In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

Molecular diagnostics of prostate cancer: impact of molecular tests.

ABSTRACT: Prostate cancer (PCa) is the second leading cause of cancer-related death among men. Prostate-specific antigen (PSA) testing is used in screening programs for early detection with a consequent reduction of PCa-specific mortality at the cost of overdiagnosis and overtreatment of the nonaggressive PCa. Recently, several assays have been commercially developed to implement PCa diagnosis, but they have not been included in both screening and diagnosis of PCa. This review aims to describe the actual and novel commercially available molecular biomarkers that can be used in PCa management to implement and tailor the screening and diagnosis of PCa.

Unraveling the Complex Nexus of Human Papillomavirus (HPV) in Extragenital Keratinocyte Skin Tumors: A Comprehensive Analysis of Bowen's Disease and In Situ Squamous-Cell Carcinoma.

This comprehensive study delves into the intricate landscape surrounding the role of human papillomavirus (HPV) in extragenital keratinocyte skin tumors, specifically exploring Bowen's disease (BD) and in situ squamous-cell carcinoma (iSCC). Through a multifaceted examination, this research study elucidates the nuanced interplay of HPV, gender dynamics, anatomical site variations, and potential implications for the etiopathogenesis of these malignancies.

Overview of Tumor Heterogeneity in High-Grade Serous Ovarian Cancers.

Ovarian cancers encompass a group of neoplasms originating from germinal tissues and exhibiting distinct clinical, pathological, and molecular features. Among these, epithelial ovarian cancers (EOCs) are the most prevalent, comprising five distinct tumor histotypes. Notably, high-grade serous ovarian cancers (HGSOCs) represent the majority, accounting for over 70% of EOC cases. Due to their silent and asymptomatic behavior, HGSOCs are generally diagnosed in advanced stages with an evolved and complex genomic state, characterized by high intratumor heterogeneity (ITH) due to chromosomal instability that distinguishes HGSOCs. Histologically, these cancers exhibit significant morphological diversity both within and between tumors. The histologic patterns associated with solid, endometrioid, and transitional (SET) and classic subtypes of HGSOCs offer prognostic insights and may indicate specific molecular profiles. The evolution of HGSOC from primary to metastasis is typically characterized by clonal ITH, involving shared or divergent mutations in neoplastic sub-clones within primary and metastatic sites. Disease progression and therapy resistance are also influenced by non-clonal ITH, related to interactions with the tumor microenvironment and further genomic changes. Notably, significant alterations occur in nonmalignant cells, including cancer-associated fibroblast and immune cells, during tumor progression. This review provides an overview of the complex nature of HGSOC, encompassing its various aspects of intratumor heterogeneity, histological patterns, and its dynamic evolution during progression and therapy resistance.

Evaluation of a Set of miRNAs in 26 Cases of Fatal Traumatic Brain Injuries.

In forensic medicine, identifying novel biomarkers for use as diagnostic tools to ascertain causes of death is challenging because of sample degradation. To that aim, a cohort (n = 26) of fatal traumatic brain injuries (TBIs) were tested for three candidate miRNAs (namely, miR-124-3p, miR-138-5p, and miR144-3p). For each case, three FFPE specimens (coup area (CA), contrecoup area (CCA), and the corpus callosum (CC)) were investigated, whereas the FFPE brain tissues of 45 subjects (deceased due to acute cardiovascular events) were used as controls. Relative quantification via the ∆∆Ct method returned significantly higher expression levels of the three candidate miRNAs (p < 0.01) in the TBI cases. No difference was detected in the expression levels of any miRNA investigated in the study among the CA, CCA, and CC. Furthermore, the analyzed miRNAs were unrelated to the TBI samples' post-mortem intervals (PMIs). On the contrary, has-miR-124-3p ahashsa-miR-144-3p were significantly correlated (p < 0.01) with the agonal time in TBI deaths. Since the RNA was highly degraded in autoptic FFPE tissues, it was impossible to analyze the mRNA targets of the miRNAs investigated in the present study, highlighting the necessity of standardizing pre-analytical processes even for autopsy tissues.

A group of three miRNAs can act as candidate circulating biomarkers in liquid biopsies from melanoma patients.

Background: Staging of melanoma and follow up after melanoma diagnosis aims at predicting risk and detecting progression or recurrence at early stage, respectively in order to timely start and/or change treatment. Tumor thickness according to Breslow, status of the sentinel node and value of the lactate dehydrogenase (LDH) are well-established prognostic markers for metastatic risk, but reliable biomarkers identifying early recurrence or candidates who may benefit best from medical treatment are still warranted. Liquid biopsy has emerged to be a suitable method for identifying biomarkers for early cancer diagnosis, prognosis, therapeutic response prediction, and patient follow-up. Liquid biopsy is a blood-based non-invasive procedure that allows analyzing circulating analytes, including extracellular vesicles. Methods: In this study we have explored the use of 7 miRNAs, namely hsa-miR-149-3p, hsa-miR-150-5p, hsa-miR-21-5p, hsa-miR-200c-3p, hsa-miR-134-5p, hsa-miR-144-3p and hsa-miR-221-3p in plasma exosomes to discriminate melanoma patients from controls without melanoma in a cohort of 92 individuals. Results and discussion: Our results showed that three out seven miRNAs, namely hsa-miR-200c-3p, hsa-miR-144-3p and hsa-miR-221-3p were differentially expressed in plasma-derived exosomes from melanoma patients and controls. Furthermore, the expression of the three miRNAs may be a promising ancillary tool as a melanoma biomarker, even for discriminating between nevi and melanoma.

Nanomechanical Characterization of Ovarian Cancer Cell Lines as a Marker of Response to 2c Treatment.

Epithelial ovarian cancers (EOCs) are a heterogeneous group of tumors with different molecular and clinical features. In past decades, few improvements have been achieved in terms of EOC management and treatment efficacy, such that the 5-year survival rate of patients remained almost unchanged. A better characterization of EOCs' heterogeneity is needed to identify cancer vulnerabilities, stratify patients and adopt proper therapies. The mechanical features of malignant cells are emerging as new biomarkers of cancer invasiveness and drug resistance that can further improve our knowledge of EOC biology and allow the identification of new molecular targets. In this study, we determined the inter and intra-mechanical heterogeneity of eight ovarian cancer cell lines and their association with tumor invasiveness and resistance to an anti-tumoral drug with cytoskeleton depolymerization activity (2c).

Long-term effect of SARS-CoV-2 infection on cardiovascular outcomes and all-cause mortality.

Since the very beginning of the coronavirus disease 2019 (COVID-19) pandemic in early 2020, it was evident that patients with cardiovascular disease (CVD) were at an increased risk of developing severe illness, and complications spanning cerebrovascular disorders, dysrhythmias, acute coronary syndrome, ischemic and non-ischemic heart disease, pericarditis, myocarditis, heart failure, thromboembolic disease, stroke, and death. Underlying these was excessive systemic inflammation and coagulopathy due to SARS-COV-2 infection, the effects of which also continued long-term as evidenced by post-COVID-19 cardiovascular complications. The acute and chronic cardiovascular effects of COVID-19 occurred even among those who were not hospitalized and had no previous CVD or those with mild symptoms. This comprehensive review summarizes the current understanding of molecular mechanisms triggered by the SARS-CoV-2 virus on various cells that express the angiotensin-converting enzyme 2, leading to endothelial dysfunction, inflammation, myocarditis, impaired coagulation, myocardial infarction, arrhythmia and a multisystem inflammatory syndrome in children or Kawasaki-like disease.

AKT Isoforms Interplay in High-Grade Serous Ovarian Cancer Prognosis and Characterization.

High-grade serous ovarian cancer (HGSOC) is among the deadliest gynecological malignancies. The acquired resistance to platinum-based therapies and the intrinsic heterogeneity of the disease contribute to the low survival rate. To improve patients' outcomes, new combinatorial approaches able to target different tumor vulnerabilities and enhance the efficacy of the current therapies are required. AKT inhibitors are promising antineoplastic agents able to act in synergy with PARP inhibitors, but the spectrum of patients who can benefit from this combination is unclear, since the role of the three different isoforms of AKT is still unknown. Here, we study the expression of AKT isoforms on a retrospective cohort of archive tissue by RT-droplet digital PCR (ddPCR) analyzing their association with the clinicopathological features of patients. Based on AKT1/AKT2 and AKT1/AKT3 ratios, we define four AKT classes which were related to patients' survival, tumor morphology and BRCA1 expression. Moreover, our results show that high AKT3 expression levels were frequently associated with tumors having classic features, a low number of mitoses and the presence of psammoma bodies. Overall, our study obtains new insights on AKT isoforms and their associations with the clinicopathological features of HGSOC patients. These evidences could help to better define the subsets of patients who can benefit from AKT and PARP inhibitors therapy in future clinical trials.

Histological patterns and intra-tumor heterogeneity as prognostication tools in high grade serous ovarian cancers.

OBJECTIVE: High grade serous ovarian carcinoma (HGSOC) is the most common type of malignant ovarian neoplasm and the main cause of ovarian cancer related deaths worldwide. Although novel biomarkers such as homologous recombination deficiency testing have been implemented into the clinical decision-making algorithm since diagnosis, morphological classification and immunohistochemistry analysis are essential for diagnostic purpose. This study aims at identifying histologic and clinical features that can be predictive of patients' prognosis. METHODS: Morphological and architectural characterization including SET (Solid-Endometroid-Transitional)/Classic features was carried out in a cohort of 234 patients analyzing 695 slides. From each slide tumor infiltrating lymphocyte (TILs), the presence of necrosis, the number of mitoses, the presence of psammoma bodies, giant cells and atypical mitoses were recorded. Morphological heterogeneity was quantified by the Shannon's diversity index (SDI) considering the percentage of each architectural pattern per patient's slide. RESULTS: The frequency of architectural patterns and morphological variables varied with respect of the surgical strategy (primary debulking surgery vs interval surgery after neoadjuvant chemotherapy). HGSOCs exhibiting SET features had a longer overall as well as progression free survival. Among SET features, pseudo-endometrioid and transitional like patterns had the best outcome, while it was heterogenous for solid pattern, that had better outcome for BRCA 1 negative and less heterogeneous tumors. In patients submitted to neoadjuvant chemotherapy a higher intratumor heterogeneity as defined by SDI was a negative independent prognostic factor. CONCLUSIONS: A comprehensive histological examination considering architectural patterns and their heterogeneity can help in prognostication of HGSOCs.

Cell-stiffness and morphological architectural patterns in clinical samples of high grade serous ovarian cancers.

High grade serous ovarian carcinoma (HGSOC) is recognized as the most frequent type of ovarian cancer and the main cause of ovarian cancer related deaths worldwide. Although homologous recombination deficiency testing has been adopted in the clinical workflow, morphological analysis remains the main diagnostic tool. In this study Atomic Force Microscopy (AFM) was tested in standard hematoxylin and eosin (H&E) stained sections to investigate the biomechanical properties of different architectural growing patterns of HGSOC. Our results showed that AFM was able to discriminate HGSOC morphological growing patterns as well as patients' stage. Micropapillary pattern, which has been associated to poor outcome, had lower Young's moduli. In addition stage IV HGSOC was significantly softer than stage III cancers. Based on our results, AFM analysis could represent an additional tool in HGSOC morphological diagnosis as the biomechanical proprieties of HGSOC were quantitatively associated to tumor staging and architectural pattern.

A Novel HPLC-Based Method to Investigate on RNA after Fixation.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Reliability of miRNA Analysis from Fixed and Paraffin-Embedded Tissues.

In clinical practice, patients' tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin's solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin's solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin's-fixed specimens, but on average, higher Ct values and lower copies/µL were found in Bouin's-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin's ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin's-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin's-fixed ones.