Telomerase: a good target in hepatocellular carcinoma? An overview of relevant preclinical data.

INTRODUCTION: The expression of telomerase reverse transcriptase (TERT) in liver is restricted to rare cells, that are able to replace senescent hepatocytes and regenerate tissue in response to hepatic damage, while becoming extinguished in differentiated progeny cells. TERT gene is permanently activated in liver neoplasms from the very early stage of the hepatocarcinogenesis mainly through the accumulation of genetic alterations, virus-related insertional mutagenesis and somatic mutations in the TERT promoter region. Several lines of evidence suggest that telomerase, beyond the canonical function of telomeres elongation, has multiple oncogenic activities in cancer cells and may represent a promising therapeutic target in hepatocellular carcinoma (HCC). AREAS COVERED: We review the mechanisms of activation of telomerase in HCC, the canonical and non-canonical functions of TERT as well as experimental strategies to directly target telomerase or to inhibit pathways associated with telomerase activity. EXPERT OPINION: TERT holoenzyme and telomerase components represent promising therapeutic targets in the treatment of liver malignancies. Several chemical agents and natural products known to alter telomerase activity are under evaluation for their potency to inhibit telomeres attrition in cirrhosis and TERT function in liver cancer. Therefore, this review outlines the current strategies pursued to suppress the multiple mechanisms of the major telomerase components in liver cancer.

Bimodal antibody-titer decline following BNT162b2 mRNA anti-SARS-CoV-2 vaccination in healthcare workers of the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy).

BACKGROUND: Both SARS-CoV-2 mRNA-based vaccines [BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)] have shown high efficacy, with very modest side effects in limiting transmission of SARS-CoV-2 and in preventing the severe COVID-19 disease, characterized by a worrying high occupation of intensive care units (ICU), high frequency of intubation and ultimately high mortality rate. At the INT, in Naples, only the BNT162b2/Pfizer vaccine has been administered to cancer patients and healthcare professionals aged 16 and over. In the present study, the antibody response levels and their decline were monitored in an interval of 6-9 months after vaccine administration in the two different cohorts of workers of the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy): the group of individuals previously infected with SARS-CoV-2 and vaccinated with a single dose; and that of individuals negative for previous exposure to SARS-CoV-2 vaccinated with two doses 21 days apart. METHODS: Specific anti-RBD (receptor-binding domain) titers against trimeric spike glycoprotein (S) of SARS-CoV-2 by Roche Elecsys Anti-SARS-CoV-2 S ECLIA immunoassay were determined in serum samples of 27 healthcare workers with a previously documented history of SARS-CoV-2 infection and 123 healthcare workers without, during antibody titers' monitoring. Moreover, geometric mean titers (GMT) and relative fold changes (FC) were calculated. RESULTS: Bimodal titer decline was observed in both previously infected and uninfected SARS-CoV-2 subjects. A first rapid decline was followed by a progressive slow decline in the 6/9 month-period before the further vaccine boost. The trend was explained by 2 different mathematical models, exponential and power function, the latter revealing as predictive of antibody titer decline either in infected or in not previously infected ones. The value of the prolonged lower vaccine titer was about 1 log below in the 6/9-month interval after the single dose for previously infected individuals with SARS-CoV-2 and the two doses for those not previously infected. The titer change, after the boost dose administration, on the other hand, was ≥ 1.5 FC higher than the titers at the 6/9-month time-points in both cohorts. A similar quantitative immune titer was observed in both cohorts 8 days after the last boost dose. The subsequent immunoresponse trend remains to be verified. DISCUSSION: The results show that a very rapid first decline, from the highest antibody peak, was followed by a very slow decline which ensured immune protection lasting more than 6 months. The apparent absence of adverse effects of the rapid decline on the vaccine's immune protective role has been related to a large majority of low avidity antibodies induced by current vaccines. High avidity antibodies with prolonged anti-transmission efficacy show a longer half-life and are lost over a longer interval period. The cellular immunity, capable of preventing severe clinical diseases, lasts much longer. The unbalanced dual activity (cellular vs humoral) while effective in limiting ICU pressure and overall mortality, does not protect against transmission of SARS-CoV-2, resulting in high circulation of the virus among unvaccinated subjects, including the younger population, and the continuous production of variants characterized by changes in transmissibility and pathogenicity. The high mutation rate, peculiar to the RNA virus, can however lead to a dual opposite results: selection of defective and less efficient viruses up to extinction; risk of more efficiently transmitted variants as the current omicron pandemic. CONCLUSIONS: In conclusion the current bimodal antibody-titer decline, following BNT162b2 mRNA anti-SARS-CoV-2 vaccination, needs a further extended analysis to verify the protective borderline levels of immunity and the optimal administration schedule of vaccine boosters. Our current results can contribute to such goal, besides a direct comparison of other FDA-approved and candidate vaccines.

Profiling the HCV Immune Response in Patients with Chronic Liver Diseases and Hepatocellular Carcinoma by Peptide Microarray Analysis.

BACKGROUND: Chronic infection with hepatitis C virus (HCV) is among the major causes of hepatic fibrosis, cirrhosis, as well as hepatocellular carcinoma (HCC), and it is associated with a significant risk of developing lymphoproliferative disorders. The rate of clinical disease progression is variable depending on multiple host and viral factors, including immune response. METHODS: To perform a comprehensive epitope mapping of anti-HCV antibodies in patients suffering from HCV-related liver or lymphoproliferative diseases, we analyzed clinical samples on a peptide microarray platform made of 5952 overlapping 15-mer synthetic peptides derived from the whole HCV proteome. We evaluated the antibody profile of 71 HCV-positive patients diagnosed with HCC, mixed cryoglobulinemia (MC), and HCV chronic infection. Antibody reactivity against virus peptides was detected in all HCVpositive patients. Importantly, the signal amplitude varied significantly within and between diverse patient groups. RESULTS: Antibody reactivity against C peptides were found generally low in HCV chronically infected asymptomatic subjects and increasingly high in HCC and MC patients. Moreover, we found a statistically significant higher IgG response in HCC and MC patients against specific domains of HCV C, E2, NS3, NS4A, NS4B, NS5A, and p7 compared to HCV-positive subjects. CONCLUSION: In conclusion, our data suggest that immune response against specific HCV protein domains may represent useful biomarkers of disease progression among HCVpositive patients and suggest that peptide microarrays are good tools for the screening of immunotherapy targets in preclinical HCV research.

Clinical Significance of Telomerase Reverse-Transcriptase Promoter Mutations in Hepatocellular Carcinoma.

Telomerase reactivation during hepatocarcinogenesis is recurrently caused by two point mutations occurring most frequently at the nucleotide -124 (95%) and occasionally at the nucleotide -146 (<5%) upstream of the TERT translational start site in hepatocellular carcinoma (HCC). In this study, we designed a droplet digital PCR (ddPCR) assay to detect TERT promoter (TERTp) nucleotide change G>A at position -124 and to quantify the mutant allele frequency (MAF) in 121 primary liver cancers, including 114 HCC along with 23 autologous cirrhotic tissues, five cholangiocarcinoma (CC), and two hepato-cholangiocarcinoma (HCC-CC). All cases were evaluated for tumour markers such as α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA). We compared the sensitivity of ddPCR and Sanger sequencing and investigated the prognostic relevance of TERTp mutations. The TERTp G>A transition was identified in 63.6% and 52.1% of HCC samples by ddPCR and Sanger sequencing, respectively. One out of 23 (4.3%) peri-tumour tissues tested positive only by ddPCR. One out of five CC (20%) and none of the HCC-CC were found concordantly mutated by the two methods. The TERTp MAF ranged from 2% to 66%, and the large majority (85.5%) of mutated samples showed a value above 20%. A statistically significant correlation was found between TERTp mutation and tumour size (p = 0.048), while an inverse correlation was observed with CA19-9 levels (p = 0.0105). Moreover, HCC patients with TERTp -124A had reduced survival. In conclusion, the single nucleotide variation G>A at position -124 in TERTp, detected either by ddPCR or by Sanger sequencing, showed a remarkable high frequency in HCC. Such mutation is associated with lower levels of CA19-9 and reduced survival in HCC patients suggesting that the TERTp status may represent a distinct signature of liver cancer subgroups.

Vaccination strategy and anti - SARS-CoV-2 S titers in healthcare workers of the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy).

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and the resulting disease, coronavirus disease 2019 (COVID-19), have spread to millions of people globally, requiring the development of billions of different vaccine doses. The SARS-CoV-2 spike mRNA vaccine (named BNT162b2/Pfizer), authorized by the FDA, has shown high efficacy in preventing SARS-CoV-2 infection after administration of two doses in individuals 16 years of age and older. In the present study, we retrospectively evaluated the differences in the SARS-CoV-2 humoral immune response after vaccine administration in the two different cohorts of workers at the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy): previously infected to SARS-CoV-2 subjects and not infected to SARS-CoV-2 subjects. METHODS: We determined specific anti-RBD (receptor-binding domain) titers against trimeric spike glycoprotein (S) of SARS-CoV-2 by Roche Elecsys Anti-SARS-CoV-2 S immunoassay in serum samples of 35 healthcare workers with a previous documented history of SARS-CoV-2 infection and 158 healthcare workers without, after 1 and 2 doses of vaccine, respectively. Moreover, geometric mean titers and relative fold changes (FC) were calculated. RESULTS: Both previously infected and not infected to SARS-CoV-2 subjects developed significant immune responses to SARS-CoV-2 after the administration of 1 and 2 doses of vaccine, respectively. Anti-S antibody responses to the first dose of vaccine were significantly higher in previously SARS-CoV-2-infected subjects in comparison to titers of not infected subjects after the first as well as the second dose of vaccine. Fold changes for subjects previously infected to SARS-CoV-2 was very modest, given the high basal antibody titer, as well as the upper limit of 2500.0 BAU/mL imposed by the Roche methods. Conversely, for naïve subjects, mean fold change following the first dose was low ([Formula: see text] =1.6), reaching 3.8 FC in 72 subjects (45.6%) following the second dose. CONCLUSIONS: The results showed that, as early as the first dose, SARS-CoV-2-infected individuals developed a remarkable and statistically significant immune response in comparison to those who did not contract the virus previously, suggesting the possibility of administering only one dose in previously SARS-CoV-2-infected subjects. FC for previously infected subjects should not be taken into account for the generally high pre-vaccination values. Conversely, FC for not infected subjects, after the second dose, were = 3.8 in > 45.0% of vaccinees, and ≤ 3.1 in 19.0%, the latter showing a potential susceptibility to further SARS-CoV-2 infection.

Lung histopathological findings in COVID-19 disease - a systematic review.

Since December 2019, the global burden of the COVID-19 pandemic has increased rapidly and has impacted nearly every country in the world, affecting those who are elderly or with underlying comorbidities or immunocompromised states. Aim of this systematic review is to summarize lung histopathological characteristics of COVID-19, not only for diagnostic purpose but also to evaluate changes that can reflect pathophysiological pathways that can inform clinicians of useful treatment strategies. We identified following histopathological changes among our patients:: hyaline membranes; endothelial cells/ interstitial cells involvement; alveolar cells, type I pneumocytes/ type II pneumocytes involvement; interstitial and/ or alveolar edema; evidence of hemorrhage, of inflammatory cells, evidence of microthrombi; evidence of fibrin deposition and of viral infection in the tissue samples.The scenario with proliferative cell desquamation is typical of Acute Respiratory Distress Syndrome (ARDS) that can be classified as diffuse alveolar damage (DAD) and not DAD-ARDS. The proposed pathological mechanism concerns the role of both innate and adaptive components of the immune system. COVID-19 lethal cases present themselves as a heterogeneous disease, characterized by the different simultaneous presence of different histological findings, which reflect histological phases with corresponding different pathological pathways (epithelial, vascular and fibrotic changes), in the same patient.

The Role of circRNAs in Human Papillomavirus (HPV)-Associated Cancers.

Circular RNAs (circRNAs) are a new class of "non-coding RNAs" that originate from non-sequential back-splicing of exons and/or introns of precursor messenger RNAs (pre-mRNAs). These molecules are generally produced at low levels in a cell-type-specific manner in mammalian tissues, but due to their circular conformation they are unaffected by the cell mRNA decay machinery. circRNAs can sponge multiple microRNAs or RNA-binding proteins and play a crucial role in the regulation of gene expression and protein translation. Many circRNAs have been shown to be aberrantly expressed in several cancer types, and to sustain specific oncogenic processes. Particularly, in virus-associated malignancies such as human papillomavirus (HPV)-associated anogenital carcinoma and oropharyngeal and oral cancers, circRNAs have been shown to be involved in tumorigenesis and cancer progression, as well as in drug resistance, and some are useful diagnostic and prognostic markers. HPV-derived circRNAs, encompassing the HPV E7 oncogene, have been shown to be expressed and to serve as transcript for synthesis of the E7 oncoprotein, thus reinforcing the virus oncogenic activity in HPV-associated cancers. In this review, we summarize research advances in the biogenesis of cell and viral circRNAs, their features and functions in the pathophysiology of HPV-associated tumors, and their importance as diagnostic, prognostic, and therapeutic targets in anogenital and oropharyngeal and oral cancers.

Evaluation of the diagnostic accuracy of a new point-of-care rapid test for SARS-CoV-2 virus detection.

BACKGROUND: The easy access to a quick diagnosis of coronavirus disease 2019 (COVID-19) is a key point to improve the management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to contain its spread. Up to now, laboratory real-time PCR is the standard of care, but requires a fully equipped laboratory and significant infrastructure. Consequently, new diagnostic tools are required. METHODS: In the present work, the diagnostic accuracy of the point-of-care rapid test "bKIT Virus Finder COVID-19" (Hyris Ltd) is evaluated by a retrospective and a prospective analysis on SARS CoV-2 samples previously assessed with an FDA "authorized for the emergency use-EUA" reference method. Descriptive statistics were used for the present study. RESULTS: Results obtained with the Hyris Kit are the same as that of standard laboratory-based real time PCR methods for all the analyzed samples. In addition, the Hyris Kit provides the test results in less than 2 h, a significantly shorter time compared to the reference methods, without the need of a fully equipped laboratory. CONCLUSIONS: To conclude, the Hyris kit represents a promising tool to improve the health surveillance and to increase the capacity of SARS-CoV-2 testing.

Modeling the epithelial-mesenchymal transition process in a 3D organotypic cervical neoplasia.

Here, we proposed an innovative organotypic cervical tumor model able to investigate the bi-directional crosstalk between epithelium and stroma as well as the key disease features of the epithelial-mesenchymal transition (EMT) process in vitro. By using a modular tissue assembling approach, we developed 3D cervical stromal models composed of primary human cervical fibroblasts (HCFs) or cervical cancer-associated fibroblasts (CCAFs) embedded in their own ECM to produce 3D normal cervical-instructed stroma (NCIS) or 3D cervical cancer-instructed stroma (CCIS), respectively. Then, we demonstrate the role of the tumor microenvironment (TME) in potentiating the intrinsic invasive attitude of cervical cancer derived SiHa cells and increasing their early viral gene expression by comparing the SiHa behavior when cultured on NCIS or CCIS (SiHa-NCIS or SiHa-CCIS). We proved the crucial role of the CCAFs and stromal microenvironment in the mesenchymalization of the cancer epithelial cells by analyzing several EMT markers. We further assessed the expression of the epithelial adhesion molecules, matricellular enzymes, non-collagenous proteins as well as ECM remodeling in terms of collagen fibers texture and assembly. This cervical tumor model, closely recapitulating key cervical carcinogenesis features, may provide efficient and relevant support to current approaches characterizing cancer progression and develop new anticancer therapy targeting stroma rather than cancer cells.

Transoral robotic surgery in head and neck district: a retrospective study on 67 patients treated in a single center.

BACKGROUND: The anatomical complexity of the oropharynx and the difficulty in reaching its distal portion have always conditioned the surgical accessibility.Robotic surgery represents an excellent alternative in the treatment of cervico-facial oncological diseases. METHODS: This series comprises all patients managed for head and neck cancer by Trans Oral Robotic Surgery TORS.The staging assessment, including neck ultrasound and total body PET/CT scan, was performed in each patient according to the TNM classification.All charts were recorded with the following data: name and surname, age, gender, date of surgery intra or post-operative hemorragia, tumor site, histology, TNM stage, robot set-up time, tumor resection time, whether or not tracheotomy was performed, whether or not neck dissection was performed, insertion of a nasogastric tube or gastrostomy, time to resumption of oral feeding, surgical margins, mean length of hospital stay, adjuvant treatment and follow-up. RESULTS: From February 2013 to February 2018, TORS was performed in 67 consecutive patients affected by head and neck tumours.We divided, our sample, in 3 subsites: supraglottic larynx, parapharyngeal space and oropharynx.Pathology reports confimed malignancy in 44 cases: 8 cases lymphomas, 36 cases of Squamous cell carcinoma (SCC), 5 cases of benign salivary glands tumors and 18 miscellaneous cases. Neck dissection was performed in 12 cases.Tracheotomy was perfomed in 3/67 cases for respiratory failures. A nasogastric tube was inserted at the end of the surgical procedure in 21 patients. The mean length of hospital stay was 10 days .Major complications included post-operative bleeding in 3 patients, 1 exitus for massive bleeding 20 days post-surgery and 1 respiratory failure treated with tracheotomy and monitoring in the Intensive Care Unit (ICU) for 3 days. CONCLUSIONS: Robotic surgery has been considered a valid alternative to traditional open treatment in many specializations with the advantages of an endoscopic procedure, with the same oncological and functional results and with fewer complications. The advantages of this type of surgical technique have been discussed, it is mandatory to focus on the indications and contraindications.

Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities.

Antimicrobial peptides (AMPs), or host defense peptides, are small cationic or amphipathic molecules produced by prokaryotic and eukaryotic organisms that play a key role in the innate immune defense against viruses, bacteria and fungi. AMPs have either antimicrobial or anticancer activities. Indeed, cationic AMPs are able to disrupt microbial cell membranes by interacting with negatively charged phospholipids. Moreover, several peptides are capable to trigger cytotoxicity of human cancer cells by binding to negatively charged phosphatidylserine moieties which are selectively exposed on the outer surface of cancer cell plasma membranes. In addition, some AMPs, such as LTX-315, have shown to induce release of tumor antigens and potent damage associated molecular patterns by causing alterations in the intracellular organelles of cancer cells. Given the recognized medical need of novel anticancer drugs, AMPs could represent a potential source of effective therapeutic agents, either alone or in combination with other small molecules, in oncology. In this review we summarize and describe the properties and the mode of action of AMPs as well as the strategies to increase their selectivity toward specific cancer cells.

Detection of a large spectrum of viral infections in conjunctival premalignant and malignant lesions.

To study the interaction between HIV and other carcinogenic infections in conjunctival squamous cell carcinoma (SCC), we evaluated the presence of a broad spectrum of human viruses in conjunctiva specimens. Beta Human papillomavirus (HPV; n = 46), gamma HPV (n = 52), polyomaviruses (n = 12) and herpes viruses (n = 3) was determined in DNA extracted from 67 neoplastic and 55 non-neoplastic conjunctival tissues of HIV-positive and HIV negative subjects by Luminex-based assays. Next-generation sequencing (NGS) was also used to further characterize the presence of cutaneous HPVs. Detection of beta-2 HPV infections was associated with the risk of neoplasia (adjusted odds ratio [aOR] 3.0; 95% confidence interval [CI] 1.3-6.8), regardless of HIV status (HIV positive, aOR 2.6, 95% CI 0.9-7.7; HIV negative, aOR 3.5, 95% CI 0.9-14.4). EBV was strongly associated with the risk of neoplasia (aOR 12.0, 95% CI 4.3-33.5; P < .01) mainly in HIV individuals (HIV positive, aOR 57.5; 95% CI: 10.1-327.1; HIV negative aOR 2.6; 95% CI: 0.2-34.7). NGS allowed to identify 13 putative novel HPVs in cases and controls. Our findings suggest a role of beta HPV types and EBV, in conjunctival SCC. However, additional studies of viral expression in tumor tissue are required to confirm the causal association.

The Role of microRNAs, Long Non-coding RNAs, and Circular RNAs in Cervical Cancer.

Prolonged infection of uterine cervix epithelium with human papillomavirus (HPV) and constitutive expression of viral oncogenes have been recognized as the main cause of the complex molecular changes leading to transformation of cervical epithelial cells. Deregulated expression of microRNAs (miRNA), long non-coding RNAs (lncRNA), and circular RNAs (circRNA) is involved in the initiation and promotion processes of cervical cancer development. Expression profiling of small RNAs in cervical neoplasia revealed up-regulated "oncogenic" miRNAs, such as miR-10a, miR-21, miR-19, and miR-146a, and down regulated "tumor suppressive" miRNAs, including miR-29a, miR-372, miR-214, and miR-218, associated with cell growth, malignant transformation, cell migration, and invasion. Also several lncRNAs, comprising among others HOTAIR, MALAT1, GAS5, and MEG3, have shown to be associated with various pathogenic processes such as tumor progression, invasion as well as therapeutic resistance and emerged as new diagnostic and prognostic biomarkers in cervical cancer. Moreover, human genes encoded circular RNAs, such as has_circ-0018289, have shown to sponge specific miRNAs and to concur to the deregulation of target genes. Viral encoded circE7 has also demonstrated to overexpress E7 oncoprotein thus contributing to cell transformation. In this review, we summarize current literature on the complex interplay between miRNAs, lncRNAs, and circRNAs and their role in cervical neoplasia.

Precision medicine in gastric cancer.

Gastric cancer (GC) is a complex disease linked to a series of environmental factors and unhealthy lifestyle habits, and especially to genetic alterations. GC represents the second leading cause of cancer-related deaths worldwide. Its onset is subtle, and the majority of patients are diagnosed once the cancer is already advanced. In recent years, there have been innovations in the management of advanced GC including the introduction of new classifications based on its molecular characteristics. Thanks to new technologies such as next-generation sequencing and microarray, the Cancer Genome Atlas and Asian Cancer Research Group classifications have also paved the way for precision medicine in GC, making it possible to integrate diagnostic and therapeutic methods. Among the objectives of the subdivision of GC into subtypes is to select patients in whom molecular targeted drugs can achieve the best results; many lines of research have been initiated to this end. After phase III clinical trials, trastuzumab, anti-Erb-B2 receptor tyrosine kinase 2 (commonly known as ERBB2) and ramucirumab, anti-vascular endothelial growth factor receptor 2 (commonly known as VEGFR2) monoclonal antibodies, were approved and introduced into first- and second-line therapies for patients with advanced/metastatic GC. However, the heterogeneity of this neoplasia makes the practical application of such approaches difficult. Unfortunately, scientific progress has not been matched by progress in clinical practice in terms of significant improvements in prognosis. Survival continues to be low in contrast to the reduction in deaths from many common cancers such as colorectal, lung, breast, and prostate cancers. Although several target molecules have been identified on which targeted drugs can act and novel products have been introduced into experimental therapeutic protocols, the overall approach to treating advanced stage GC has not substantially changed. Currently, surgical resection with adjuvant or neoadjuvant radiotherapy and chemotherapy are the most effective treatments for this disease. Future research should not underestimate the heterogeneity of GC when developing diagnostic and therapeutic strategies aimed toward improving patient survival.

Prevalence of "unclassified" HPV genotypes among women with abnormal cytology.

BACKGROUND: High risk human papillomaviruses (HPVs) have been unequivocally recognised as the necessary cause of squamous intraepithelial lesions (SIL) and invasive carcinoma of the cervix. The distribution and the role of unclassified risk HPV genotypes in cervical neoplasia has not been fully elucidated. METHODS: Liquid-based cytological samples were collected from 337 women referred for colposcopy following an abnormal cytological diagnosis. HPV DNA was detected by broad-spectrum PCR and genotypes identified by nucleotide sequencing analysis and reverse line blot (RLB). RESULTS: The overall frequency of HPV infection was 36.5% (35 out of 96) in samples negative for intraepithelial lesions or malignancy (NILM), 80% (181 out of 226) in low grade SIL and 93.3% (14 out of 15) in high grade SIL (P < 0.001). Thirty-five different genotypes were identified among the 230 HPV-positive cases. The Group 1 oncogenic viruses (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) were found in 21.9, 46.5, and 86.7% of NILM, low grade SIL and high grade SIL, respectively. The Group 2A, including the probably oncogenic virus HPV68, was found in 1 and 0.8% of NILM and low grade SIL, respectively. The Group 2b possibly oncogenic HPVs (HPV34, 53, 66, 67, 70, 73, 82 and 85) were found in 4.2, 21.7 and 26.7% of NILM, low grade SIL and high grade SIL, respectively. The unclassified viruses (HPV12, 42, 54, 55, 61, 62, 81, 83, 84, 89, 90, 91) were detected in 8.3 and 14.6% of NILM and low grade SIL, respectively, and never in high grade SIL. CONCLUSIONS: Group 1 HPVs were mainly prevalent in high grade SIL and low grade SIL while Group 2B were equally distributed among the two groups. The dominant frequency of unclassified HPVs in low grade SIL and NILM and their rarity in high grade SIL suggests their marginal role in cervical neoplasia of the studied population.

Human Oncoviruses and p53 Tumor Suppressor Pathway Deregulation at the Origin of Human Cancers.

Viral oncogenesis is a multistep process largely depending on the complex interplay between viruses and host factors. The oncoviruses are capable of subverting the cell signaling machinery and metabolic pathways and exploit them for infection, replication, and persistence. Several viral oncoproteins are able to functionally inactivate the tumor suppressor p53, causing deregulated expression of many genes orchestrated by p53, such as those involved in apoptosis, DNA stability, and cell proliferation. The Epstein⁻Barr virus (EBV) BZLF1, the high-risk human papillomavirus (HPV) E6, and the hepatitis C virus (HCV) NS5 proteins have shown to directly bind to and degrade p53. The hepatitis B virus (HBV) HBx and the human T cell lymphotropic virus-1 (HTLV-1) Tax proteins inhibit p53 activity through the modulation of p300/CBP nuclear factors, while the Kaposi's sarcoma herpesvirus (HHV8) LANA, vIRF-1 and vIRF-3 proteins have been shown to destabilize the oncosuppressor, causing a decrease in its levels in the infected cells. The large T antigen of the Merkel cell polyomavirus (MCPyV) does not bind to p53 but significantly reduces p53-dependent transcription. This review describes the main molecular mechanisms involved in the interaction between viral oncoproteins and p53-related pathways as well as in the development of therapeutic strategies targeting such interactions.

The Role of Circulating Free DNA and MicroRNA in Non-Invasive Diagnosis of HBV- and HCV-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third and the fifth leading cause of cancer related death worldwide in men and in women, respectively. HCC generally has a poor prognosis, with a very low 5-year overall survival, due to delayed diagnosis and treatment. Early tumour detection and timely intervention are the best strategies to reduce morbidity and mortality in HCC patients. Histological evaluation of liver biopsies is the gold standard for cancer diagnosis, although it is an invasive, time-consuming and expensive procedure. Recently, the analysis of circulating free DNA (cfDNA) and RNA molecules released by tumour cells in body fluids, such as blood serum, saliva and urine, has attracted great interest for development of diagnostic assays based on circulating liver cancer molecular biomarkers. Such "liquid biopsies" have shown to be useful for the identification of specific molecular signatures in nucleic acids released by cancer cells, such as gene mutations and altered methylation of DNA as well as variations in the levels of circulating microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Body fluids analysis may represent a valuable strategy to monitor liver disease progression in subjects chronically infected with hepatitis viruses or cancer relapse in HCC treated patients. Several studies showed that qualitative and quantitative assays evaluating molecular profiles of circulating cell-free nucleic acids could be successfully employed for early diagnosis and therapeutic management of HCC patients. This review describes the state of art on the use of liquid biopsy for cancer driver gene mutations, deregulated DNA methylation as well as miRNA levels in HCC diagnosis.

Role of gut microbiota and oxidative stress in the progression of non-alcoholic fatty liver disease to hepatocarcinoma: Current and innovative therapeutic approaches.

Non-alcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in industrialized countries. NAFLD progresses through the inflammatory phase of non-alcoholic steatohepatitis (NASH) to fibrosis and cirrhosis, with some cases developing liver failure or hepatocellular carcinoma (HCC). Liver biopsy remains the gold standard approach to a definitive diagnosis of NAFLD and the distinction between simple steatosis and NASH. The pathogenesis of NASH is still not clear. Several theories have been proposed ranging from the "Two Hit Theory" to the "Multiple Hit Theory". However, the general consensus is that the gut microbiota, oxidative stress, and mitochondrial damage play key roles in the pathogenesis of NASH. The interaction between the gut epithelia and some commensal bacteria induces the rapid generation of reactive oxygen species (ROS). The main goal of any therapy addressing NASH is to reverse or prevent progression to liver fibrosis/cirrhosis. This problem represents the first "Achilles' heel" of the new molecules being evaluated in most ongoing clinical trials. The second is the inability of these molecules to reach the mitochondria, the primary sites of energy production and ROS generation. Recently, a variety of non-pharmacological and pharmacological treatment approaches for NASH have been evaluated including vitamin E, the thiazolidinediones, and novel molecules related to NASH pathogenesis (including obeticholic acid and elafibranor). Recently, a new isoform of human manganese superoxide dismutase (MnSOD) was isolated and obtained in a synthetic recombinant form designated rMnSOD. This protein has been shown to be a powerful antioxidant capable of mediating ROS dismutation, penetrating biological barriers via its uncleaved leader peptide, and reducing portal hypertension and fibrosis in rats affected by liver cirrhosis. Based on these distinctive characteristics, it can be hypothesized that this novel recombinant protein (rMnSOD) potentially represents a new and highly efficient adjuvant therapy to counteract the progression from NASH to HCC.