Comprehensive characterization and effective combinatorial targeting of high-grade serous ovarian cancer via single-cell analysis

Ovarian cancer kills more than 40 000 women in Europe and more than 150 000 women globally each year. High-grade serous ovarian cancer (HGSOC) is the most common and most difficult to treat subtype of the disease. The high-grade serous tumors are highly heterogeneous, therefore, though most of the patients respond well to surgery and chemotherapy initially, more than half experience relapse. HERCULES is a research project funded by the EU H2020 program with the target to characterize comprehensively high grade serous ovarian cancers to find novel therapeutic strategies to fight them. The aim of my PhD thesis was to validate biomarkers identified within HERCULES project in a retrospective case study of patients affected by HGSOC, studying tumor heterogeneity and evaluating the effects of pre-analytical variables, in particular fixation, in the validation process. High grade serous ovarian cancer samples were characterized validating selected biomarkers at both RNA and protein level. Molecular analysis and in situ analysis were performed on multiple tissue biopsies in order to detect spatial heterogeneity and, moreover, biomechanical proprieties of fixed tumor tissues were measured. Lastly, the reliability of molecular analyses on archive tissues were assessed, determining the effect of formalin and Bouin\u2019s fixation at RNA level and evaluating their impact on gene expression using different platforms. Our results showed that detail morphological and immunophenotypical analyses, at the level of the entire tissue slide and not of TMA spots (Tissue micro array), of HGSOC tumors are paramount for the differential diagnosis as well as for both prognostication and therapy. In this view, biomechanical properties, by AFM can support the morphological findings. Among the immunohistochemical markers, Ki67 and BRCA1 have been shown their predictive value for response to first line chemotherapy and overall survival in HGSOC patients. Furthermore, neo adjuvant chemotherapy seems to have a detrimental effect on patient in our cohort. At the RNA level, cyclin C and HLA-B biomarkers showed their prognostic value indicating longer overall survival, while AKTs isoforms have shown a different impact on patients\u2019 outcome. Regarding the pre-analytical variables, fixation confirmed to have a deep impact on molecular analyses, especially in RNA expression. Tissues with highly fragmented RNA such those fixed in Bouin\u2019s can lead to analytical bias in both ddPCR, RT-qPCR, Nanostring and RNAscope technologies. A careful selection of samples with proper nucleic acids quality and integrity is of paramount importance before starting any molecular analysis. Also in that case, to minimize the effect of sample to sample variability a proper sample size should be used. Bouin\u2019s fixed samples because of their high level of nucleic acids fragmentation are not recommended for mRNA expression analyses, especially for low expressed targets. Contrarily, miRNAs, giving their length, are more resistant to fixation procedures and can be used for RNA expression analyses in both formalin and Bouin\u2019s tissues after a proper method of normalizatio

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

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

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)

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

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

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 CVS complications. The acute and chronic CVS 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