Plasma Circular RNAs as Biomarkers for Breast Cancer

Breast cancer (BC) is currently the most common neoplasm, the second leading cause of cancer death in women worldwide, and is a major health problem. The discovery of new biomarkers is crucial to improve our knowledge of breast cancer and strengthen our clinical approaches to diagnosis, prognosis, and follow-up. In recent decades, there has been increasing interest in circulating RNA (circRNA) as modulators of gene expression involved in tumor development and progression. The study of circulating circRNAs (ccircRNAs) in plasma may provide new non-invasive diagnostic, prognostic, and predictive biomarkers for BC. This review describes the latest findings on BCassociated ccircRNAs in plasma and their clinical utility. Several ccircRNAs in plasma have shown great potential as BC biomarkers, especially from a diagnostic point of view. Mechanistically, most of the reported BC-associated ccircRNAs are involved in the regulation of cell survival, proliferation, and invasion, mainly via MAPK/AKT signaling pathways. However, the study of circRNAs is a relatively new area of research, and a larger number of studies will be crucial to confirm their potential as plasma biomarkers and to understand their involvement in BC

Next-Generation Sequencing and Triple-Negative Breast Cancer: Insights and Applications

The poor survival of triple-negative breast cancer (TNBC) is due to its aggressive behavior, large heterogeneity, and high risk of recurrence. A comprehensive molecular investigation of this type of breast cancer using high-throughput next-generation sequencing (NGS) methods may help to elucidate its potential progression and discover biomarkers related to patient survival. In this review, the NGS applications in TNBC research are described. Many NGS studies point to TP53 mutations, immunocheckpoint response genes, and aberrations in the PIK3CA and DNA repair pathways as recurrent pathogenic alterations in TNBC. Beyond their diagnostic and predictive/prognostic value, these findings suggest potential personalized treatments in PD -L1-positive TNBC or in TNBC with a homologous recombination deficit. Moreover, the comprehensive sequencing of large genomes with NGS has enabled the identification of novel markers with clinical value in TNBC, such as AURKA, MYC, and JARID2 mutations. In addition, NGS investigations to explore ethnicity-specific alterations have pointed to EZH2 overexpression, BRCA1 alterations, and a BRCA2-delaAAGA mutation as possible molecular signatures of African and African American TNBC. Finally, the development of long-read sequencing methods and their combination with optimized short-read techniques promise to improve the efficiency of NGS approaches for future massive clinical use

A Case–Control Study by ddPCR of ALU 260/111 and LINE-1 266/97 Copy Number Ratio in Circulating Cell-Free DNA in Plasma Revealed LINE-1 266/97 as a Potential Biomarker for Early Breast Cancer Detection

Background: In Western countries, breast cancer (BC) is the most common cancer in women. Early detection has a positive impact on survival, quality of life, and public health costs. Mammography screening programs have increased early detection rates, but new approaches to more personalized surveillance could further improve diagnosis. Circulating cell-free DNA (cfDNA) in blood could provide a potential tool for early diagnosis by analyzing cfDNA quantity, circulating tumor DNA mutations, or cfDNA integrity (cfDI). Methods: Plasma was obtained from the blood of 106 breast cancer patients (cases) and 103 healthy women (controls). Digital droplet PCR was used for the determination of ALU 260/111 bp and LINE-1 266/97 bp copy number ratio and cfDI. cfDNA abundance was calculated using copies of the EEF1A2 gene. The accuracy of biomarker discrimination was analyzed with receiver operating characteristic curve (ROC). Sensitivity analyses were performed to account for age as a potential confounder. Results: Cases had significantly lower ALU 260/111 or LINE-1 266/97 copy number ratios (median; ALU 260/111 = 0.08, LINE-1 266/97 = 0.20), compared with control (median; ALU 260/111 = 0.10, LINE-1 266/97 = 0.28) (p < 0.001). ROC analysis showed that copy number ratio discriminated cases from controls (area under the curve, AUC = 0.69, 95% CI: 0.62–0.76 for ALU and 0.80, 95% CI: 0.73–0.86 for LINE-1). ROC from cfDI confirmed the better diagnostic performance of LINE-1 compared with ALU. Conclusions: Analysis of LINE-1 266/97 copy number ratio or cfDI by ddPCR appears to be a useful noninvasive test that could aid in early BC detection. Further studies in a large cohort are needed to validate the biomarker

Annali storici di Principato Citra A. 4, n. 1.1 (2006)

A. 4, n. 1.1 (2006): G. Guardia, Editoriale, P. 3 ; D. Ienna, Menhir a "la Mannina". Un sito megalitico a San Nazario di San Mauro La Bruca? Materiali e ipotesi interpretative, P. 5 ; A. Tierno, Il Vaticano Borgiano gr 27: un rotolo liturgico in lingua greca prodotto a Salerno, P. 44 ; G. Palmisciano, Baronissi nei moti del 1848, P. 54 ; C. Cerone, L’'arrivo dell'illuminazione a Capaccio e Agropoli. Dalle lampade a gas alla nazionalizzazione dell'energia. La centrale idroelettrica Maida, P. 68 ; F. La Greca, Paestanae valles: un antico nome per il Cilento?, P. 104 ; A. Giudice, Da Capo Palinuro alla conca di Sapri: la romanizzazione di un territorio, P. 110 ; Pietro III Paleologo di Bisanzio, Note storiche sulla vita del Sacro Militare Ordine Costantiniano di S. Giorgio, con la Regola di S. Basilio, dalla sua fondazione al gran magistero della Imperiale Famiglia dei Paleologo di Bisanzio, P. 124 ; M. Cerrato – P. Zoccoli, Elementi per la gestione del marketing strategico del prodotto tipico. Il caso di un formaggio caprino, P. 140 ; E. Frescani, "Lò scritto meno del successo". I racconti di Antonio Sessa, un notaio salernitano del XVII secolo, P. 153 ; A. Capano, Sapri, note storiche e il suo Catasto "provvisorio" del 1815, P. 162 ; M. Di Pasquale, 1815 - San Martino Cilento. Un processo per tentato omicidio, P. 178 ; A. Tortorella Bracco, Da un cassettone dell'800 una romantica storia d'amore, P. 202

Defining Kawasaki disease and pediatric inflammatory multisystem syndrome-temporally associated to SARS-CoV-2 infection during SARS-CoV-2 epidemic in Italy: results from a national, multicenter survey

Background: There is mounting evidence on the existence of a Pediatric Inflammatory Multisystem Syndrome-temporally associated to SARS-CoV-2 infection (PIMS-TS), sharing similarities with Kawasaki Disease (KD). The main outcome of the study were to better characterize the clinical features and the treatment response of PIMS-TS and to explore its relationship with KD determining whether KD and PIMS are two distinct entities. Methods: The Rheumatology Study Group of the Italian Pediatric Society launched a survey to enroll patients diagnosed with KD (Kawasaki Disease Group - KDG) or KD-like (Kawacovid Group - KCG) disease between February 1st 2020, and May 31st 2020. Demographic, clinical, laboratory data, treatment information, and patients' outcome were collected in an online anonymized database (RedCAP®). Relationship between clinical presentation and SARS-CoV-2 infection was also taken into account. Moreover, clinical characteristics of KDG during SARS-CoV-2 epidemic (KDG-CoV2) were compared to Kawasaki Disease patients (KDG-Historical) seen in three different Italian tertiary pediatric hospitals (Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste; AOU Meyer, Florence; IRCCS Istituto Giannina Gaslini, Genoa) from January 1st 2000 to December 31st 2019. Chi square test or exact Fisher test and non-parametric Wilcoxon Mann-Whitney test were used to study differences between two groups. Results: One-hundred-forty-nine cases were enrolled, (96 KDG and 53 KCG). KCG children were significantly older and presented more frequently from gastrointestinal and respiratory involvement. Cardiac involvement was more common in KCG, with 60,4% of patients with myocarditis. 37,8% of patients among KCG presented hypotension/non-cardiogenic shock. Coronary artery abnormalities (CAA) were more common in the KDG. The risk of ICU admission were higher in KCG. Lymphopenia, higher CRP levels, elevated ferritin and troponin-T characterized KCG. KDG received more frequently immunoglobulins (IVIG) and acetylsalicylic acid (ASA) (81,3% vs 66%; p = 0.04 and 71,9% vs 43,4%; p = 0.001 respectively) as KCG more often received glucocorticoids (56,6% vs 14,6%; p &lt; 0.0001). SARS-CoV-2 assay more often resulted positive in KCG than in KDG (75,5% vs 20%; p &lt; 0.0001). Short-term follow data showed minor complications. Comparing KDG with a KD-Historical Italian cohort (598 patients), no statistical difference was found in terms of clinical manifestations and laboratory data. Conclusion: Our study suggests that SARS-CoV-2 infection might determine two distinct inflammatory diseases in children: KD and PIMS-TS. Older age at onset and clinical peculiarities like the occurrence of myocarditis characterize this multi-inflammatory syndrome. Our patients had an optimal response to treatments and a good outcome, with few complications and no deaths

Comprehensive Characterization and Effective Combinatorial Targeting of Epithelial Ovarian Cancer and 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. The epithelial ovarian cancer (EOC) is the most common subtype and encompasses a collection of neoplasms with distinct clinical-pathological, molecular features and prognosis; among them the most common are the clear cells (CCOC), the mucinous (MOC), the endometrioid (ENOC), the low grade serous (LGSOC) and the high grade serous ovarian cancer (HGSOC). The HGSOC, in particular, is the most deadly form of EOC due to its high intratumor heterogeneity and lack of early diagnosis. The mechanical properties can be useful as new possible biomarkers of EOC subtypes in association to the classic ones used in clinical routine. In this thesis, the mechanical characterization of 9 ovarian cancer cell lines with different histological and morphological classification was carried out by AFM. Then, the achieved mechanical properties were evaluated on the same cell lines as markers of metastatic potential and drug sensitivity against 2C, a compound synthesized by the group of prof. Benedetti at the University of Trieste with anti-tumoral and cytoskeleton depolymerizing activity. Finally, an assessment of the effects of the culture medium composition on mechanical properties was performed. The mechanical characterization showed that the HGSOC cell lines had a high variability in the average Young’s modulus and resulted stiffer than the other cell lines with different histological classification. Moreover, the cell lines displayed two distinct single cell Young’s moduli distribution patterns: unimodal and bimodal. The cell lines with bimodal pattern showed two different populations with distinct mechanical behaviors. The invasion assay indicated a correlation between the stiffness decrease and the increase of invasion capacity. Accordingly, in cell lines with bimodal pattern only the “softer” population showed eventually the metastatic potential to invade. The cell lines with bimodal pattern were more resistant to 2C than the ones with unimodal pattern. For cell lines with bimodal pattern, the “stiffer” population had tendentially a higher resistance to 2C than the “softer” one. The F-actin network organization could influence the 2C resistance and the stiffness of cell lines: the HEY and OVCAR4 (high 2C resistance and high average Young’s modulus) had an Actin cytoskeleton more distributed over the cell than the TYKNU (low 2C resistance and low average Young’s modulus). Finally, variations in the culture medium components had an impact on the achieved Young’s moduli. This highlighted the need to develop optimized culture protocols for elasticity measurements, able to overcome the effects of different media on the mechanical properties.Ovarian cancer kills more than 40 000 women in Europe and more than 150 000 women globally each year. The epithelial ovarian cancer (EOC) is the most common subtype and encompasses a collection of neoplasms with distinct clinical-pathological, molecular features and prognosis; among them the most common are the clear cells (CCOC), the mucinous (MOC), the endometrioid (ENOC), the low grade serous (LGSOC) and the high grade serous ovarian cancer (HGSOC). The HGSOC, in particular, is the most deadly form of EOC due to its high intratumor heterogeneity and lack of early diagnosis. The mechanical properties can be useful as new possible biomarkers of EOC subtypes in association to the classic ones used in clinical routine. In this thesis, the mechanical characterization of 9 ovarian cancer cell lines with different histological and morphological classification was carried out by AFM. Then, the achieved mechanical properties were evaluated on the same cell lines as markers of metastatic potential and drug sensitivity against 2C, a compound synthesized by the group of prof. Benedetti at the University of Trieste with anti-tumoral and cytoskeleton depolymerizing activity. Finally, an assessment of the effects of the culture medium composition on mechanical properties was performed. The mechanical characterization showed that the HGSOC cell lines had a high variability in the average Young’s modulus and resulted stiffer than the other cell lines with different histological classification. Moreover, the cell lines displayed two distinct single cell Young’s moduli distribution patterns: unimodal and bimodal. The cell lines with bimodal pattern showed two different populations with distinct mechanical behaviors. The invasion assay indicated a correlation between the stiffness decrease and the increase of invasion capacity. Accordingly, in cell lines with bimodal pattern only the “softer” population showed eventually the metastatic potential to invade. The cell lines with bimodal pattern were more resistant to 2C than the ones with unimodal pattern. For cell lines with bimodal pattern, the “stiffer” population had tendentially a higher resistance to 2C than the “softer” one. The F-actin network organization could influence the 2C resistance and the stiffness of cell lines: the HEY and OVCAR4 (high 2C resistance and high average Young’s modulus) had an Actin cytoskeleton more distributed over the cell than the TYKNU (low 2C resistance and low average Young’s modulus). Finally, variations in the culture medium components had an impact on the achieved Young’s moduli. This highlighted the need to develop optimized culture protocols for elasticity measurements, able to overcome the effects of different media on the mechanical properties

Angiogenesis and Ovarian Cancer: What Potential Do Different Subtypes of Circulating Endothelial Cells Have for Clinical Application?

Ovarian cancer (OC) remains the most fatal disease of gynaecologic malignant tumours. The neovasculature in the tumour microenvironment principally comprises endothelial cells. Haematogenous cancer metastases are significantly impacted by tumour neovascularisation, which predominantly depends on the tumour-derived endothelial vasculogenesis. There is an urgent need for biomarkers for the diagnosis, prognosis and prediction of drug response. Endothelial cells play a key role in angiogenesis and other forms of tumour vascularisation. Subtypes of circulating endothelial cells may provide interesting non-invasive biomarkers of advanced OC that might have the potential to be included in clinical analysis for patients’ stratification and therapeutic management. In this review, we summarise the reported studies on circulating endothelial subtypes in OC, detailing their isolation methods as well as their potential diagnostic, prognostic, predictive and therapeutic utility for clinical application. We highlight key biomarkers for the identification of circulating endothelial cell subtypes and their targets for therapies and critically point out future challenges

An Overview of Circulating Cell-Free Nucleic Acids in Diagnosis and Prognosis of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer due to its molecular heterogeneity and poor clinical outcomes. Analysis of circulating cell-free tumor nucleic acids (ctNAs) can improve our understanding of TNBC and provide efficient and non-invasive clinical biomarkers that may be representative of tumor heterogeneity. In this review, we summarize the potential of ctNAs to aid TNBC diagnosis and prognosis. For example, tumor fraction of circulating cell-free DNA (TFx) may be useful for molecular prognosis of TNBC: high TFx levels after neoadjuvant chemotherapy have been associated with shorter progression-free survival and relapse-free survival. Mutations and copy number variations of TP53 and PIK3CA/AKT genes in plasma may be important markers of TNBC onset, progression, metastasis, and for clinical follow-up. In contrast, the expression profile of circulating cell-free tumor non-coding RNAs (ctncRNAs) can be predictive of molecular subtypes of breast cancer and thus aid in the identification of TBNC. Finally, dysregulation of some circulating cell-free tumor miRNAs (miR17, miR19a, miR19b, miR25, miR93, miR105, miR199a) may have a predictive value for chemotherapy resistance. In conclusion, a growing number of efforts are highlighting the potential of ctNAs for future clinical applications in the diagnosis, prognosis, and follow-up of TNBC

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