRNAs. In addition, the web-tool allows for the selection of patients, which can be filtered by receptors status, lymph node involvement, histological grade, and treatments. The complete analysis tool can be accessed online at: www.kmplot.com/mirpower . We used this tool to analyze a large number of deregulated miRNAs associated with breast cancer features and outcome, and confirmed the prognostic value of 26 miRNAs. A significant correlation in three out of four datasets was validated only for miR-29c and miR-101. CONCLUSIONS: In summary, we established an integrated platform capable to mine all available miRNA data to perform a survival analysis for the identification and validation of prognostic miRNA markers in breast cancer

MicroRNA-21 links epithelial-to-mesenchymal transition and inflammatory signals to confer resistance to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast cancer patients

Patients with primary HER2-positive breast cancer benefit from HER2-targeted therapies. Nevertheless, a significant proportion of these patients die of disease progression due to mechanisms of drug resistance. MicroRNAs (miRNAs) are emerging as critical core regulators of drug resistance that act by modulating the epithelial- to-mesenchymal transition (EMT) and cancer-related immune responses. In this study, we investigated the association between the expression of a specific subset of 14 miRNAs involved in EMT processes and immune functions and the response to neoadjuvant trastuzumab and chemotherapy in 52 patients with HER2-overexpressing breast tumors. The expression of only a single miRNA, miR-21, was significantly associated with residual disease (p = 0.030) and increased after trastuzumab-chemotherapy (p = 0.012). A target prediction analysis coupled with in vitro and in vivo validations revealed that miR-21 levels inversely correlated with the expression of PTEN (rs = −0.502; p = 0.005) and PDCD4 (rs = −0.426; p = 0.019), which differentially influenced the drug sensitivity of HER2-positive breast cancer cells. However, PTEN expression was only marginally associated with residual disease. We further demonstrated that miR-21 was able to affect the response to both trastuzumab and chemotherapy, triggering an IL-6/STAT3/NF-κB-mediated signaling loop and activating the PI3K pathway. Our findings support the ability of miR-21 signaling to sustain EMT and shape the tumor immune microenvironment in HER2-positive breast cancer. Collectively, these data provide a rationale for using miR-21 expression as a biomarker to select trastuzumab-chemotherapy-resistant HER2-positive breast cancer patients who may benefit from treatments containing PI3K inhibitors or immunomodulatory drugs

Clinical Relevance of the Immune Contexture and AXL Kinase in Triple-Negative Breast Cancer

Triple-negative breast cancers (TNBC) are usually associated with an aggressive phenotype, an increased risk of early relapse, and poor outcome. Burgeoning evidence demonstrates that TNBC encompasses distinct molecular entities that are differentially characterized by specific hallmarks of cancer, including chromosomal instability (CIN), epithelial-to-mesenchymal transition (EMT), and cancer-related immune responses. In particular, the tumour immune microenvironment and the interaction between immune and cancer cells are emerging as crucial factors in tumour progression, prognosis, and response to therapy in TNBC. In this study, we aimed to evaluate the relationship between these three major hallmarks of TNBC. In particular, we assessed the composition and functionality of immune infiltrates in TNBC samples with different levels of CIN and investigated their clinical relevance in patients treated with adjuvant chemotherapy. Additionally, we explored the interactions between tumour-associated macrophages (TAM) and TNBC cells, particularly those with mesenchymal traits. For this purpose, we integrated in vivo analysis of human TNBC tissues, in vitro experiments, and bioinformatics data. To assess the tumour immune microenvironment heterogeneity, we first identified different gene signatures from mRNA expression data, representing distinct immune components, and then evaluated their expression in different molecular subtypes of TNBC and in tumour samples characterized by low and high levels of CIN. To further explore the composition and functionality of immune infiltration in TNBC in vivo, formalin-fixed, paraffin-embedded tissues were retrospectively collected from large cohorts of early-stage TNBC patients treated with anthracycline-based chemotherapy. Stromal tumour-infiltrating lymphocytes (TIL) were evaluated on haematoxylin and eosin-stained sections. The density of CD4+, CD8+, CD103+, and FOXP3+ lymphocytes, CD68+ and CD163+ macrophages, and the expression of the immune checkpoints PD-1 and LAG-3 were assessed by immunohistochemistry. Furthermore, to understand the biological and clinical relevance of the interaction between TNBC cells and innate immune cells, we investigated several kinases functioning in the EMT process and their association with TAM in patients treated with adjuvant chemotherapy and in several TNBC cell lines. We demonstrated that immune expression signatures were differentially expressed in TNBC characterized by varying levels of CIN and that TNBC molecular subgroups with a mesenchymal phenotype were enriched for immune signatures related to pro-tumour M2 macrophages. Conjunctly, by analysing human TNBC tissues, we showed that the presence of elevated TIL positively correlated with the density of all T cell subtypes, especially cytotoxic CD8+ lymphocytes. Among immune subpopulations, CD8+ lymphocytes were the main effectors of anti-tumour immune responses. We also found that PD-1 and LAG-3 were concurrently expressed in nearly 15% of TNBC. The expression of both checkpoint receptors positively correlated with the presence of TIL, but was not significantly associated with patient outcome. Furthermore, we showed that intraepithelial CD8+ cells frequently expressed the integrin CD103, which mediates the localization of cytotoxic lymphocytes within epithelial tissues. Importantly, the massive intraepithelial infiltration of cytotoxic CD103+ TIL co-expressing PD-1, correlated with prolonged survival in TNBC. In addition to TIL, we have demonstrated that the activation of the innate immune cells within the TNBC tumour stroma had a crucial role in tumour progression and chemoresistance, especially through the modulation of EMT. Accordingly, the EMT-related kinase AXL was highly associated with the presence of CD163+ TAM. Tumours from relapsing patients presented a high expression of AXL and CD163, although only AXL retained independent prognostic significance in multivariate analysis. In vitro analysis demonstrated that AXL-expressing TNBC cells were able to polarize human macrophages toward an M2-like phenotype. A selective inhibition of AXL impaired the activity of M2-like macrophages, reducing cancer cell invasiveness and restoring the sensitivity of breast cancer cells to chemotherapeutic drugs, especially anthracyclines. Overall, our data indicate that TNBC subgroups with different biological and genomic features are characterized by distinct compositions of the immune microenvironment. Our results confirm that the evaluation of stromal TIL is the most reliable immune prognostic marker in TNBC patients. Our data also support the pharmacological and clinical evaluation of anti-PD-1/PD-L1 and anti-LAG-3 in a specific subset of TNBC patients and the inhibition of AXL as a novel strategy to simultaneously target TNBC cells and tumour promoting TAM

Integrated MicroRNA–mRNA Profiling Identifies Oncostatin M as a Marker of Mesenchymal-Like ER-Negative/HER2-Negative Breast Cancer

MicroRNAs (miRNAs) simultaneously modulate different oncogenic networks, establishing a dynamic system of gene expression and pathway regulation. In this study, we analyzed global miRNA and messenger RNA (mRNA) expression profiles of 17 cell lines representing different molecular breast cancer subtypes. Spearman’s rank correlation test was used to evaluate the correlation between miRNA and mRNA expression. Hierarchical clustering and pathway analysis were also performed. Publicly available gene expression profiles (n = 699) and tumor tissues (n = 80) were analyzed to assess the relevance of key miRNA-regulated pathways in human breast cancer. We identified 39 significantly deregulated miRNAs, and the integration between miRNA and mRNA data revealed the importance of immune-related pathways, particularly the Oncostatin M (OSM) signaling, associated with mesenchymal-like breast cancer cells. OSM levels correlated with genes involved in the inflammatory response, epithelial-to-mesenchymal transition (EMT), and epidermal growth factor (EGF) signaling in human estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Our results suggest that the deregulation of specific miRNAs may cooperatively impair immune and EMT pathways. The identification of the OSM inflammatory pathway as an important mediator of EMT in triple-negative breast cancer (TNBC) may provide a novel potential opportunity to improve therapeutic strategies

Studio del processo di sterilizzazione di materiali e liquidi mediante l'impiego di vapore saturo

La sterilizzazione effetuata mediante vapore saturo può essere influenzata da: tipo, quantità e disposizione dei materiali presenti nella camera dell’autoclave, tipologia e resistenza dei microrganismi, durata del ciclo, temperatura e pressione. Questa segue una cinetica di primo ordine, quindi solo con un processo di durata infinita si raggiunge la sterilità assoluta. Non potendo portare avanti un trattamento all’infinito, le aziende farmaceutiche utilizzano un SAL minore o uguale a 10-6: su un milione di unità trattate al massimo una può risultare non sterile. Per arrivare a ciò è necessario che i parametri relativi a un ciclo siano valutati accuratamente, altrimenti si potrebbe sovrastimare l’intero processo. Lo scopo di questo lavoro è quello di analizzare i fattori da cui dipende una sterilizzazione. Riferendoci a tre autoclavi, sono valutati i requisiti della strumentazione critica, è specificata la compatibilità al vapore dei materiali, l’omogeneità e la configurazione dei carichi, i requisiti microbiologici degli elementi prima del trattamento, la collocazione e i criteri degli indicatori biologici nonchè la letalità raggiunta in ogni parte del carico. Inoltre sono rivisitati lavori abbastanza recenti relativi all’argomento. I risultati ottenuti dalle analisi effettuate permettono di chiarire gli aspetti necessari per garantire il corretto funzionamento delle autoclavi: un ciclo, se correttamente disegnato, dà risultati soddisfacenti che ci pemettono di raggiungere valori di SAL minori a 10-6 e che quindi vanno ad avvalorare studi già condotti in passato. Steam sterilization is influenced by different parameters such as: type, quality and disposition of the elements of the load, type and resistance of microorganisms, temperature, pressure and time of a cycle. Sterilization follows a first chemical reaction, in which only with the infinite time of exposure to steam, all microorganisms are destroyed. It is impossible sterilize elements for infinite time, so pharmaceutical companies, use a Sterility Assurance Level equal or lower to 10-6: it denotes the probability of not more than one viable microorganism in 106 sterilized items. In order to reach this value it is necessary that all parameters must be analyzed: if they are ignored, it is possible overvalue the whole process. The aim of this work is to analyze all parameters influencing a sterilization cycle, during a validation process of three autoclaves. In this study is considered the calibration of the critical instruments, the compatibility to steam of the elements, the homogeneity of loads, its configurations in the chamber of sterilization, the microbiological characteristics of the elements before the treatment, the requirements and the position of biological indicators and the lethality reached in each part of the load. Furthermore, with this study, relatively recent works is revisited. All results confirmed the effectiveness of sterilization: if the cycle is correctly designed, it reaches a SAL lower to 10-6, so this strengthens past works

Femoral fractures in the extremely elderly

At the Trauma Unit of Pisa we performed an observational study reviewing nineties that about 200 patients were treated and underwent surgery for femoral neck fracture from 1998 to 2005. The clinical and radiographic results obtained were discrete, with a mortality of 42.5%, the survivors are still having a good quality of life

Indices of risk assessment of fracture of the proximal humerus

Osteoporotic fractures are now a social problem for incidence and costs. Fractures of the proximal humerus events are frequent and constantly increasing. It is estimated that they are 20% of all osteoporotic fractures. Bone densitometry in most cases underestimates the real humeral bone density. There is little information about osteoporotic changes in the proximal humerus and their association with the cortical thickness of the humeral shaft. The ratio between the thickness of the cortical and the total diameter of the humeral diaphysis is the cortical index. Fracture risk limit value is 0.231. Convinced of the need to quantify in a reproducible way the real local humerus bone density, we performed a comparative evaluation of bone density of the humerus and femur in patients admitted to our clinic for fractures of the humerus and femur. We evaluated 28 women treated surgically for a fragility fracture of the proximal humerus or femur neck in 2010. All cortical index obtained were lower than the limit for fracture risk set at 0.231, so the IC was more predictive of neck medial fractures of the femur than had DEXA and the U.S. The information about the cortical index may provide a simple way of determining the bone quality of the proximal humerus and of facilitating decision-making in the surgical treatment of patients with fractures of the humerus. So we want to emphasize the importance of therapy for osteoporosis even in patients with fractures of the proximal humerus, which often have not critical densitometric values of femur or column, but they are at risk of new fractures

Humeral bone fragility in patients with shoulder prosthesis: a case of humeral periprosthetic refracture

In recent decades there has been an increase in upper limb prosthetic surgery, primarily for the shoulder, for osteoarthritis disease and for traumatic pathology. It is occurring in parallel an increase in periprosthetic fractures of the humerus, although with less impact than other anatomical districts such as the hip. We report a case of humeral periprosthetic refracture in a 66-years-old female patient. The humerus bone quality is worse than in other districts in patient of the same age. The fragility humerus fracture are increasing, affecting relatively younger individuals than those with femoral neck fractures and represent an independent risk factor for the occurrence of subsequent fractures. Actually humeral BMD is underestimated by traditional densitometric evaluation technique