Microcalcifications in breast cancer: an active phenomenon mediated by epithelial cells with mesenchymal characteristics.

BACKGROUND: Mammary microcalcifications have a crucial role in breast cancer detection, but the processes that induce their formation are unknown. Moreover, recent studies have described the occurrence of the epithelial-mesenchymal transition (EMT) in breast cancer, but its role is not defined. In this study, we hypothesized that epithelial cells acquire mesenchymal characteristics and become capable of producing breast microcalcifications. METHODS: Breast sample biopsies with microcalcifications underwent energy dispersive X-ray microanalysis to better define the elemental composition of the microcalcifications. Breast sample biopsies without microcalcifications were used as controls. The ultrastructural phenotype of breast cells near to calcium deposits was also investigated to verify EMT in relation to breast microcalcifications. The mesenchymal phenotype and tissue mineralization were studied by immunostaining for vimentin, BMP-2, ß2-microglobulin, ß-catenin and osteopontin (OPN). RESULTS: The complex formation of calcium hydroxyapatite was strictly associated with malignant lesions whereas calcium-oxalate is mainly reported in benign lesions. Notably, for the first time, we observed the presence of magnesium-substituted hydroxyapatite, which was frequently noted in breast cancer but never found in benign lesions. Morphological studies demonstrated that epithelial cells with mesenchymal characteristics were significantly increased in infiltrating carcinomas with microcalcifications and in cells with ultrastructural features typical of osteoblasts close to microcalcifications. These data were strengthened by the rate of cells expressing molecules typically involved during physiological mineralization (i.e. BMP-2, OPN) that discriminated infiltrating carcinomas with microcalcifications from those without microcalcifications. CONCLUSIONS: We found significant differences in the elemental composition of calcifications between benign and malignant lesions. Observations of cell phenotype led us to hypothesize that under specific stimuli, mammary cells, which despite retaining a minimal epithelial phenotype (confirmed by cytokeratin expression), may acquire some mesenchymal characteristics transforming themselves into cells with an osteoblast-like phenotype, and are able to contribute to the production of breast microcalcifications.

Transglutaminase 2 as a biomarker of osteoarthritis: an update.

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca(2+)-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-ß seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.

Propionyl-L-carnitine improves postischemic blood flow recovery and arteriogenetic revascularization and reduces endothelial NADPH-oxidase 4-mediated superoxide production.

OBJECTIVE: The beneficial effect of the natural compound propionyl-l-carnitine (PLC) on intermittent claudication in patients with peripheral arterial disease is attributed to its anaplerotic function in ischemic tissues, but inadequate information is available concerning action on the vasculature. METHODS AND RESULTS: We investigated the effects of PLC in rabbit hind limb collateral vessels after femoral artery excision, mouse dorsal air pouch, chicken chorioallantoic membrane, and vascular cells by angiographic, Doppler flow, and histomorphometrical and biomolecular analyses. PLC injection accelerated hind limb blood flow recovery after 4 days (P<0.05) and increased angiographic quadriceps collateral vascularization after 7 days (P<0.001) Histomorphometry confirmed the increased vascular area (P<0.05), with unchanged intramuscular capillary density. PLC-induced dilatative adaptation, and growth was found associated with increased inducible nitric oxide synthase and reduced arterial vascular endothelial growth factor and intracellular adhesion molecule-1 expression. PLC also increased vascularization in air pouch and chorioallantoic membrane (P<0.05), particularly in large vessels. PLC increased endothelial and human umbilical vascular endothelial cell proliferation and rapidly reduced inducible nitric oxide synthase and NADPH-oxidase 4-mediated reactive oxygen species production in human umbilical vascular endothelial cells; NADPH-oxidase 4 also regulated NF-kappaB-independent intracellular adhesion molecule-1 expression. CONCLUSIONS: Our results provided strong evidence that PLC improves postischemic flow recovery and revascularization and reduces endothelial NADPH-oxidase-related superoxide production. We recommend that PLC should be included among therapeutic interventions that target endothelial function.

Difficulty diagnosing chronic cryptococcal meningitis in idiopathic CD4+ lymphocytopenia.

A 64-year-old man with idiopathic CD4(+) lymphocytopenia developed cognitive impairment and gait ataxia with isolated obstructive hydrocephalus, which was fatal. Cerebrospinal fluid showed mild pleocytosis, but the etiology was not revealed by extensive analysis. At autopsy, inflammatory cells, CD8(+) lymphocytes and abundant macrophages but not CD4(+) lymphocytes were infiltrating the meninges at the base of the brain. Electron microscopy demonstrated that inflammation was caused by Cryptococcus neoformans, which was localized exclusively within macrophages, where it grew with budding. Our study suggests that, in idiopathic CD4(+) lymphocytopenia, macrophages can efficiently phagocytize but inefficiently digest C. neoformans, thus representing a vehicle of chronic intracellular infection.

Alpha actin isoforms expression in human and rat adult cardiac conduction system.

In the adult heart, cardiac muscle comprises the working myocardium and the conduction system (CS). The latter includes the sinoatrial node (SAN), the internodal tract or bundle (IB), the atrioventricular node (AVN), the atrioventricular bundle (AVB), the bundle branches (BB) and the peripheral Purkinje fibers (PF). Most of the information concerning the phenotypic features of CS tissue derives from the characterization of avian and rodent developing hearts; data concerning the expression of actin isoforms in adult CS cardiomyocytes are scarce. Using specific antibodies, we investigated the distribution of alpha-skeletal (alpha-SKA), alpha-cardiac (alpha-CA), alpha-smooth muscle (alpha-SMA) actin isoforms and other muscle-typical proteins in the CS of human and rat hearts at different ages. SAN and IB cardiomyocytes were characterized by the presence of alpha-SMA, alpha-CA, calponin and caldesmon, whereas alpha-SKA and vimentin were absent. Double immunofluorescence demonstrated the co-localisation of alpha-SMA and alpha-CA in I-bands of SAN cardiomyocytes. AVN, AVB, BB and PF cardiomyocytes were alpha-SMA, calponin, caldesmon and vimentin negative, and alpha-CA and alpha-SKA positive. No substantial differences in actin isoform distribution were observed in human and rat hearts, except for the presence of isolated subendocardial alpha-SMA positive cardiomyocytes co-expressing alpha-CA in the ventricular septum of the rat. Aging did not influence CS cardiomyocyte actin isoform expression profile. These findings support the concept that cardiomyocytes of SAN retain the phenotype of a developing myogenic cell throughout the entire life span.

Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations.

Archival formalin-fixed paraffin-embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI-TOF imaging MS (MALDI-IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo-preserved tissues. We have developed an in vitro approach using "tissue surrogate" samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI-TOF MS and nLC-MS(E) analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI-IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.

High-resolution multicontrast-weighted MR imaging from human carotid endarterectomy specimens to assess carotid plaque components.

The American Heart Association modified classification for atherosclerotic plaque lesions has defined vulnerable plaques as those prone to rupture. The aim of our study was to assess the sensitivity and specificity of 1.5-T magnetic resonance imaging (MRI) in the evaluation of the characteristics of plaque components. Twelve carotid endarterectomy specimens were imaged by ex-vivo high-resolution 1.5-T MRI. Thirty-four cross-section axial images were selected for pixel-by-pixel basis analysis to demonstrate the most significant tissue features. Data were then submitted for histopathological examination and each specimen analysed in the light of the histological components (lipid core, fibrous tissue, fibrous/loose connective tissue, calcifications). The overall sensitivity and specificity rates for each tissue type were, respectively, 92% and 74% for the lipid core, 82% and 94% for the fibrous tissue, 72% and 87% for the fibrous/loose connective tissue, and 98% and 99% for calcification. The use of 1.5-T MRI appears to be a reliable tool to characterise plaque components and could help in the screening of patients with high risk of plaque rupture. The possibility of applying MRI in clinical daily practice may change the non-invasive approach to carotid artery diagnostic imaging, thus allowing an early identification of patients with vulnerable plaques.

Fluorescent in situ hybridization as a screening test for HER2 amplification in G2 and G3 breast cancers of lobular and ductal histotype and metastases.

The aim of the present study was to evaluate the effectiveness of fluorescence in situ hybridisation (FISH), as a screening test, in moderately- (G2) or poorly- (G3) differentiated breast cancers of the ductal (IDC) and lobular (ILC) histotypes and distant metastases. HER2 FISH was performed on 486 G2 and 477 G3 both of IDC and ILC histotypes and in 241 metastases. A significant difference in the HER2 amplification was observed between G2 (14.8%) and G3 (31.9%), with no difference according to the histotype. However, the rate of amplification increased to 36% in the G2/hormone receptor-negative cases as compared to 10.6% in the G2/receptor-positive cases (p<0.0001). HER2 was amplified in 17% of metastases with some differences depending on the location. These data suggest that the HER2 FISH analysis may be an effective screening test in breast cancer metastases and G3 tumors, irrespective of the hormone receptor status or presence of lymphovascular invasion.

Correction: Evaluation of tenascin-C by tenatumomab in T-cell non-Hodgkin lymphomas identifies a new target for radioimmunotherapy.

[This corrects the article DOI: 10.18632/oncotarget.23919.].

Evaluation of tenascin-C by tenatumomab in T-cell non-Hodgkin lymphomas identifies a new target for radioimmunotherapy.

The clinical outcome of T-cell non-Hodgkin lymphoma (NHL) is poor and innovative treatments are needed. Tenascin-C is a large extracellular glycoprotein not expressed under physiological conditions, but overexpressed in cancer. Aim of the study was to evaluate tenascin-C expression within pathologic tissue of T-cell NHL and determine its clinical significance. We used an immunohistochemistry approach using the anti-tenascin-C monoclonal antibody Tenatumomab in 75 systemic T-cell NHL (including 72 mature and 3 precursor T-cell NHL), and 25 primary cutaneous T-cell NHL. Data were analyzed in terms of staining intensity, proportion of involved areas and histologic pattern, and results were correlated with clinical characteristics and outcome. Ninety-three percent of the cases were tenascin-C positive and 59% of systemic diseases were characterized by a predominant involvement (>50%). Stromal expression was detected in all the cases while vascular and vascular plus cytoplasmic expression was present in 49% and 23%. The constant overexpression of the tenascin-C gene was observed in two independent publicly available T-cell NHL gene expression datasets. In conclusions, tenascin-C represents an attractive target that sets the rationale to investigate the therapeutic activity of radiolabeled Tenatumomab in T-cell NHL.

Role of inflammation in atherosclerosis.

Inflammation plays a major role in all phases of atherosclerosis. Stable plaques are characterized by a chronic inflammatory infiltrate, whereas vulnerable and ruptured plaques are characterized by an "active" inflammation involved in the thinning of the fibrous cap, predisposing the plaque to rupture. Although a single vulnerable atherosclerotic plaque rupture may cause the event, there are many other types of plaques, several of which are vulnerable. The existence of multiple types of vulnerable plaques suggests that atherosclerosis is a diffuse inflammatory process. A current challenge is to identify morphologic and molecular markers able to discriminate stable plaques from vulnerable ones, allowing the stratification of patients at high risk for acute cardiovascular and cerebrovascular events before clinical syndromes develop. With that aim in mind, this article summarizes the natural history of atherosclerotic plaques, focusing on molecular mechanisms affecting plaque progression and serum markers correlated with plaque inflammation.

Stool test for colorectal cancer screening: what is going on?

Given the increasing incidence of colorectal cancer (CRC), performing new and cost-effective stool tests is of particular importance for early diagnosis and treatment. In the present review, we describe the main characteristics, and the performance of the most recently developed stool tests in the screening setting of colorectal tumoral diseases. Most of the studies reported high sensitivity both for adenomas and CRC diagnosis; less than half studies reported also high specificity with respect to stage and localization of the tumor. However, the performance of every single test was extremely variable, reaching >95% specificity for most of DNA-based markers, although lacking sensitivity even in case of invasive CRC. A new potential stool marker of colon cancer is clusterin, a protein of particular interest for its high sensitivity and positive predictive value in patients with highly aggressive CRC.

Diffuse and active inflammation occurs in both vulnerable and stable plaques of the entire coronary tree: a histopathologic study of patients dying of acute myocardial infarction.

OBJECTIVES: This study was undertaken to define and compare geographic coronary artery inflammation in patients who were dying of acute myocardial infarction (AMI), chronic stable angina (SA), and noncardiac causes (CTRL). BACKGROUND: Biochemical markers and flow cytometry provide indirect evidence of diffuse coronary inflammation in patients dying of acute coronary syndromes. Yet no histopathologic studies have corroborated these findings. A key unanswered question is whether the inflammatory burden involves the entire coronary tree or is limited to a few plaques. METHODS: We examined 544 coronary artery segments from 16 patients with AMI, 109 segments from 5 patients with SA, and 304 coronary segments from 9 patients with CTRL. RESULTS: An average of 6.8 +/- 0.5 vulnerable segments per patient were found in the AMI group (in addition to culprit lesions) compared with an average of 0.8 +/- 0.3 and 1.4 +/- 0.3 vulnerable lesions/patient in the SA and CTRL groups, respectively. The AMI group, independent of the type of plaque observed, showed significantly more inflammatory infiltrates compared with the SA and CTRL groups (121.6 +/- 12.4 cell x mm2 vs. 37.3 +/- 11.9 cell x mm2 vs. 26.6 +/- 6.8 cell x mm2, p = 0.0001). In AMI patients, active inflammation was not only evident within the culprit lesion and vulnerable plaques but also involved stable plaques. These showed a three- to four-fold higher inflammation than vulnerable and stable plaques from the SA and CTRL groups, respectively. CONCLUSIONS: This histopathologic study found that both vulnerable and stable coronary plaques of patients dying of AMI are diffusely infiltrated by inflammatory cells.

Aging, smooth muscle cells and vascular pathobiology: implications for atherosclerosis.

Epidemiological and autopsy studies suggest a close link between aging and the clinical manifestation of atherosclerosis. Several experiments show increased arterial susceptibility to atherogenetic stimuli in aged subjects. All together, these findings support the concept that aging represents an independent atherogenetic risk factor, intimately associated to other parietal, microenvironmental and systemic noxae. Smooth muscle cells (SMCs) represent the major arterial cell population. As aging occurs, SMCs progressively migrate from the tunica media and accumulate into the tunica intima. Myointimal thickening may represent the site where low-grade atherogenic stimuli cause early development and more severe lesion progression. Intimal SMC accumulation is characterized from a switch, from a differentiated to a synthetic phenotype, with reduced myocytic cytoskeletal markers and the expression of new proteins. Aging also associates to changes of SMC proliferative and apoptotic behavior and response to growth factors, such as transforming growth factor-beta1. The alteration of SMC properties represents a crucial event in the pathobiology of arterial wall, since it contributes to the vascular remodeling and decline of function with aging and favors the progression of atherosclerosis. Increased knowledge of biomolecular mechanisms regulating these events helps to develop new strategies aimed at contrasting the adverse effect of vascular aging.

Modulation of clusterin isoforms is associated with all-trans retinoic acid-induced proliferative arrest and apoptosis of intimal smooth muscle cells.

OBJECTIVE: Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs). METHODS AND RESULTS: Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased alpha-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform. CONCLUSIONS: Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all-trans retinoic acid-induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis. Clusterin was detected in human arterial myointimal thickening and absent in the underlying media. Rat neointimal cells overexpressed clusterin and clusterin antisense oligonucleotide reduced proliferation and increased apoptosis. All-trans retinoic acid-induced proliferative arrest showed association with s-CLU reduction and n-CLU overexpression with apoptosis, supporting a different biological role of these isoforms.

Extracellular proteases in atherosclerosis and restenosis.

Extracellular proteolysis plays a key role in many pathophysiologic processes including cancer, inflammatory diseases, and cardiovascular conditions such as atherosclerosis and restenosis. Whereas matrix metalloproteinases are their best known member, many others are becoming better known. The extracellular proteases are a complex and heterogeneous superfamily of enzymes. They include metalloproteinases (matrix metalloproteinases, adamalysins, or pappalysins), serine proteases (elastase, coagulation factors, plasmin, tissue plasminogen activator, urokinase plasminogen activator), and the cysteine proteases (such cathepsins). In addition to their matrix degradation capabilities, they have other less well known biologic functions that include angiogenesis, growth factor bioavailability, cytokine modulation, receptor shedding, enhancing cell migration, proliferation, invasion, and apoptosis. This review discusses extracellular proteases relevant to the vasculature, their classification and function, and how protease disorders contribute to arterial plaque growth, including chronic atherosclerosis, acute coronary syndromes, restenosis, and vascular remodeling. These broad extracellular protease functions make them potentially interesting therapeutic targets.

Intra-tumor AvidinOX allows efficacy of low dose systemic biotinylated Cetuximab in a model of head and neck cancer.

For locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC), the current clinical use of Cetuximab in chemo/radiotherapy protocols is often associated to severe systemic toxicity. Here we report in vitro data in human FaDu pharynx SCC cells, showing that inactive concentrations of biotinylated Cetuximab (bCet) become active upon anchorage to AvidinOX on the surface of tumor cells. AvidinOX-anchored bCet induces apoptosis and DNA damage as well as specific inhibition of signaling, degradation and abrogation of nuclear translocation of EGFR. In the mouse model of FaDu cancer, we show that intra-tumor injection of AvidinOX allows anti-tumor activity of an otherwise inactive, intraperitoneally delivered, low dose bCet. Consistently with in vitro data, in vivo tumor inhibition is associated to induction of apoptosis, DNA damage and reduced angiogenesis. AvidinOX is under clinical investigation for delivering radioactive biotin to inoperable tumors (ClinicalTrials.gov NCT02053324) and present data support its use for the local treatment of HNSCC in combination with systemic administration of low dose bCet.

Modulation of different clusterin isoforms in human colon tumorigenesis.

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell-cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell-cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells.

BACKGROUND: Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. METHODS AND RESULTS: O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. CONCLUSIONS: Our data show that hyperglycemia, through the hexosamine pathway, impairs activation of the IR/IRS/PI3-K/Akt pathway, resulting in deregulation of eNOS activity.