Heart failure impairs the mechanotransduction properties of human cardiac pericytes.

The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically.

miRNA expression profiles in liver grafts of HCV and HIV/HCV-infected recipients, 6 months after liver transplantation.

In hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients, HIV enhances HCV replication and liver damage. Several microRNAs (miRNAs), active in pro-fibrotic and inflammatory pathways, have been implicated in the pathogenesis of this phenomenon. However, these miRNAs have been tested only in explanted cirrhotic livers, when the liver damage has become chronic and irreversible. No data are available on the early phase of viral infection, such as early after liver transplantation (LT). In the present study, the expression of miR-101, miR-122, miR-155, miR-192, miR-200c, miR-338, and miR-532 was determined by quantitative real-time polymerase chain reaction in liver biopsies of HCV (n = 19) and HCV/HIV-infected (n = 20) LT recipients, as well as in a control group (n = 18) of noninfected patients, transplanted for alcoholic cirrhosis. The timing of liver biopsy was 6 months post-LT. None of the patients was treated with direct-acting anti-HCV drugs. All co-infected recipients had suppressed HIV viral load. Grading and staging were assessed according to the Ishak Classification. HCV and HIV viral load were measured in the sera. miR-101 (p = .03), miR-122 (p = .012), and miR-192 (p = .038) were significantly downregulated in HCV/HIV co-infected and HCV mono-infected recipients when compared with noninfected recipients, and such downregulation was more pronounced in co-infected ones. Moreover, in co-infected recipients but not in mono-infected ones, miR-101 inversely correlated with the peripheral HCV-RNA levels (r = .41, p = .04) and miR-122 inversely correlated with peripheral HCV-RNA levels (r = .49, p = .03) and with the histological grading (r = .51, p = .02).  In conclusion, as early as 6 months after LT, the presence of HIV-HCV co-infection enhanced a significant downregulation of certain miRNAs that showed a direct correlation with HCV viral load and liver inflammation.

TFEB, SIRT1, CARM1, Beclin-1 expression and PITX2 methylation in breast cancer chemoresistance: a retrospective study.

BACKGROUND: Breast cancer chemoresistance is attributed to a wide variety of mechanisms, including autophagy. Transcription factor EB (TFEB) has been recently identified and characterized as one major regulator of autophagy and lysosomal genesis. OBJECTIVE: This study aims to evaluate the prognostic impact of TFEB and its pathway in breast cancer chemoresistance. METHODS: This retrospective study analyzes the expression of TFEB, CARM1, SIRT1, and Beclin-1 and the methylation of PITX2 in breast carcinoma. A group of breast cancer patients treated with chemotherapy, who relapsed within 12 months from treatment initiation, were compared to a sub-cohort of chemo-treated patients who did not recur within 12 months of follow-up. The expression of TFEB, CARM1, SIRT1, and Belcin-1 was analyzed using immunohistochemistry or RT-PCR on formalin-fixed paraffin-embedded samples. PITX2 methylation was tested with the diagnostic CE-marked kit Therascreen PITX2 RGQ PCR. In the final model, 136 cases of chemo-treated breast cancer were included. RESULTS: A higher TFEB and Beclin-1 expression correlate with shorter survival in patients with chemo-treated invasive breast cancer (respectively HR 3.46, CI.95 1.27-9.47, p < 0.05 and 7.11, CI.95 2.54-19.9). TFEB, CARM1, and SIRT1 are positively correlated with Beclin-1. The protein expression of SIRT1 is significantly associated with TFEB and CARM1 so that a very low SIRT1 expression (lower than the first quartile of the H-score distribution) correlates with a low expression of TFEB and CARM1 and with longer survival. SIRT1 seems to have a lower H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. Beclin-1 and TFEB seem to have a higher H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. PITX2 methylation analysis was feasible only in 65% of the selected samples, but no significant differences between cases and controls were found, and there was also no correlation with the expression of the TFEB pathway. CONCLUSIONS: TFEB, SIRT1, and Beclin-1 seem to have a potential prognostic significance in patients with chemo-treated breast cancer, likely because of their role in the regulation of autophagy. In addition, no correlation between TFEB and PITX2 methylation was found, likely because they perform two different roles within the autophagy process.

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements.

INTRODUCTION: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). AIM: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. METHODS: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. RESULTS AND CONCLUSIONS: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.

High Viral Load in Upper Respiratory Specimens of Persons With SARS-CoV-2 Infection in a Northern Italian Area.

BACKGROUND: An assessment of viral load in biologic specimens of subjects with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may have important implications for public health planning. The aim of this study was to estimate the prevalence of high viral load in upper respiratory specimens of patients with SARS-CoV-2 infection during the first Italian wave (spring) and at the beginning of the second wave (summer) of the COVID-19 epidemic, through the measurement of cycle threshold (Ct) values from real-time reverse transcription-polymerase chain reaction tests conducted at the University Hospital of Udine, Italy, serving 530,000 inhabitants. METHODS: We compared the prevalence of high viral load, defined as Ct ≤ 20 at the first positive test result, in 262 subjects from the spring and 453 from the summer period. Logistic regression was used to account for potential confounding due to sex, age, and severity of infection. RESULTS: In the spring, 9.2% of subjects had Ct ≤ 20 versus 21.4% in the summer. After adjusting for confounders, the likelihood of having high viral load was 2.9 times higher in the summer than in the spring (95% confidence interval, 1.7-5.0). CONCLUSIONS: In this Italian area, more COVID-19 patients had high viral load in the spring epidemic wave than at the beginning of the second, during the summer. Cycle threshold values may represent useful information to monitor viral load at a population level in subjects with SARS-CoV-2 infection.

Thymic stromal lymphopoietin expression from benign lymphoproliferation to malignant B-cell lymphoma in primary Sjögren's syndrome.

OBJECTIVES: To investigate the expression of thymic stromal lymphopoietin (TSLP) in primary Sjögren's syndrome (pSS), stratified according to the lymphoproliferative status, from a fully benign (fbSS) stage to myoepithelial sialadenitis (MESA) and to B-cell non-Hodgkin's lymphoma (NHL). METHODS: After initial serum studies in large numbers of pSS patients and in controls, TSLP was investigated also in pathologic salivary glands (SG) biopsies from 38 stratified pSS patients (13 fbSS; 13 MESA; 12 NHL) and from 13 controls with non-autoimmune sicca syndrome (nSS) by RT-PCR, immunohistochemistry and immunofluorescence. RESULTS: Significantly higher TSLP serum levels were shown in pSS than controls, increasing from fbSS to MESA and to NHL. In SG biopsies, TSLP-positive B lymphocytes increased with increasing lymphoproliferation, maximally in NHL, consistent with the detection of inducible TSLP long isoform (lfTSLP) mRNA only in MESA and NHL. By contrast, the constitutive TSLP short isoform (sfTSLP) mRNA showed no difference among subgroups. The TSLP expression by glandular epithelium declined with the progression from fbSS to MESA and to NHL. CONCLUSIONS: TSLP progressively increases from benign to malignant B-cell lymphoproliferation in pSS. The salivary epithelium expresses TSLP but, with the progression of lymphoproliferation, the B-cells may represent the major source of TSLP, in its long inducible isoform. A possible pathogenetic role of TSLP is herein hypothesised in pSS for the first time. Further analyses on TSLP, also as a biomarker of pSS and related lymphoproliferation, are worthwhile.

Expression and modulation of S100A4 protein by human mast cells.

S100A4 protein is expressed in fibroblasts during tissue remodelling and in cancer stem cells and it induces the metastatic spread of tumor cells. In mast cells (MCs) S100A4 have been found in some pathological conditions, but its function in normal MCs remains to be described. The purpose of this study was to characterize the cellular localization of the S100A4 protein in MCs of human tissues with inflammatory or tumor disorders and, to determine the consequence of reducing its expression in MC response. We found that tissue resident MCs stained positive to S100A4. Both human HMC-1 cell line and resting CD34+-derived MCs expressed S100A4, whose levels were differentially modulated upon MC activation. Downregulation of the S100A4 protein resulted in MC growth inhibition, enhanced apoptosis and deregulation of MMP-1 and MMP-10 production. Our results suggest that S100A4 is also playing a role in the MC life cycle and functions.

Functional expression of aryl hydrocarbon receptor on mast cells populating human endometriotic tissues.

Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The aryl hydrocarbon receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: (i) examined the cytokine expression profile; (ii) counted AhR-expressing MCs; (iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; (iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); (v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and (vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors released by MCs on AhR activation. These results suggest a new mechanistic role of MCs in the pathogenesis of endometriosis.

In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis.

BACKGROUND: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. METHODS: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. RESULTS: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. CONCLUSION: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.

Dissecting the Heterogeneity of Circulating Tumor Cells in Metastatic Breast Cancer: Going Far Beyond the Needle in the Haystack.

Although the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM⁺/CK⁺) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a "surrogate" of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well.

A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single-Cell Metabolism in Droplet-Based Microfluidics.

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients.

Verification of a Rapid Analytical Method for the Qualitative Detection of Listeria spp. and Listeria monocytogenes by a Real-Time PCR Assay according to EN UNI ISO 16140-3:2021.

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

A streamlined workflow for a fast and cost-effective count of tyndallized probiotics using flow cytometry.

The use of dead probiotics and their cellular metabolites seems to exhibit immunomodulatory and anti-inflammatory properties, providing protection against pathogens. These inanimate microorganisms, often referred to as tyndallized or heat-killed bacteria, are a new class of probiotics employed in clinical practice. Safety concerns regarding the extensive use of live microbial cells have increased interest in inactivated bacteria, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. Culture-dependent methods are not suitable for the quality assessment of these products, and alternative methods are needed for their quantification. To date, bacterial counting chambers and microscopy have been used for tyndallized bacteria enumeration, but no alternative validated methods are now available for commercial release. The aim of the present study is to design a new method for the qualitative and quantitative determination of tyndallized bacterial cells using flow cytometric technology. Using a live/dead viability assay based on two nucleic acid stains, thiazole orange (TO) and propidium iodide (PI), we optimized a workflow to evaluate bacterial viability beyond the reproduction capacity that provides information about the structural properties and metabolic activities of probiotics on FACSVerse without using beads as a reference. The data obtained in this study represent the first analytical application that works effectively both on viable and non-viable cells. The results provided consistent evidence, and different samples were analyzed using the same staining protocol and acquisition settings. No significant discrepancies were highlighted between the declared specification of commercial strain and the analytical data obtained. For the first time, flow cytometry was used for counting tyndallized bacterial cells as a quality control assessment in probiotic production. This aspect becomes important if applied to medical devices where we cannot boast metabolic but only mechanical activities.

COVID-19-Related Myocarditis: Are We There Yet? A Case Report of COVID-19-Related Fulminant Myocarditis.

There is increasing evidence of cardiac involvement in COVID-19 cases, with a broad range of clinical manifestations spanning from acute life-threatening conditions such as ventricular dysrhythmias, myocarditis, acute myocardial ischemia and pulmonary thromboembolism to long-term cardiovascular sequelae. In particular, acute myocarditis represents an uncommon but frightening complication of SARS-CoV-2 infection. Even if many reports of SARS CoV-2 myocarditis are present in the literature, the majority of them lacks histological confirmation of cardiac injury. Here, we report a case of a young lady, who died suddenly a few days after testing positive for SARS-CoV-2, whose microscopic and genetics features suggested a direct cardiac involvement compatible with fulminant myocarditis.

An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements.

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination.

Reinfection of Transplanted Livers in HCV- and HCV/HIV-Infected Patients Is Characterized by a Different MicroRNA Expression Profile.

BACKGROUND: After liver transplantation, HCV/HIV co-infected patients present, compared to the HCV mono-infected ones, increased HCV viral load, rapid progression to liver fibrosis and higher mortality. Liver biopsies (LB), obtained routinely 6 months after transplantation, represent a unique model to assess the early events related to graft re-infection. Here, we used miRNA sequencing of LB obtained from both HCV-and HCV/HIV-infected recipients, to identify transcriptional profiles able to explain the more severe outcome of these latter. METHODS: miRNAs of 3 healthy livers, 3 HCV-LB and 3 HCV/HIV-LB were sequenced by Illumina HiSeq2500 platform. The DIANA-miRPath v3.0 webserver and DIANA-microT-CDS algorithm (v5.0) were used to characterize the functions of differentially expressed (DE-) miRNAs, querying the KEGG and Gene Ontology-Biological Process databases. RESULTS: LB obtained from infected patients were characterized, with respect to controls, by a miRNA profile related to viral infection, immune system signaling and DNA damage in HCV-induced carcinogenesis. Instead, HCV-LB and HCV/HIV-LB differed in the expression of miRNAs involved in immunological and apoptotic processes and in extracellular matrix remodeling. CONCLUSIONS: liver reinfection processes are associated with early miRNA changes. Further studies are necessary to establish their prognostic role and possible actionability.

Intrauterine fetal death in a COVID positive pregnant woman: a case report and literature review.

SARS-CoV-2 disease mechanisms are not yet fully understood, especially in cases affecting pregnant women. In fact, although they suffer from the same symptoms as non-pregnant women, they are more susceptible to adverse outcomes of COVID-19 as well as pregnancy complications leading to stillbirth, premature rupture of membrane, or intrauterine fetal demise (IUFD). The consequences on pregnancy are not clearly correlated with the severity of the infection. We present a case of fetal death in a 30-year-old woman with a confirmed SARS-CoV-2 infection without any other significant clinical or obstetric disorders. A histopathological examination of the placenta indicated massive and diffuse intervillar fibrinoid deposits associated with chronic histiocytic intervillositis that can be associated with SARS-CoV-2 placentitis. Given the high rates of SARS-CoV-2 infections in pregnancy, it is important to understand the factors that determine negative pregnancy outcomes, regardless of the severity of the patient's symptoms.

One-Step Nucleic Acid Amplification System in Comparison to the Intraoperative Frozen Section and Definitive Histological Examination Among Breast Cancer Patients: A Retrospective Survival Study.

Simple Summary: Implementing intraoperative assessment of sentinel lymph nodes by one-step nucleic acid amplification in early breast cancer can reduce the surgical burden to the patient and the costs to the health system. However, only limited data are available in terms of long-term disease-free survival and overall survival. Therefore, this study aims to compare disease-free survival and overall survival between one-step nucleic acid amplification, frozen section, and definitive histology. These results could impact the healthcare community, adding further proof to the body of evidence supporting the broader adoption of this innovative technology that enables a safe reduction in patient surgical burden and healthcare costs. Background: The one-step nucleic acid amplification (OSNA) system is a novel molecular technique, which consents to quick intraoperative detection of sentinel lymph node metastases by the amplification of cytokeratin 19 mRNA. Our study aims to evaluate the OSNA method in comparison with frozen section (FS) and definitive histological examination of the sentinel lymph node biopsy among early breast cancer patients considering disease-free survival (DFS) and overall survival (OS). Methods: In this study, we included all women who underwent sentinel lymph node biopsy (SLNB) for breast cancers classified as TNM stage I and II in our center between January 2005 and January 2017, and the follow-up was collected up to January 2019. We divided patients among three groups based on SLNB evaluation: definitive histological examination, intra-operative FS, or OSNA. Results: We included 2412 SLNBs: 727 by definitive histological examination, 697 by FS, and 988 by OSNA. Isolated tumor cells were found in 2.32% of cases, micrometastasis in 9.12%, and macrometastases in 13.64%. Surgical procedure duration was significantly shorter in OSNA than in FS (42.1 minutes ±5.1 vs. 70.1 minutes ±10.5, p <0.05). No significant differences have been observed among the three groups regarding OS, DSF, cumulative local, or distant metastases. In particular 5-year DFS was 96.38% in definitive histology (95% C.I. 95.02-97.75%), 96.37% in FS (95% C.I. 94.98-97.78%), and 96.51% in OSNA group (95% C.I. 95.32-97.72%). Conclusions: No difference in OS and DFS was found comparing OSNA, FS, and definitive histology. Furthermore, reduced operative time was found in the OSNA group.

Incidence and Risk Factors for Venous Thromboembolism in Female Patients Undergoing Breast Surgery.

Albeit it does not have the highest venous thromboembolism (VTE) incidence compared to other neoplasms, breast cancer contributes to many VTE events because it is the most diagnosed tumor in women. We aim to analyze the occurrence and timing of VTE during the follow-up of patients who underwent breast surgery, the possible correlated factors, and the overall survival. This retrospective study included all female patients diagnosed with mammary pathology and surgically treated in our clinic between January 2002 and January 2012. Of 5039 women who underwent breast surgery, 1056 were found to have no evidence of malignancy, whereas 3983 were diagnosed with breast cancer. VTE rate resulted significantly higher in patients with invasive breast cancer than in women with benign breast disease or carcinoma in situ. Invasive cancers other than lobular or ductal were associated with a higher VTE rate. In addition, chronic hypertension, high BMI, cancer type, and evidence of metastasis turned out to be the most significant risk factors for VTE in women who underwent breast surgery. Moreover, VTE occurrence significantly impacted survival in invasive breast cancer patients. Compared to women with benign mammary pathology, VTE prevalence in women with breast cancer is significantly higher. The knowledge about the risk factors of VTE could be helpful as prognostic information, but also to eventually target preventive treatment strategies for VTE, as far as the co-existence of invasive breast cancer and VTE has a significantly negative impact on survival.