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Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

BackgroundA new strain of West Nile virus lineage 1 (WNV-1) emerged in the Veneto Region, northern Italy, in 2021, 8 years after the last WNV-1 outbreak in Italy. The virus, which co-circulates with West Nile virus lineage 2 (WNV-2), has become endemic in the region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred.MethodsComparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from 16 May to 21 August 2022, to determine if the high number of WNND cases was associated with WNV-1.Results222 human cases of WNV infection were confirmed by molecular testing, including 103 with West Nile fever (WNF) and 119 with WNND. The WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. In addition, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that WNND patients were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% confidence interval: 1.54–8.24; P = 0.0043). As observed in humans, in wild birds, WNV-1 had a higher infection rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2.ConclusionsComparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1-infected patients might have an increased risk to develop severe disease.

Neurological manifestations are common in COVID-19, the disease caused by SARS-CoV-2. Despite reports of SARS-CoV-2 detection in the brain and cerebrospinal fluid of COVID-19 patients, it’s still unclear whether the virus can infect the central nervous system, and which neuropathological alterations can be ascribed to viral tropism, rather than immune-mediated mechanisms.Here, we assess neuropathological alterations in 24 COVID-19 patients and 18 matched controls who died due to pneumonia / respiratory failure. Aside from a wide spectrum of neuropathological alterations, SARS-CoV-2-immunoreactive neurons were detected in specific brainstem nuclei of 5 COVID-19 subjects. Viral RNA was also detected by real-time RT-PCR. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation within affected brainstem regions, and was higher when compared to controls. While the results of this study support the neuroinvasive potential of SARS-CoV-2, the role of SARS-CoV-2 neurotropism in COVID-19 and its long-term sequelae require further investigation.

Neurological manifestations are common in COVID-19, the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Despite some reports of detection of SARS-CoV-2 in the brain and cerebrospinal fluid of patients with COVID-19, it is still unclear whether the virus can infect the central nervous system (CNS), and which neuropathological alterations can be ascribed to viral tropism rather than immune-mediated mechanisms. Available autopsy reports are often conflictual, reporting a heterogeneous spectrum of neuropathological alterations, while viral proteins and RNA were detected only in sparse cells within the brainstem; furthermore, there appears to be no consistent correlation between viral invasion and neuropathological alterations to date. Here, we assess the neuropathological changes occurring in 24 patients who died following a diagnosis of SARS-CoV-2 infection in Italy during the COVID-19 pandemic (from March 2020 to May 2021) and 10 age-matched controls with comparable medical conditions. Aside from a wide spectrum of neuropathological alterations, including astrogliosis, sparse lympho-monocytic infiltrations and several instances of small vessel thromboses, we identified 5 COVID-19 subjects presenting SARS-CoV-2-immunoreactive neurons within the boundaries of the solitary tract nucleus, nucleus ambiguus and substantia nigra in the brainstem. In these subjects, viral RNA was also detected by real-time RT-PCR. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation targeting mainly the medulla oblongata and the mesencephalon, and was significantly higher when compared to controls. However, SARS-CoV-2 direct invasion did not appear to correlate with the severity of neuropathological changes. The results of this study support the neuroinvasive potential of SARS-CoV-2 by demonstrating the presence of viral proteins and genome sequences within the human brainstem, but further investigation is required to identify the link between invasion and consequent neuropathological alterations in humans.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that belong to the Japanese encephalitis virus serocomplex within the genus Flavivirus. Due to climate change and the expansion of mosquito vectors, flaviviruses are becoming endemic in increasing numbers of countries. WNV infections are reported with symptoms ranging from mild fever to severe neuro-invasive disease. Until now, only a few USUV infections have been reported in humans, mostly with mild symptoms. The serological diagnosis and differentiation between flavivirus infections, in general, and between WNV and USUV, in particular, are challenging due to the high degree of cross-reacting antibodies, especially of those directed against the conserved fusion loop (FL) domain of the envelope (E) protein. We have previously shown that E proteins containing four amino-acid mutations in and near the FL strongly reduce the binding of cross-reactive antibodies leading to diagnostic technologies with improved specificities. Here, we expanded the technology to USUV and analyzed the differentiation of USUV- and WNV-induced antibodies in humans. IgG ELISAs modified by an additional competition step with the heterologous antigen resulted in overall specificities of 93.94% for WNV Equad and 92.75% for USUV Equad. IgM antibodies against WNV could be differentiated from USUV IgM in a direct comparison using both antigens. The data indicate the potential of the system to diagnose antigenically closely related flavivirus infections.

Introduction: West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus, which is endemic in many countries, especially in Europe and in North America, where the virus has increased its activity in the recent years. No vaccines nor antiviral drugs are available for the prevention and treatment of WNV infection in humans.Areas covered: This review article describes viral and host targets that have been addressed by anti-WNV drug discovery studies and summarizes the most relevant anti-WNV candidate compounds identified so far, focusing on those showing antiviral efficacy in in vivo models and broad-spectrum anti-flavivirus activity.Expert opinion: The most promising anti-WNV drug candidates target conserved enzymatic motifs in viral NS3 protease and NS5 polymerase and are effective against different flaviviruses. Targeting host factors required for viral infection and replication and modulation of host innate antiviral response are also promising approaches, which may lead to the development of compounds with broad-spectrum antiviral activity, a desirable feature for an antiviral drug.

Background In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy. Methods Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood. Results The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014–2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site. Conclusions Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.

Background Usutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years. Aim To describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018. Methods From 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel. Results Eight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2. Conclusions Clinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

Generation of human induced pluripotent stem cells (hiPSCs) and their differentiation into a variety of cells and organoids have allowed setting up versatile, non-invasive, ethically sustainable, and patient-specific models for the investigation of the mechanisms of human diseases, including viral infections and host&ndash, pathogen interactions. In this study, we investigated and compared the infectivity and replication kinetics in hiPSCs, hiPSC-derived neural stem cells (NSCs) and undifferentiated neurons, and the effect of viral infection on host innate antiviral responses of representative flaviviruses associated with diverse neurological diseases, i.e., Zika virus (ZIKV), West Nile virus (WNV), and dengue virus (DENV). In addition, we exploited hiPSCs to model ZIKV infection in the embryo and during neurogenesis. The results of this study confirmed the tropism of ZIKV for NSCs, but showed that WNV replicated in these cells with much higher efficiency than ZIKV and DENV, inducing massive cell death. Although with lower efficiency, all flaviviruses could also infect pluripotent stem cells and neurons, inducing similar patterns of antiviral innate immune response gene expression. While showing the usefulness of hiPSC-based infection models, these findings suggest that additional virus-specific mechanisms, beyond neural tropism, are responsible for the peculiarities of disease phenotype in humans.

In 2018, there was a large West Nile virus (WNV) outbreak in northern Italy. We observed five atypical cases of WNV infection that were characterised by the presence of WNV RNA and WNV IgG at the time of diagnosis, but no IgM response during follow-up. Neutralisation assays demonstrated pre-existing Usutu virus immunity in all patients. Besides challenging diagnosis, the immunological crosstalk between the two viruses warrants further investigation on possible cross-protection or infection enhancement effects.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod-borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV-endemic regions co-circulation of both viruses is increasing. Flaviviruses are structurally highly similar, which leads to cross-reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false-positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross-reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV- and WNV-assays with improved specificities.

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

After the WHO declared Zika virus (ZIKV) as a public health emergency of international concern, intense research for the development of vaccines and drugs has been undertaken, leading to the development of several candidates. Areas covered: This review discusses the developments achieved so far by computational methods in the discovery of candidate compounds targeting ZIKV proteins, i.e. the envelope and capsid structural proteins, the NS3 helicase/protease, and the NS5 methyltransferase/RNA-dependent RNA polymerase. Expert opinion: Research for effective drugs against ZIKV is still in a very early discovery phase. Notwithstanding the intense efforts for the development of new drugs and the identification of several promising candidates by using different approaches, including computational methods, so far only a few candidates have been experimentally tested. An important caveat of anti-flavivirus drug development is represented by the difficult of reproducing the in vivo microenvironment of the replication complex, which may lead to discrepancies between in vitro results and experimental evaluation in vivo. Moreover, anti-ZIKV drugs have the additional requirement of an excellent safety profile in pregnancy and ability to diffuse to different tissues, including the central nervous system, the testis, and the placenta.

Persistent infection by high-risk human papillomaviruses (HPVs) is associated with the development of cervical cancer and a subset of anogenital and head and neck squamous cell carcinomas. Abnormal expression of cellular microRNAs (miRNAs) plays an important role in the development of cancer, including HPV-related tumors. In this study, we demonstrated that miR-146a-5p was down-regulated by E6 and, less efficiently, by E7 of high-risk HPV16 in keratinocytes and the presence of low levels of this miRNA in cervical carcinoma cell lines and in high-risk HPV-positive cervical specimens. Down-regulation of miR-146a-5p was mediated at least in part by the transcription repressor c-MYC, through binding sites in the miR-146a promoter. Overexpression of miR-146a-5p significantly inhibited proliferation and migration of keratinocytes and cervical cancer cells. The histone demethylase KDM2B was validated as a new direct target of miR-146a-5p and two putative binding sites for miR-146a-5p were identified in its 3'UTR sequence. Western blot analysis and immunohistochemistry showed that KDM2B was overexpressed in HPV16 E6/E7-positive keratinocytes, in cervical cancer cell lines, and in a subset of invasive cervical carcinomas and HPV-positive laryngeal squamous cell carcinomas. In these tumors, KDM2B overexpression was associated with c-MYC copy number gain. In vitro, silencing of KDM2B inhibited proliferation of cervical cancer cells. In conclusion, this study identified a novel player, the hystone demethylase KDM2B, in HPV-mediated tumorigenesis. E6 and, less efficiently, E7 of high-risk HPV16 up-regulated KDM2B expression in human keratinocytes through a pathway involving overexpression of c-MYC, which in turn downregulated miR-146a-5p.

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy., Author summary Mycobacterium tuberculosis (Mtb) is one of the most successful pathogens in human history and remains the second leading cause of death from an infectious agent worldwide. The major reason of Mtb success relies on its ability to evade host immunity. Autophagy, a cellular mechanism involved in intracellular pathogen elimination, is one of the pathways hijacked by Mtb to elude the control of dendritic cells (DC), major cellular effectors of immune response. Recently, it has become clear that Mtb infection not only alters cellular gene expression, but also controls the level of small RNA molecules, namely microRNAs (miRNAs), which function as negative regulators of mRNA translation into protein. In the present study, we observed that the infection of human DC with Mtb leads to a strong induction of host miR-155, a critical regulator of host immune response. By mean of miR-155 induction, Mtb reduces Atg3 protein content, a crucial enzyme needed for the initial phase of the autophagic process. Interestingly, miR-155 silencing during Mtb infection restores Atg3 level and rescues autophagy. These findings contribute to better elucidate Mtb-triggered escape mechanisms and highlight the importance to develop host-directed therapies to combat tuberculosis based on autophagy boosting.

Background & Aims MicroRNAs (miRNAs) have been involved in hepatocarcinogenesis, but little is known on their role in the progression of chronic viral hepatitis. Aim of this study was to identify miRNA signatures associated with stages of disease progression in patients with chronic viral hepatitis. Methods MiRNA expression profile was investigated in liver biopsies from patients with chronic viral hepatitis and correlated with clinical, virological and histopathological features. Relevant miRNAs were further investigated. Results Most of the significant changes in miRNA expression were associated with liver fibrosis stages and included the significant up-regulation of a group of miRNAs that were demonstrated to target the master regulators of epithelial–mesenchymal transition ZEB1 and ZEB2 and involved in the preservation of epithelial cell differentiation, but also in cell proliferation and fibrogenesis. In agreement with miRNA data, immunostaining of liver biopsies showed that expression of the epithelial marker E-cadherin was maintained in severe fibrosis/cirrhosis while expression of ZEBs and other markers of epithelial–mesenchymal transition were low or absent. Severe liver fibrosis was also significantly associated with the down-regulation of miRNAs with antiproliferative and tumour suppressor activity. Similar changes in miRNA and target gene expression were demonstrated along with disease progression in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, suggesting that they might represent a general response to liver injury. Conclusion Chronic viral hepatitis progression is associated with the activation of miRNA pathways that promote cell proliferation and fibrogenesis, but preserve the differentiated hepatocyte phenotype.

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross- reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA- format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre- incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.

Dysregulation of host microRNA expression has been involved in the development and progression of human papillomavirus (HPV)-related tumors. Analysis of miR-146a expression in a series of 59 penile squamous cell carcinomas (PSCCs) showed that its levels were lower in high-risk HPV-positive than in HPV-negative PSCCs and inversely correlated with expression of epidermal growth factor receptor (EGFR), a known target for miR-146a. Analysis of genotype distribution for rs2910164, a common functional polymorphism of miR-146a, did not identify correlations with miR-146a levels and EGFR expression in PSCCs. In vitro experiments demonstrated that E6 of HPV type 16, but not low-risk HPV-6, down-regulated miR-146a in human foreskin keratinocytes and up-regulated EGFR. Ectopic expression of miR-146a decreased expression of EGFR and inhibited proliferation of keratinocytes and cervical carcinoma cells. EGFR is commonly overexpressed in penile cancer and in other squamous cell carcinomas. Molecular mechanisms leading to EGFR overexpression and activation are known for HPV-negative cancers and include amplification or mutations of the EGFR gene. The results of this study indicate that down-regulation of miR-146a may represent another mechanism of EGFR overexpression in PSCCs, which can be mediated by high-risk HPV E6 in HPV-related tumors.

This study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified.

We report the isolation of infectious Zika virus (ZIKV) in cell culture from the saliva of a patient who developed a febrile illness after returning from the Dominican Republic to Italy, in January 2016. The patient had prolonged shedding of viral RNA in saliva and urine, at higher load than in blood, for up to 29 days after symptom onset. Sequencing of ZIKV genome showed relatedness with strains from Latin America.

West Nile virus (WNV) is an emerging flavivirus responsible for an increasing number of outbreaks of neuroinvasive disease in North America, Europe, and neighboring countries. Almost all WNV infections in humans are transmitted through the bite of infected mosquitoes. Transmission during pregnancy and through breastfeeding has been reported, but the risk seems to be very low. West Nile disease in children is less common (1-5% of all WNV cases) and associated with milder symptoms and better outcome than in elderly individuals, even though severe neuroinvasive disease and death have been reported also among children. However, the incidence of WNV infection and disease in children is probably underestimated and the disease spectrum is not fully understood because of lack of reporting and underdiagnosis in children. Infection is diagnosed by detection of WNV-specific antibodies in serum and WNV RNA in plasma and urine. Since no effective WNV-specific drugs are available, therapy is mainly supportive.

MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS), host’s repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG) in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death. Tissue samples obtained either at surgery, [neoplastic (HCC) and adjacent non-cancerous cirrhotic tissues (NCCT)] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy) were analysed for 8-OHdG (HPLC-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), telomerase activity, telomere length (T/S, by RT-PCR), Taqman microRNA assay and Bad/Bax mRNA (RT-PCR). Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues), 22 from chronic hepatitis (CH) and 10 controls (cholecystectomy patients - CON) were examined. Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001). Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002); conversely T/S was significantly lower in HCC (p=0.05). MiR-199a-b, -195, -122, -92a and −145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA. The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS-mediated oxidative DNA damage, and suggest that this correlates with induction of telomerase activity and, as a novel finding, with over-expression of miR-92, a microRNA that plays a role in both the apoptotic process and in cellular proliferation pathways.

Mutations of the Cell Division Cycle 73 (CDC73) tumor suppressor gene (previously known as HRPT2), encoding for parafibromin, are associated with the Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, an autosomal dominant disease whose clinical manifestations are mainly parathyroid tumors and, less frequently, ossifying fibromas of the jaws, uterine and renal tumors. Most mutations of CDC73 are nonsense or frameshift, while missense mutations are rare and generally affect the N-terminal domain of parafibromin, a region that is still poorly characterized. The aim of this study was to characterize a novel somatic CDC73 missense mutation (Ile60Asn) identified in the mandibular tumor of a HPT-JT patient carrying a germline CDC73 inactivating mutation. Immunostaining of the tumor showed reduced nuclear parafibromin immunoreactivity. Western blotting and confocal microscopy of transfected cells demonstrated that the Ile60Asn mutant parafibromin was less expressed than the wild-type protein and exhibited impaired nucleolar localization. Treatment of transfected cells with translation and proteasome inhibitors demonstrated a decreased stability of the Ile60An mutant, partially due to an increase in proteasomal degradation. Overexpression of the Ile60Asn mutant led to increased cell proliferation and to accumulation in the G2/M phase of cell cycle. Moreover, mutant parafibromin lost the ability to down-regulate c-myc expression. In conclusion, our study shows that a missense mutation in the N-terminus of parafibromin, identified in an ossifying fibroma from a HPT-JT patient, stimulated cell proliferation and impaired parafibromin expression and nucleolar localization, suggesting a relevant role of the N-terminal domain for parafibromin function.

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16 INK4a , Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV + PSCCs overexpressed p16 INK4a in 88% cases and p53 in 35% of cases, whereas HR-HPV − PSCCs were positive for p16 INK4a and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53 − cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV + PSCCs and in HR-HPV − PSCCs without immunohistochemical detection of p53.