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We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

Bone development-related genes are enriched in healthy femoral arteries, which are more prone to calcification, as documented by the predominance of fibrocalcific plaques at the femoral location. We undertook a prospective histological study on the presence of calcifications in normal femoral arteries collected from donors. Since endothelial-to-mesenchymal transition (EndMT) participates in vascular remodeling, immunohistochemical (IHC) and molecular markers of EndMT and chondro-osteogenic differentiation were assessed. Transmission electron microscopy (TEM) was used to describe calcification at its inception. Two hundred and fourteen femoral arteries were enrolled. The mean age of the donors was 39.9 ± 12.9 years; male gender prevailed (M: 128). Histology showed a normal architecture; calcifications were found in 52 (24.3%) cases, without correlations with cardiovascular risk factors. Calcifications were seen on or just beneath the inner elastic lamina (IEL). At IHC, SLUG was increasingly expressed in the wall of focally calcified femoral arteries (FCFA). ETS-related gene (ERG), SLUG, CD44, and SOX-9 were positive in calcifications. RT-PCR showed increased levels of BPM-2, RUNX-2, alkaline phosphatase, and osteocalcin osteogenic transcripts and increased expression of the chondrogenic marker, SOX-9, in FCFA. TEM documented osteoblast-like cells adjacent to the IEL, releasing calcifying vesicles from the cell membrane. The vesicles were embedded in a proteoglycan-rich matrix and were entrapped in IEL fenestrations. In this study, ERG- and CD44-positive cell populations were found in the context of increased SLUG expression, thus supporting the participation of EndMT in FCFA; the increased transcript expression of osteochondrogenic markers, particularly SOX-9, reinforced the view that EndMT, osteochondrogenesis, and neoangiogenesis interact in the process of arterial calcification. Given its role as a transcription factor in the regulation of endothelial homeostasis, arterial ERG expression can be a clue of endothelial dysregulation and changes in IEL organization which can ultimately hinder calcifying vesicle diffusion through the IEL fenestrae. These results may have a broader implication for understanding arterial calcification within a disease context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vasuri, Valente, Motta, Degiovanni, Ciavarella and Pasquinelli.)

Introduction and Objectives: Conventional transarterial chemoembolization (cTACE) has several limitations due to the lack of standardization. The aim of this study was to evaluate the chemical and physical characteristics and behaviors over time of emulsions for cTACE and to assess intra- and inter-operator variabilities in the preparation processes., Materials and Methods: This in vitro study involved evaluation of emulsions for cTACE prepared using two methods: water-in-oil (WiO) and chemotherapeutic-in-oil (CiO). Three emulsions were prepared with each method and obtained after 20, 50, and 100 pumping exchanges. A drop from each final mixture was analyzed via light microscopy (time 1) and after 5, 10, 15, and 20min since the end of preparation. After 20min, all preparations were re-mixed and new drops were re-evaluated. The intra- and inter-operator variabilities were analyzed., Results: The mean droplet diameter decreased non-significantly when the number of pumping exchanges increased and increased significantly over time for both WiO and CiO. The droplets returned to their initial diameters after re-mixing. There were no significant differences in the intra- and inter-operator variabilities (P>0.01)., Conclusions: Any interventional radiologist, regardless of their experience, may prepare these emulsions. These data may represent a set of instructions to standardize cTACE., (Copyright © 2020 Fundación Clínica Médica Sur, A.C. Published by Elsevier España, S.L.U. All rights reserved.)

We describe a case of a patient with anemia referring to our Digestive Endoscopy Unit. Upper GI endoscopy revealed a polypoid lesion with an ulcerated central depression. Histopathological examination of the biopsy specimen taken during endoscopy revealed a gastric metastatic melanoma. The dermatologic inspection failed in finding the primary melanoma. The importance of endoscopic examination in the diagnostic process of metastatic patients with unknown primaries is highlited by this case.

The endothelial to mesenchymal transition (End-MT) can be associated with vascular calcification, by providing mesengenic progenitors. In this study, we investigated a link between End-MT and the osteogenic process and explored the involvement of miR-30a-5p and miR-30d as potential regulators of these processes. End-MT was induced in Human Umbilical Vein Endothelial Cells (HUVEC) through transforming growth factor-β1 (TGF-β1), TGFβ-3 and tumor necrosis factor-α (TNF-α), for 24 h and 6 days. End-MT mediators, mesenchymal and osteo/chondrogenic markers were analyzed through Real-Time PCR, immunofluorescence, flow cytometry and Western Blot. miR-30a-5p and miR-30d over-expression was carried out in HUVEC to explore their effects on End-MT and osteogenic differentiation. HUVEC at 24 h and 6 days gained mesenchymal morphology markers, including matrix metalloproteinase 9 (MMP-9), SLUG, VIMENTIN and α-smooth muscle actin (α-SMA), and a significant migratory potential, notably with TNF-α. After 6 days, the osteo/chondrogenic markers runt-related transcription factor 2 (RUNX-2) and SRY box transcription factor 9 (SOX-9) were upregulated. At this time point, miR-30a-5p and miR-30d decreased. Over-expression of miR-30a-5p and miR-30d affected End-MT mediators and the osteogenic potency in HUVEC, by reducing SLUG, VIMENTIN and RUNX-2. Our data suggest that End-MT represents a key link between inflammation and vascular calcification. Further, miR-30a-5p and miR-30d can regulate both the End-MT and the osteogenic processes, prompting future studies for exploring their potential use as therapeutic targets or biomarkers in vascular diseases.

Background: We report the findings of a single Italian center in the evaluation of renal lesions in deceased donors from 2001 to 2017. In risk evaluation, we applied the current Italian guidelines, which include donors with small (< 4 cm, stage pT1a) renal carcinomas in the category of non-standard donors with a negligible risk of cancer transmission., Methods: From the revision of our registries, 2,406 donors were considered in the Emilia Romagna region of Italy; organs were accepted from 1,321 individuals for a total of 3,406 organs., Results: The evaluation of donor safety required frozen section analysis for 51 donors, in which a renal suspicious lesion was detected by ultrasound. Thirty-two primary renal tumors were finally diagnosed: 26 identified by frozen sections and 6 in discarded kidneys. The 32 tumors included 13 clear cell renal cell carcinomas (RCCs), 6 papillary RCCs, 6 angiomyolipomas, 5 oncocytomas, 1 chromophobe RCC, and 1 papillary adenoma. No cases of tumor transmission were recorded in follow-up of the recipients., Conclusion: Donors with small RCCs can be accepted to increase the donor pool. Collaboration in a multidisciplinary setting is fundamental to accurately evaluate donor candidate risk assessment and to improve standardized protocols for surgeons and pathologists.

Pancreatic adenocarcinoma (PDAC) is the most frequent histological type of malignancy in the pancreas. Extracellular matrix (ECM), plays a critical role during the process of human carcinogenesis and the possible diversity in matricellular proteins composition of ECM may have a significant impact on the clinical course of PDAC. Aim of this paper was to evaluate the expression of three matricellular proteins, including Periostin (POSTN), Tenascin (TNS) and Osteopontin (OPN), in PDAC from long-survival (LS) and non-long survival (NLS) patients. A total of 30 PDAC were analyzed, 15 from patients that survived more than 60 months after surgery (LS) and 15 that died from the disease within 24 (NLS). RNA was extracted and OPN, TNS and POSTN mRNA levels were evaluated by qRT-PCR. LS and NLS samples showed the same type of POSTN and TN isoforms. On the contrary, OPN seems to be preferentially expressed in NLS PDAC. Moreover, OPNb and OPNc isoforms were expressed exclusively in NLS samples. In conclusion, Our data led to hypothesize a possible relationship between the expression of different isoforms of each of these proteins and the clinical outcome of patients with PDAC.

Background and Aims: HCV eradication by direct-acting antiviral agents (DAAs) reduces de novo hepatocellular carcinoma (HCC) incidence in cirrhosis; however, contrasting evidence about beneficial or detrimental effects still exists in patients who have already developed HCC., Methods: we investigated whether sofosbuvir and daclatasvir modulate cell proliferation, invasion capability and gene expression (RNA-seq) in HCC-derived cell lines, hypothesizing possible off-target effects of these drugs. Results observed in HCC cell lines were validated in non-HCC cancer-derived cell lines and a preliminary series of human HCC tissues by qPCR and IHC., Results: DAAs can affect HCC cell proliferation and migration capability by either increasing or reducing them, showing transcriptomic changes consistent with some unexpected drug-associated effects. Off-target gene modulation, mainly affecting ribosomal genes, mitochondrial functions and histones, points to epigenetics and proliferation as relevant events, consistent with matched phenotypic changes. A preliminary validation of in vitro findings was performed in a restricted cohort of HCC patients previously treated with DAAs, with immunohistochemical correlations suggesting DAA-treated HCCs to be more aggressive in terms of migration and epidermal-to-mesenchymal transition., Conclusions: Our findings suggested the possible occurrence of off-target effects ultimately modulating cell proliferation and/or migration and potentially justified previous findings showing some instances of particularly aggressive HCC recurrence as well as reduced incidence of recurrence of HCC following treatment with DAAs.

We evaluated the long-term effects of sirolimus on three different cell in vitro models, cultured in physiological conditions mimicking sirolimus-eluted stent, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. Three cells lines (WPMY-1 myofibroblasts, HT-29 colorectal adenocarcinoma, and U2OS osteosarcoma) were selected and growth in 10 ml of Minimum Essential Medium for 5 weeks with serial dilutions of sirolimus. The number of colonies and the number of cells per colony were counted. As main result, the number of WPMY-1 surviving colonies increased in a dose-dependent manner when treated with sirolimus ( p = 0.0011), while the number of U2OS colonies progressively decreased ( p = 0.0011). The clonal capacity of HT-29 was not modified by the exposure to sirolimus ( p = 0.6679). In conclusion sirolimus showed the well-known cytostatic effect, but with an effect on clonogenic potential different among the different cell types. In the practice, the plaque typology and composition may influence the response to sirolimus and thus the effectiveness of eluted stent., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Atherosclerosis is considered a chronic inflammatory disease of arteries associated with the aging process. Many risk factors have been identified and they are mainly related to life-styles, gene-environment interactions and socioeconomic status. Carotid and coronary artery diseases are the two major atherosclerotic conditions, being the primary cause of stroke and heart attack, respectively. Nevertheless, carotid plaque assumes particular aspects not only for the specific molecular mechanisms, but also for the types of atheroma which may be associated with a better or a worst prognosis. The identification of circulating blood biomarkers able to distinguish carotid plaque types (stable or vulnerable) is a crucial step for the improvement of adequate therapeutic approaches avoiding or delaying endarterectomy in the oldest old individuals (> 80 years), a population predicted to growth in the next years. The review highlights the most recent knowledge on carotid plaque molecular mechanisms, focusing on microRNAs (miRs), as a site-specific accelerated aging within the conceptual framework of Geroscience for new affordable therapies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Malignant aortic tumors (MATs) are exceedingly rare, and a comprehensive review of clinical and therapeutic aspects is lacking in the literature. The aim of this study was to analyze all known cases of MATs and to identify predictors of patients' survival., Methods: All patients diagnosed with an aortic tumor treated in a single center along with all case reports and reviews available in the literature through a specific PubMed search using keywords such as "malignant" and "aorta" or "aortic," "tumor," or "sarcoma" or "angiosarcoma" were analyzed. The tumor's primary location, clinical presentation, histologic subtype, and treatment choice were examined. Survival at 1 year, 3 years, and 5 years and the possible preoperative and operative outcome predictors were evaluated using Kaplan-Meier analysis with a log-rank test and by Cox regression for multivariate analysis., Results: In addition to the 5 cases treated in our center, 218 other cases of MAT were reported in the literature from 1873 to 2017. The mean age of the patients was 60.1 ± 11.9 years, and the male to female ratio was 1.59:1. The median overall survival from diagnosis was 8 (7-9) months; 1-, 3-, and 5-year survival rates were 26%, 7.6%, and 3.5%, respectively. Chronic hypertension (P = .03), fever (P = .03), back pain (P = .01), asthenia (P = .04), and signs of peripheral embolization (P = .007) were significant predictors of a poor result. Histologic subtypes had a different impact on survival, with no statistical significance. Compared with other treatment strategies, combined surgical-medical therapy had the best impact on the median survival rate (surgical-medical, 12 [8-24] months; medical, 8 [5-10] months; surgical 7 [2-16] months; no treatment, 2 [0.5-15] months; P = .001). Analyzing exclusively medical approaches, chemotherapy and radiotherapy had the best impact on median survival rate compared with untreated patients (chemotherapy-radiotherapy, 18 [10-26] months; radiotherapy, 16 [8-20] months; chemotherapy, 10 [7-24] months; no medical treatment, 6 [2-16] months; P = .005); these data were not sustained by multivariate analysis., Conclusions: Aortic tumors are a malignant pathologic condition with a short survival rate after initial diagnosis. Survival is further diminished in the presence of clinical factors such as hypertension, fever, back pain, asthenia, and signs of peripheral embolization. Combined surgical and medical treatment, particularly with chemotherapy and radiotherapy, has shown the highest survival rate., (Copyright © 2019 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

The molecular background of hepatocellular carcinoma (HCC) is highly heterogeneous, and biomarkers predicting response to treatments are an unmet clinical need. We investigated miR-30e-3p contribution to HCC phenotype and response to sorafenib, as well as the mutual modulation of TP53/MDM2 pathway, in HCC tissues and preclinical models. MiR-30e-3p was downregulated in human and rat HCCs, and its downregulation associated with TP53 mutations. TP53 contributed to miR-30e-3p biogenesis, and MDM2 was identified among its target genes, establishing an miR-30e-3p/ TP53 / MDM2 feedforward loop and accounting for miR-30e-3p dual role based on TP53 status. EpCAM , PTEN , and p27 were demonstrated as miR-30e-3p additional targets mediating its contribution to stemness and malignant features. In a preliminary cohort of patients with HCC treated with sorafenib, increased miR-30e-3p circulating levels predicted the development of resistance. In conclusion, molecular background dictates miR-30e-3p dual behavior in HCC. Mdm2 targeting plays a predominant tumor suppressor function in wild-type TP53 contexts, whereas other targets such as PTEN , p27 , and EpCAM gain relevance and mediate miR-30e-3p oncogenic role in nonfunctional TP53 backgrounds. Increased circulating levels of miR-30e-3p predict the development of sorafenib resistance in a preliminary series of patients with HCC and deserve future investigations. SIGNIFICANCE: The dual role of miR-30e-3p in HCC clarifies how the molecular context dictates the tumor suppressor or oncogenic function played by miRNAs., (©2020 American Association for Cancer Research.)

Background: Extended criteria donors (ECD) are widely utilized due to organ shortage, but they may increase the risk of graft dysfunction and poorer outcomes. Hypothermic oxygenated perfusion (HOPE) is a recent organ preservation strategy for marginal kidney and liver grafts, allowing a redirect from anaerobic metabolism to aerobic metabolism under hypothermic conditions and protecting grafts from oxidative species-related damage. These mechanisms may improve graft function and survival., Objective: With this study, we will evaluate the benefit of end-ischemic HOPE on ECD grafts for livers and kidneys as compared to static cold storage (SCS). The aim of the study is to demonstrate the ability of HOPE to improve graft function and postoperative outcomes of ECD kidney and liver recipients., Methods: This is an open-label, single-center randomized clinical trial with the aim of comparing HOPE with SCS in ECD kidney and liver transplantation. In the study protocol, which has been approved by the ethics committee, 220 patients (110 liver recipients and 110 kidney recipients) will be enrolled. Livers and kidneys assigned to the HOPE group undergo machine perfusion with cold Belzer solution (4-10°C) and continuous oxygenation (partial pressure of oxygen of 500-600 mm Hg). In the control group, livers and kidneys undergoing SCS are steeped in Celsior solution and stored on ice. Using the same perfusion machine for both liver and kidney grafts, organs are perfused from the start of the back-table procedure until implantation, without increasing the cold ischemia time. For each group, we will evaluate clinical outcomes, graft function tests, histologic findings, perfusate, and the number of allocated organs. Publication of the results is expected to begin in 2021., Results: Dynamic preservation methods for organs from high-risk donors should improve graft dysfunction after transplantation. To date, we have recruited 108 participants. The study is ongoing, and recruitment of participants will continue until January 2020., Conclusions: The proposed preservation method should improve ECD graft function and consequently the postoperative patient outcomes., Trial Registration: ClinicalTrials.gov NCT03837197; https://clinicaltrials.gov/ct2/show/NCT03837197 ; Archived by WebCite® at http://www.webcitation.org/76fSutT3R., International Registered Report Identifier (irrid): DERR1-10.2196/13922., (©Matteo Ravaioli, Lorenzo Maroni, Andrea Angeletti, Guido Fallani, Vanessa De Pace, Giuliana Germinario, Federica Odaldi, Valeria Corradetti, Paolo Caraceni, Maurizio Baldassarre, Francesco Vasuri, Antonia D'Errico, Gabriela Sangiorgi, Antonio Siniscalchi, Maria Cristina Morelli, Anna Rossetto, Vito Marco Ranieri, Matteo Cescon, Massimo Del Gaudio, Chiara Zanfi, Valentina Bertuzzo, Giorgia Comai, Gaetano La Manna. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 19.03.2020.)

Arterial calcification is an actively regulated process, with different morphological manifestations. Micro-RNAs emerged as potential regulators of vascular calcification; they may become novel diagnostic tools and be used for a finest staging of the carotid plaque progression. The present study aimed at characterizing the different miRNA-mRNA axes in carotid plaques according to their histological patterns of calcification. Histopathological analysis was performed on 124 retrospective carotid plaques, with clinical data and preoperatory angio-CT. miRNA analysis was carried out with microfluidic cards. Real-time PCR was performed for selected miRNAs validation and for RUNX-2 and SOX-9 mRNA levels. CD31, CD68, SMA, and SOX-9 were analyzed by immunohistochemistry. miRNA levels on HUVEC cells were analyzed for confirming results under in vitro osteogenic conditions. Histopathological analysis revealed two main calcification subtypes of plaques: calcific cores (CC) and protruding nodules (PN). miRNA array and PCR validation of miR-1275, miR-30a-5p, and miR-30d indicated a significant upregulation of miR-30a-5p and miR-30d in the PN plaques. Likewise, the miRNA targets RUNX-2 and SOX-9 resulted poorly expressed in PN plaques. The inverse correlation between miRNA and RUNX-2 levels was confirmed on osteogenic-differentiated HUVEC. miR-30a-5p and miR-30d directly correlated with calcification extension and thickness at angio-CT imaging. Our study demonstrated the presence of two distinct morphological subtypes of calcification in carotid atheromatous plaques, supported by different miRNA signatures, and by different angio-CT features. These results shed the light on the use of miRNA as novel diagnostic markers, suggestive of plaque evolution.

Aims: Primary mixed liver cancers (PLCs), combined hepatocellular-cholangiocellular (cHCC-CC) and intermediate-cell carcinomas are rare tumours characterised by different molecular mechanisms. Nestin is a marker of progenitor cells with a promising application in human tumours. The aims of the present paper are (i) to determine the expression of Nestin in mixed PLCs; and (ii) to correlate the PLC immunoprofile with the gene expression in each tumour component., Methods and Results: We selected 28 mixed PLCs, 13 (46.4%) cHCC-CC and 15 (53.6%) intermediate-cell carcinomas. The immunohistochemistry panel consisted of keratin 7, keratin 19, CD56 and Nestin. Next-generation sequencing analysis was performed on 17 cases (27 specimens) using a multi-gene custom panel. The differentiated HCC and CC components of cHCC-CC were negative for Nestin in all cases. The intermediate areas of cHCC-CC were immunoreactive for Nestin in 92.3% of cases, for CD56 in 76.9% and for K7/K19 in all cases. The immunoprofile of the intermediate-cell carcinomas showed 73.3% of cases positive for Nestin and 66.7% for CD56. TP53 and TERT were the most frequently mutated genes (31.3% and 17.6% of samples, respectively). Mutations were also found in IDH1, IDH2, PIK3CA and NRAS genes. Intermediate and HCC areas of cHCC-CC seemed to share the same mutational profile, and both harboured different mutations than the CC component., Conclusions: According to our preliminary data, Nestin was not expressed by hepatocellular or cholangiocellular-cell components, but was expressed by most of the intermediate cells in PLCs, and therefore could be considered in the differential diagnosis of PLCs, together with mutational profile., (© 2019 John Wiley & Sons Ltd.)

Many advances have been made in the imaging diagnosis and in the histopathological evaluation of HCC. However, the classic imaging and histopathological features of HCC are still inadequate to define patient's prognosis. We aimed to find the link between new proposed morphovascular patterns of hepatocellular carcinoma (HCC) and magnetic resonance imaging (MRI) features to identify pre-operatory markers of biologically aggressive HCC. Thirty-nine liver nodules in 22 patients were consecutively identified. Histopathological analysis and immunohistochemistry for CD34 and Nestin were performed to identify the four different HCC morphovascular patterns. MRI was performed using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Three out of four morphovascular HCC patterns showed peculiar MRI features: in particular Pattern D (solid aggressive HCCs with CD34+/Nestin+ new-formed arteries) were isointense on T1-WI in 83% of cases and hyperintense on T2-WI in 50%. Five histologically-diagnosed HCC were diagnosed as non-malignant nodules on MRI due to their early vascularization and low aggressiveness (Pattern A). The comparison between histology and MRI confirms that a subclassification of HCC is possible in a pre-operatory setting. MRI seems to reinforce once more the identity of the different morphovascular HCC patterns and the possibility to pre-operatively identify HCCs with features of biological aggressiveness.

We describe three cases of liver lesions, characterized by a discrepancy between presurgical imaging and histological features, in which the final histological diagnosis was quite different from what the surgeons expected. We present (1) a case of primary liver angiomyolipoma associated with focal nodular hyperplasia, (2) a case of perivascular epithelioid cell tumor, and (3) a case of liver splenosis associated with focal nodular hyperplasia. In all cases, a presurgical diagnosis of hepatocellular adenoma was made. Due to nonspecific clinical and radiological features, these rare liver lesions are often presurgically misdiagnosed, especially in young noncirrhotic patients. The association among different lesions represents one additional diagnostic challenge., Competing Interests: Conflict of interest statement: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2019.)

Background: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results., Methods: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers., Results: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo., Conclusion: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

Case Presentation:: A 55-year-old man with a history of basaloid squamous cell carcinoma of the oropharynx with laterocervical lymph node metastases 6 years before (and treated with chemoradiation) presented with flank pain and hematuria. Computed tomography scan found a renal lesion, with radiological features more suspicious for primitive renal neoplasia. Histopathological and immunohistochemical examination after surgical excision revealed a basaloid squamous cell carcinoma involving renal parenchyma., Conclusion:: Basaloid squamous cell carcinoma is a rare tumor but with a high percentage of distant metastasis, and it is mandatory, also for a general pathologist, to know this disease. Moreover, in a patient with renal metastases, any type of cancer should be taken into account, and this case is emblematic of why the previous medical history is crucial for differential diagnosis.

Objective: Many improvements have been made in diagnosing hepatocellular carcinoma (HCC), but the radiological hallmarks of HCC have remained the same for many years. We prospectively evaluated the imaging criteria of HCC, early HCC and high-grade dysplastic nodules (HGDNs) in patients under surveillance for chronic liver disease, using gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) MRI and diffusion-weighted imaging., Design: Our study population included 420 nodules >1 cm in 228 patients. The MRI findings of each nodule were collected in all sequences/phases. The diagnosis of HCC was made according to the American Association for the Study of Liver Diseases (AASLD) criteria; all atypical nodules were diagnosed using histology., Results: A classification and regression tree was developed using three MRI findings which were independently significant correlated variables for early HCC/HCC, and the best sequence of their application in a new diagnostic algorithm (hepatobiliary hypointensity, arterial hyperintensity and diffusion restriction) was suggested. This algorithm demonstrated, both in the entire study population and for nodules ≤2 cm, higher sensitivity (96% [95% CI 93.5% to 97.6%] and 96.6% [95% CI 93.9% to 98.5%], P<0.001, respectively) and slightly lower specificity (91.8% [95% CI 88.6% to 94.1%], P=0.063, and 92.7% [95% CI 88.9% to 95.4%], P=0.125, respectively) than those of the AASLD criteria. Our new diagnostic algorithm also showed a very high sensitivity (94.7%; 95% CI 92% to 96.6%) and specificity (99.3%; 95% CI 97.7% to 99.8%) in classifying HGDN., Conclusion: Our new diagnostic algorithm demonstrated significantly higher sensitivity and comparable specificity than those of the AASLD imaging criteria for HCC in patients with cirrhosis evaluated using Gd-EOB-DTPA MRI, even for lesions ≤2 cm. Moreover, this diagnostic algorithm allowed evaluating other lesions which could arise in a cirrhotic liver, such as early HCC and HGDN., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Hepatocellular carcinoma (HCC) is the most common primary liver malignant neoplasia. HCC is characterized by a poor prognosis. The need to find new molecular markers for its diagnosis and prognosis has led to a progressive increase in the number of scientific studies on this topic. MicroRNAs (miRNAs) are small non-coding RNA that play a role in almost all main cellular pathways. miRNAs are involved in the regulation of expression of the major tumor-related genes in carcinogenesis, acting as oncogenes or tumor suppressor genes. The aim of this review was to identify papers published in 2017 investigating the role of miRNAs in HCC tumorigenesis. miRNAs were classified according to their role in the main molecular pathways involved in HCC tumorigenesis: (1) mTOR; (2) Wnt; (3) JAK/STAT; (4) apoptosis; and (5) MAPK. The role of miRNAs in prognosis/response prediction was taken into consideration. Bearing in mind that the analysis of miRNAs in serum and other body fluids would be crucial for clinical management, the role of circulating miRNAs in HCC patients was also investigated. The most represented miRNA-regulated pathway in HCC is mTOR, but apoptosis, Wnt, JAK/STAT or MAPK pathways are also influenced by miRNA expression levels. These miRNAs could thus be used in clinical practice as diagnostic, prognostic or therapeutic targets for HCC treatment., Competing Interests: Conflict-of-interest statement: The authors declare no financial or other conflicts of interest related to the submitted manuscript.

The choice of surgical treatment for hepatocellular carcinoma (HCC) depends on several prognostic variables, among which histological features, like microvascular invasion and tumor grade, are well established. This study aims to identify the tissue miRNAs predictive of recurrence after liver resection in "histologically advanced" HCC. We selected 54 patients: 15 retrospective resected patients without recurrence (group A), 19 retrospective resected patients with HCC recurrence (group B), and 20 prospective patients (group C), with 4 recurrence cases. All selected HCC were "histologically advanced" (high Edmondson grade and/or presence of microvascular invasion). A wide spectrum of miRNAs was studied with TaqMan Human microRNA Arrays; qRT-PCR assays were used to validate results on selected miRNAs; immunohistochemistry for IGF2 was applied to study the mechanism of miR-483-3p. As a result, a significant differential expression between group A and B was found for 255 miRNAs. Among them we selected miR-483-3p and miR-548e (P<0.001). As a single variable (group C), HCC with miR-483-3p downregulation (mean fold increase 0.21) had 44.4% of recurrence cases; HCC with miR-483-3p upregulation (mean fold increase 5.94) showed no recurrence cases (P=0.011). At immunohistochemistry (group C), the HCC with loss of cytoplasmic IGF2 expression showed a down-regulation of miR-483-3p (fold increase 0.57). In conclusion, in patients with "histologically advanced" HCC, the analysis of specific tissue miRNAs (particularly miR-483-3p) could help identify the recurrence risk and choose which treatment algorithm to implement (follow-up, resection or transplantation). This could have an important impact on patient survival and transplantation outcome, improving organ allocation., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Background: Pancreatic adenocarcinoma (PDAC) is one of the deadliest human malignancies. Although surgery is currently the only effective treatment for PDAC, most patients survive less than 20 months after tumor resection., Objective: The primary goal was to investigate alterations in KRAS, TP53, SMAD4 and CDKN2A/p16 in tumors from patients with exceptionally long survival after surgery., Methods: Tumors from 15 patients with PDAC that survived more than 55 months after surgery ("LS") were analyzed for KRAS, TP53, IDH1, NRAS and BRAF using next-generation sequencing. SMAD4 and CDKN2A/p16 was tested using immunohistochemistry. MGMT promoter methylation was investigated., Results: Tumors from "LS" have a lower prevalence of KRAS and TP53 mutations and had more frequently SMAD4 retained expression, if compared with that of patients died within 24 months from surgery. The survival of patients with wild-type KRAS and TP53 tumors was more than twice longer than that of patients bearing KRAS and TP53 mutations (90.2 vs. 41.1 months). Patients with KRAS wild-type tumors and that retained SMAD4 expression had a survival twice longer than cases with alterations in both genes (83.8 vs. 36.7 months). Eleven tumors (39.3%) showed MGMT methylation., Conclusions: Our data indicate that absence of KRAS, TP53 and SMAD4 genetic alterations may identify a subset of pancreatic carcinomas with better outcome.

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, due to a high recurrence rate after curative treatments and a drug resistance phenotype. In this scenario, the identification of innovative and effective therapeutic strategies is an unmet clinical need. The safety and efficacy of microRNA (miRNA) mediated approaches in preclinical models and clinical trials have been widely described in cancer. MicroRNA-199a downregulation is a common feature of HCC where its reduced expression contributes to mTOR and c-Met pathways activation. Notch1 activation is also a common event in HCC, influencing epithelial-to-mesenchymal transition, tumor invasion and recurrence at least in part through E-cadherin regulation. Here we identified a negative correlation between miR-199a-3p and Notch1 or E-cadherin protein levels in HCC patients and demonstrated that miR-199a-3p regulates E-cadherin expression through Notch1 direct targeting in in vitro models. Moreover, we showed that a strong correlation exists between miR-199a-5p and miR-199a-3p in HCC specimens and that miR-199a-5p contributes to E-cadherin regulation as well, underlying the complex network of interaction carried out by miR-199a and its influence on tumor aggressiveness. In conclusion, our findings suggest the restoration of miR-199a-3p physiologic levels as a possible therapeutic strategy for the treatment of HCC., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Aims: The aims of this study were to: validate the use of the immunohistochemical (IHC) markers glutamine synthetase (GS), glypican-3 (GPC3), heat shock protein-70 (HSP70) and enhancer of zeste homologue 2 (EZH2) in liver biopsies for the differential diagnosis between small hepatocellular carcinoma (HCC) and non-neoplastic liver nodules, with special attention to <10-mm nodules; and assess the actual sensitivity and specificity of the single markers, and their combination, in needle biopsies., Methods and Results: One hundred liver nodules, i.e. 66 HCCs and 34 non-neoplastic nodules, were prospectively collected from 43 consecutive orthotopic liver transplantation patients, and subjected to 'backtable' needle biopsies directly on surgical specimens. IHC evaluation was semi-automatically performed with a Benchmark Ultra immunostainer. The morphological and IHC diagnosis in surgical specimens was considered to be the gold standard. GS, GPC3, HSP70 and EZH2 showed 16.6%, 10.7%, 28.8% and 62.1% decreases in sensitivity, respectively, from surgical specimen to needle biopsy. Higher decreases were observed in <10-mm nodules. In 18 HCCs with no morphological diagnostic features of malignancy in biopsies, GPC3 or GS were positive in 16; in seven HCCs, neither morphology nor IHC evaluation ruled out the differential diagnosis made on the basis of needle biopsy., Conclusions: We present for the first time a direct comparison between surgical specimens and needle biopsies to confirm the usefulness and reproducibility of the most widely used antibodies for the diagnosis of small liver nodules. Our results support the use of IHC evaluation in biopsies for the diagnosis of small liver lesions, although the IHC panel could also give negative results in the presence of obvious HCC, and the possibility of false positives should always be considered., (© 2017 John Wiley & Sons Ltd.)

Background: Carotid plaques with a high degree of calcification are usually considered at low embolic risk. However, since a precise evaluation of the extent of calcification is not possible preoperatively through duplex ultrasound and postoperatively by conventional histological examination due to the decalcification process, the relationship between the amount of calcium involvement and plaque vulnerability has not been evaluated yet. This study aims to correlate the extent of carotid plaque calcification with clinical, radiological, and histological complications., Methods: Symptomatic and asymptomatic consecutive patients submitted to carotid endarterectomy between January to December 2014 were included in the study. The amount of carotid calcification was assessed at preoperative computed tomography (CT) through measurement of thickness and circumferential calcium extension and graded from 1 to 8 accordingly (Babiarz classification). Patients were then categorized into 2 groups (low-level group: grade 1-5; high-level group: grade 6-8) and correlated with clinical characteristics and ipsilateral cerebral ischemic lesions at CT. Vulnerability of the plaque was assessed histologically according with American Heart Association (AHA) Classification. Results were overall blindly correlated., Results: One hundred five patients (81% male; age: 73 ± 8 years) were enrolled in the study. Forty (38%) were symptomatic and 43 (40%) had an ipsilateral focal lesion at preoperative cerebral CT. Thirty-six (38%) patients had high-level carotid calcification degree at CT scan. At histological analysis, 56 (56%) plaques were considered complicated (AHA type VI). Patients with high-level and low-level carotid calcification had similar epidemiological risk factors, preoperative neurological symptoms, and histological complications (17% vs. 15%, P = 0.76 and 50% vs. 55%, P = 0.62, respectively). The high-level calcification group showed a significantly higher incidence of ipsilateral cerebral lesions at preoperative CT (56% vs. 32%, P = 0.01)., Conclusions: A high level of calcification of the carotid plaque is not necessarily associated with lower vulnerability: the incidence of preoperative neurological symptoms and histological complications is similar in patients with and without extensive carotid plaque calcification. Cerebral ischemic lesions may be even more frequent in the presence of highly calcified plaques., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The surgical treatment of radio-induced carotid stenosis (RICS) is challenging and burdened with high risk of complications. Carotid stenting (CAS) may be a valid alternative, but better approach is still not defined. Two approaches have been assessed in this an observational monocentric study, focusing on perioperative and follow-up complications., Methods: From 2005 to 2013, data on patients treated for extracranial carotid stenosis with previous radiotherapy (RT) for head or neck tumor were prospectively collected according to the procedure performed (open repair [OR], endovascular stenting [CAS]). Patient demographics, clinical risk factors, organ of cancer origin, elapsed time from RT, duplex ultrasound (DUS) and clinical presentation of carotid stenosis were preoperatively gathered. Primary outcomes were technical success, 30-day mortality, transient ischemic attack (TIA)/stroke and myocardial infarction (MI). In OR group, laterocervical hematoma and cranial nerve injury were evaluated and, in CAS group, arterial access site complications were considered as well. Secondary outcomes were the mortality, TIA/stroke, restenosis and reintervention during follow-up., Results: Fifteen RICS were treated in 12 patients (M/F=9/3; median age: 71 years, range: 51-80). OR was performed to treat 8 RICS in 5 patients (M/F=3/2; median age: 65, range: 50-76) and CAS to treat 7 RICS in 7 patients (M/F=6/1; median age: 75, range 59-80). In OR group, all patients were asymptomatic and all plaques were hypoechogenic at DUS with histological features of vulnerable plaque. Technical success was 100%. Thirty-day mortality was 12.5% (1 death for upper airways complication in patient with previous neck surgery). TIA/stroke and MI rate were 0% and cranial nerve injury 12.5%. In CAS group, carotid stenosis was symptomatic in 2(28.6%) cases, all plaques resulted hypoechogenic at DUS. Technical success was 100%. Thirty-day mortality was 0%. TIA occurred in 4 (57.1%) patients. No stroke, MI or access site hematoma occurred in this group. Mean follow-up was 35.5±18.2 months. At 12 and 36 month, freedom from neurological events, restenosis and reintervention was 100%. At 1 and 3 years, survival was 91.7% and 81.5%, respectively. No patient died for carotid-related or cardiovascular causes., Conclusions: According with the plaque morphology and the high rate of cerebrovascular complications of CAS, OR should be suggested as the first choice for treatment of RICS in the majority of cases. In patients with previous neck surgery, assessment of airways is necessary before OR to avoid respiratory complications.

Background: miRNA deregulation and vascular modifications constitute promising predictors in the study of hepatocellular carcinoma (HCC). In the literature, the relative miRNA abundance in HCC is usually determined using as control non-matched tumoral tissue, healthy liver, or cirrhotic liver. However, a common standard RNA control for the normalization toward the tissue gene expression was not settled yet., Aim: To assess the differences existing in the quantitative miRNA gene expression in HCC on tissue according to two different liver controls., Methods: A wide array of miRNAs was analyzed on 22 HCCs arisen in cirrhotic and non-cirrhotic livers by means of microfluidic cards. Control samples included total RNA extracted from healthy and cirrhotic livers. Immunohistochemistry for CD34 and Nestin was performed to assess the pattern of intratumoral vascular modifications., Results: Six miRNAs were deregulated in HCCs using either controls: miR-532, miR-34a, miR-93, miR-149#, miR-7f-2#, and miR-30a-5p. Notably, the miRNA expression changed significantly between HCCs arisen in cirrhotic and non-cirrhotic livers, according to the control used for normalization. Different miRNA profiles were found also in HCCs with different vascular patterns, according to the control used for normalization., Conclusions: Our data confirm that the choice of the methodology, and particularly the control used for normalization, represents the main concern in miRNA evaluation, particularly in a heterogeneous model such as liver pathology. Still we observed the deregulation of some common miRNAs as promising in HCC cancerogenesis and progression. A standardized control will be a crucial achievement to compare miRNA expression among different laboratories.

Penile metastasis is an extremely rare event and mainly originate from primary pelvic tumor sites such us urinary bladder, gastro-intestinal tract and prostate and more rarely from respiratory system, bone tumors and melanoma. Here we describe the unusual presentation of two bladder urothelial cancer metastatic to the penis with no relevant clinical symptoms. Namely, a 69 years-old man with a warthy lesions of the foreskin and the glans misunderstood for a condylomata that at histological and immunohistochemical analysis showed a bladder urothelial carcinoma; and a 71 years-old man with reddish skin lesion of the glans, a previous history of bladder and urethral carcinoma and histological pagetoid spread of urothelial cancer to the glans. Recurrent bladder urothelial carcinoma is usually a visceral disease that rarely presents as a superficial asymptomatic skin lesion. The two reported cases were asymptomatic superficial penis metastases with a relatively slow growth and a fairy good prognosis after conservative surgical approach. Accurate clinical examination of the penis is mandatory for males with history of bladder cancer., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Epidemiological and biological evidence indicates a causal relationship between the presence of proliferative atrophic lesions and the development of prostatic intraepithelial neoplasia (PIN) and prostate cancer. The presence of inflammatory and atrophic lesions of the prostate is widely underestimated and they are not generally mentioned in pathology reports. We performed a histopathological concordance study among eight genitourinary specialists and seven generalist pathologists, using 116 histological slides of prostate lesions, including proliferative atrophic lesions, PIN, and cancer. The overall agreement between all possible pairs of reviewers was 80% for prostate cancer, 67% for PIN, and 49% for proliferative atrophic lesions. When using as gold standard the assessment of a single genitourinary pathologist, the mean agreement percentage increased to 97% for prostate cancer, 92% for PIN, and 72% for proliferative atrophic lesions.

To understand why livers from aged donors are successfully used for transplants, we looked for markers of liver aging in 71 biopsies from donors aged 12-92 years before transplants and in 11 biopsies after transplants with high donor-recipient age-mismatch. We also assessed liver function in 36 age-mismatched recipients. The major findings were the following: (i) miR-31-5p, miR-141-3p, and miR-200c-3p increased with age, as assessed by microRNAs (miRs) and mRNA transcript profiling in 12 biopsies and results were validated by RT-qPCR in a total of 58 biopsies; (ii) telomere length measured by qPCR in 45 samples showed a significant age-dependent shortage; (iii) a bioinformatic approach combining transcriptome and miRs data identified putative miRs targets, the most informative being GLT1, a glutamate transporter expressed in hepatocytes. GLT1 was demonstrated by luciferase assay to be a target of miR-31-5p and miR-200c-3p, and both its mRNA (RT-qPCR) and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively; (iv) miR-31-5p, miR-141-3p and miR-200c-3p expression was significantly affected by recipient age (older environment) as assessed in eleven cases of donor-recipient extreme age-mismatch; (v) the analysis of recipients plasma by N-glycans profiling, capable of assessing liver functions and biological age, showed that liver function recovered after transplants, independently of age-mismatch, and recipients apparently 'rejuvenated' according to their glycomic age. In conclusion, we identified new markers of aging in human liver, their relevance in donor-recipient age-mismatches in transplantation, and offered positive evidence for the use of organs from old donors., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Background: Calcifications of atherosclerotic plaques represent a controversial issue as they either lead to the stabilization or rupture of the lesion. However, the cellular key players involved in the progression of the calcified plaques have not yet been described. The primary reason for this lacuna is that decalcification procedures impair protein and nucleic acids contained in the calcified tissue. The aim of our study was to preserve the cellular content of heavily calcified plaques with a new rapid fixation in order to simplify the study of calcifications., Methods: Here we applied a fixation method for fresh calcified tissue using the Carnoy's solution followed by an enzymatic tissue digestion with type II collagenase. Immunohistochemistry was performed to verify the preservation of nuclear and cytoplasmic antigens. DNA content and RNA preservation was evaluated respectively with Feulgen staining and RT-PCR. A checklist of steps for successful image analysis was provided. To present the basic features of the F-DNA analysis we used descriptive statistics, skewness and kurtosis. Differences in DNA content were analysed with Kruskal-Wallis and Dunn's post tests. The value of P < 0.05 was considered significant., Results: Twenty-four vascular adult tissues, sorted as calcified (14) or uncalcified (10), were processed and 17 fetal tissues were used as controls (9 soft and 8 hard). Cells composing the calcified carotid plaques were positive to Desmin, Vimentin, Osteocalcin or Ki-67; the cellular population included smooth muscle cells, osteoblasts and osteoclasts-like cells and metakaryotic cells. The DNA content of each cell type found in the calcified carotid artery was successfully quantified in 7 selected samples. Notably the protocol revealed that DNA content in osteoblasts in fetal control tissues exhibits about half (3.0 ng) of the normal nuclear DNA content (6.0 ng)., Conclusion: Together with standard histology, this technique could give additional information on the cellular content of calcified plaques and help clarify the calcification process during atherosclerosis.

Background: The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine., Methods: Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score., Results: Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77)., Conclusion: hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting., Implications for Practice: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting., (©AlphaMed Press.)

Aims: In this paper we aimed to analyse the typology and the phenotype of the different vascular modifications in human hepatocellular carcinomas (HCCs) with a new immunomorphological and gene expression approach. We also attempted to correlate these modifications with the histological parameters of tumour aggressiveness and the surrounding liver parenchyma., Methods: Ninety-six HCCs (from 80 patients) were retrospectively enrolled, 46 occurring in non-cirrhotic livers, and 50 in livers transplanted for cirrhosis. Histopathological analysis, immunohistochemistry for CD34, Nestin and WT1 and RT-PCR for Nestin, transforming growth factor-β1 (TGFβ1) and insulin-like growth factor 1 (IGF1R) mRNA were performed in all nodules., Results: By correlating the CD34 and Nestin immunoreactivity in HCC vasculature with the tumorous architecture, we identified four vascular patterns (named from 'a' to 'd'). Each of them was characterised by different expressions of TGFβ1 and IGF1R mRNA. Pattern a showed CD34-positive/Nestin-negative sinusoids, and was prevalent in microtrabecular lesions. Pattern b showed similar morphology and architecture as pattern a, but with Nestin-positive sinusoids and a significant 'boost' in IGF1R and TGFβ1 mRNAs. In patterns c and d a progressive sinusoid loss and a gain of newly formed arterioles were seen. Notably, HCCs with pattern a arose more frequently in cirrhosis (p=0.024), and showed lower incidence of microvascular invasion (p=0.002) and infiltration (p=0.005) compared with HCCs with other patterns., Conclusions: Although future studies are surely required, the identification of different vascular profiles in HCCs from cirrhotic and non-cirrhotic livers may help clarify the relationship between HCC progression and aggressiveness., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Fibromuscular dysplasia (FMD) of the splenic artery is a rare underdiagnosed condition. Here, we report two cases of FMD affecting the splenic artery: one alone and one concomitantly with the renal artery. Histology revealed fibromuscular thickening of the media layer alternating with a circumferential calcification of the whole artery thickness. Ultrastructurally, FMD showed matrix vesicles and dense bodies in the extracellular matrix. A diagnosis of FMD with calcification was made. This is the first report to document circumferential lamellar calcifications alternating with the more typical fibrotic medial areas in the rare FMD localized to splenic artery.

The proteasome is the core of the ubiquitin-proteasome system and is involved in synaptic protein metabolism. The incorporation of three inducible immuno-subunits into the proteasome results in the generation of the so-called immunoproteasome, which is endowed of pathophysiological functions related to immunity and inflammation. In healthy human brain, the expression of the key catalytic β5i subunit of the immunoproteasome is almost absent, while it is induced in the epileptogenic foci surgically resected from patients with pharmaco-resistant seizures, including temporal lobe epilepsy. We show here that the β5i immuno-subunit is induced in experimental epilepsy, and its selective pharmacological inhibition significantly prevents, or delays, 4-aminopyridine-induced seizure-like events in acute rat hippocampal/entorhinal cortex slices. These effects are stronger in slices from epileptic vs normal rats, likely due to the more prominent β5i subunit expression in neurons and glia cells of diseased tissue. β5i subunit is transcriptionally induced in epileptogenic tissue likely by Toll-like receptor 4 signaling activation, and independently on promoter methylation. The recent availability of selective β5i subunit inhibitors opens up novel therapeutic opportunities for seizure inhibition in drug-resistant epilepsies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) with multilineage potential and anti-inflammatory property can be isolated from different human tissues, representing promising candidates in regenerative medicine. Despite the common criteria of characterization, many factors contribute to MSC heterogeneity (i.e., tissue origin, coexistence of cell subsets at different stage of differentiation, epigenetic) and no standard methods have been approved to characterize MSCs in cell culture., Aim: The present study aimed to test whether MSCs resist adverse chemical and physical culture conditions, surviving MSC subpopulations are endowed with the stemness abilities; to characterize MMP expression in AAA-MSCs under the adverse experimental conditions., Methods and Results: MSCs enzymatically isolated from human abdominal aortic aneurysm (AAA-MSCs) were exposed to media acidification, hypoxia, starving, drying and hypothermia through the following strategies: (1) low-density seeding in closed flasks; (2) exposure to a chemical hypoxia inducer, cobalt chloride; (3) exposure to a dry environment with growing medium deprivation and culture at 4°C. None of these conditions affected MSC viability and stemness profile, as evidenced by NANOG, OCT-4, and SOX-2 mRNA expression in surviving cells. A significant MMP-9 decrease, especially when AAA-MSCs were exposed to hypothermia, was associated with stress resistant stem cells., Conclusions: AAA-MSCs survive to extremely adverse culture conditions, keeping their morphology and stemness features. Besides MMP-9 role in pathological tissue remodeling, this protease may be related to MSC survival. Future studies on MSCs derived from other tissues will be necessary to refine our culture protocol, which can represent an empirical method to demonstrate MSC stemness, with potential implications for their clinical use.

Sarcopenia, the progressive loss of muscle mass and strength, is a phenomenon characterizing human aging whose etiology is still not clear. While there is increasing evidence for the influence of inter-muscular adipose tissue infiltration in the development of sarcopenia, much less is known about a possible role for intra-muscular triglycerides (IMTG). IMTG accumulate in form of lipid droplets decorated by proteins such as Perilipins (Plins). In skeletal muscle the most abundant are Plin2 and Plin5. In this study we compared the expression of these two Plins in Vastus lateralis muscle samples of subjects of different age, both healthy donors (HD) and patients with limited lower limb mobility (LLMI). These latter are characterized by a condition of chronic physical inactivity. Plin2 expression resulted higher in old age for both HD and LLMI patients, while Plin5 slightly decreased only in LLMI patients. Moreover, in these patients, only Plin2 was associated with the decrease of muscle strength and the expression of factors related to muscle atrophy (MuRF1, Atrogin and p53). An increase in Plin2 and a concomitant decrease of Plin5 was also observed when we considered animal model of disuse-induced muscle atrophy. As a whole, these data indicate that Plin2 and Plin5 have a different expression pattern during muscle aging and inactivity, and only Plin2 appears to be associated with functional alterations of the muscle.

Background: Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group showed that Nestin-positive intraplaque neovessels correlated with histological complications. The aim of the present work is to evaluate the relationship between neoangiogenesis, plaque morphology, and clinical instability of the plaque., Materials and Methods: Seventy-three patients (53 males and 20 females, mean age 71 years) were consecutively enrolled. Clinical data and 14 histological variables, including intraplaque hemorrhage and calcifications, were collected. Immunohistochemistry for CD34 and Nestin was performed. RT-PCR was performed to evaluate Nestin mRNA (including 5 healthy arteries as controls)., Results: Diffusely calcified plaques (13/73) were found predominantly in females (P = 0.017), with a significantly lower incidence of symptoms (TIA/stroke (P = 0.019) than noncalcified plaques but with the same incidence of histological complications (P = 0.156)). Accordingly, calcified and noncalcified plaques showed similar mean densities of positivity for CD34 and Nestin. Nestin density, but not CD34, correlated with the occurrence of intraplaque hemorrhage., Conclusions: Plaques with massive calcifications show the same incidence of histological complications but without influencing symptomatology, especially in female patients, and regardless of the amount of neoangiogenesis. These results can be applied in a future presurgical identification of patients at major risk of developing symptoms.

Aims: To investigate the immunohistochemical expression of dyskerin, a biomarker involved in ribosome production and telomere maintenance, in human fetal, adult and neoplastic bile ducts, and possible correlations with cholangiocarcinoma aggressiveness., Methods and Results: Sixty consecutive intrahepatic cholangiocarcinomas were collected and used for tissue microarray construction (total: 176 cores); clinical data and follow-up were also collected. Five fetal and 10 normal adult livers were included as controls. Automated immunohistochemistry for dyskerin, p53, and Ki67, and nucleolar silver staining, were performed. In normal livers, dyskerin expression was negative in smaller bile ducts (mean 44.8 μm) and positive in bile ducts of larger diameter (mean 116.1 μm; P < 0.001). Expression was positive in 56.7% of cholangiocarcinomas, and correlated with p53 mutation (P = 0.008) and a higher proliferative (Ki67) index (P = 0.003), which were included as markers of tumour aggressiveness. Finally, dyskerin-positive cholangiocarcinomas showed a negative trend in disease-free survival (P = 0.078) on univariate analysis., Conclusions: The non-neoplastic biliary tree seems to progressively lose dyskerin expression from the major branches to the peripheral portal bile ducts. Similarly, intrahepatic cholangiocarcinomas showed two patterns of dyskerin expression, and the dyskerin-positive phenotype seemed to characterize more aggressive cholangiocarcinomas., (© 2014 John Wiley & Sons Ltd.)

Introduction: Neoangiogenesis is crucial for the progression and vulnerability of atheromasic lesions. Since adult vasa vasorum, which represent the neoangiogenetic burden of healthy arteries, constitutively express Nestin and Wilms Tumor (WT1), the aims of the present study are: i) to describe and quantify Nestin and WT1 in plaque neovessels; ii) to investigate the relationship between neovessel phenotype and plaque instability., Methods: We prospectively evaluated 49 consecutive carotid endarterectomy specimens. Histopathological characteristics were separately collected, particularly the intraplaque histological complications. Immunohistochemistry was carried out for CD34, Nestin and WT1; the density of positivity was evaluated for each marker. RT-PCR was performed to assess Nestin and WT1 mRNA levels on the first 10 plaques and on 10 control arteries., Results: Six (12.2%) plaques showed no neoangiogenesis. In the others, the mean immunohistochemical densities of CD34, Nestin, and WT1-positive structures were 41.88, 28.84 and 17.68/mm2. Among the CD34+ neovessels, 68% and 42% expressed Nestin and WT1 respectively, i.e., nearly 36% of the neovessels resulted to be Nestin+/WT1-. Furthermore, complicated plaques (n=30) showed significantly more CD34 and Nestin-positive vessels than uncomplicated plaques (n=13; P=0.045 and P=0.009), while WT1 was not increased (P=0.139). RT-PCR confirmed that WT1 gene expression was 3-fold lower than Nestin gene in plaques (p=0.001)., Conclusions: Plaque neoangiogenesis shows both a Nestin+/WT1- and a Nestin+/WT1+ phenotype. The Nestin+/WT1- neovessels are significantly more abundant in complicated (vulnerable) plaques. The identification of new transcription factors in plaque neoangiogenesis, and their possible regulation, can open new perspectives in the therapy of vulnerable plaques.

Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of β5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Aim: To define the histopathological features predictive of post-transplant hepatocellular carcinoma (HCC) recurrence after transarterial chemoembolization, applicable for recipient risk stratification., Methods: We retrospectively reviewed the specimens of all suspicious nodules (total 275) from 101 consecutive liver transplant recipients which came to our Pathology Unit over a 6-year period. All nodules were sampled and analyzed, and follow-up data were collected. We finally considered 11 histological variables for each patient: total number of nodules, number of viable nodules, size of the major nodule, size of the major viable nodule, occurrence of microscopic vascular invasion, maximum Edmondson's grade, clear cell/sarcomatous changes, and the residual neoplastic volume. Survival data were computed by means of the Kaplan-Meier procedure and analyzed by means of the Cox proportional hazards model. The multivariate linear regression and a k-means cluster analysis were also used in order to compute the standardized histological score., Results: The total number of nodules, the residual neoplastic volume (the total volume of all evaluated nodules minus the necrotic portion) and the microvascular invasion entered the Cox multivariate hazard model with HCC recurrence as dependent variable. The histological score was therefore computed and a cluster analysis sorted recipients into 3 risk groups, with 3.3%, 18.5% and 53.8% respectively of tumor recurrence rates and 1.6%, 11.1% and 38.5% of tumor-related mortality respectively at the end of follow-up., Conclusion: The histological score allows a reliable stratification of HCC recurrence risk, especially in those recipients found out to be beyond the Milan criteria after orthotopic liver transplantation (OLT).

The aim of the present study is to describe the histological and mutational characteristics of a series of both large and small bowel adenocarcinomas in patients with Crohn's disease from a tertiary referral centre of inflammatory bowel disease. Bowel adenocarcinoma was diagnosed in 11 (1.7%) of 660 consecutive patients submitted to surgery for histologically proven Crohn's disease in 5 years. The following data were collected: tumour site, stage and grade, intracellular/extracellular mucin, lymphovascular invasion, immunohistochemistry for keratin 7, keratin 20 and CDX-2, mutation analyses of KRAS, B-RAF, PI3K and microsatellite instability. A strong predominance of male gender was observed (10/11). Four (36.4%) adenocarcinomas arose in the small bowel, five (45.4%) in the anus/rectum, and two (18.2%) in anastomosis. Furthermore, all cases of anorectal adenocarcinoma showed >50% of extracellular mucin, with associated KRAS mutations in three of five. No influence in cancer incidence by infliximab therapy was observed.Our series, one of the largest on the topic with immunomorphological and molecular deepening, showed that bowel adenocarcinomas in Crohn's disease have an aggressive behaviour and a strong predominance of extracellular mucin. In surgical specimens from Crohn's disease patients, mucinous-looking anal fistulas and ileal areas of adhesion/retraction should always be extensively sampled.

Although the morphological features of hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) have been well established in the last decades, the differential diagnosis still represents a challenge for the pathologist, especially early recurrent hepatitis C vs mild acute cellular rejection. The present review focuses on the role of the pathologist and the pathology laboratory in the management of recipients with recurrent hepatitis C, the usefulness of early and late post-OLT liver biopsies, and the potential role of ancillary techniques (immunohistochemistry and reverse transcription-polymerase chain reaction, RT-PCR). The English literature on the topic is reviewed, focusing on the histopathology, the immunohistochemistry and the use of RT-PCR on HCV-positive post-OLT biopsies. The different histopathological illustrations of early and chronic recurrent hepatitis C are presented, with special focus on the main differential diagnoses and those features with prognostic relevance (cholestasis above all). The usefulness of ancillary techniques are discussed, especially HCV RNA quantitation by RT-PCR. Finally, the usefulness of long-term protocol biopsies is addressed: their usefulness for the study of allograft disease progression is clear, but their meaning in the long term is still debated. The significance of plasma cell infiltrate in HCV-positive allografts, the prognostic weight of graft steatosis, and the impact of donor age in recurrent hepatitis C also represent additional open issues.

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only β5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.