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Hereditary breast and ovarian cancer syndrome is caused by germline mutations in BRCA1/2 genes. These genes are very large and their mutations are heterogeneous and scattered throughout the coding sequence. In addition to the above-mentioned mutations, variants of uncertain/unknown significance (VUSs) have been identified in BRCA genes, which make more difficult the clinical management of the patient and risk assessment. In the last decades, several laboratories have developed different databases that contain more than 2000 variants for the two genes and integrated strategies which include multifactorial prediction models based on direct and indirect genetic evidence, to classify the VUSs and attribute them a clinical significance associated with a deleterious, high/low or neutral risk. This review provides a comprehensive overview of literature studies concerning the VUSs, in order to assess their impact on the population and provide new insight useful for the appropriate patient management in clinical practice.

Recent advances in the detection of germline pathogenic variants (PVs) in BRCA1/2 genes have allowed a deeper understanding of the BRCA-related cancer risk. Several studies showed a significant heterogeneity in the prevalence of PVs across different populations. Because little is known about this in the Sicilian population, our study was aimed at investigating the prevalence and geographic distribution of inherited BRCA1/2 PVs in families from this specific geographical area of Southern Italy. We retrospectively collected and analyzed all clinical information of 1346 hereditary breast and/or ovarian cancer patients genetically tested for germline BRCA1/2 PVs at University Hospital Policlinico &ldquo, P. Giaccone&rdquo, of Palermo from January 1999 to October 2019. Thirty PVs were more frequently observed in the Sicilian population but only some of these showed a specific territorial prevalence, unlike other Italian and European regions. This difference could be attributed to the genetic heterogeneity of the Sicilian people and its historical background. Therefore hereditary breast and ovarian cancers could be predominantly due to BRCA1/2 PVs different from those usually detected in other geographical areas of Italy and Europe. Our investigation led us to hypothesize that a higher prevalence of some germline BRCA PVs in Sicily could be a population-specific genetic feature of BRCA-positive carriers.

Despite a proportion of renal cancer patients can experiment marked and durable responses to immune-checkpoint inhibitors, the treatment efficacy is widely variable and identifying the patient who will benefit from immunotherapy remains an issue. We performed a prospective study to investigate if soluble forms of the immune-checkpoints PD-1 (sPD-1), PD-L1 (sPD-L1), pan-BTN3As, BTN3A1, and BTN2A1, could be candidate to predict the response to immune-checkpoint blockade therapy. We evaluated the plasma levels in a learning cohort of metastatic clear cell renal carcinoma (mccRCC) patients treated with the anti-PD-1 agent nivolumab by ad hoc developed ELISA’s. Using specific cut-offs determined through ROC curves, we showed that high baseline levels of sPD-1 (>2.11ng/ml), sPD-L1 (>0.66ng/ml), and sBTN3A1 (>6.84ng/ml) were associated with a longer progression-free survival (PFS) to nivolumab treatment [median PFS, levels above thresholds: sPD-1, 20.7months (p 20%. The results were confirmed in a validation cohort of 20 mccRCC patients. The analysis of plasma dynamic changes after nivolumab showed a statistically significant decrease of sPD-1 after 2 cycles (Day 28) in the long-responder patients. Our study revealed that the plasma levels of sPD-1, sPD-L1, and sBTN3A1 can predict response to nivolumab, discriminating responders from non-responders already at therapy baseline, with the advantages of non-invasive sample collection and real-time monitoring that allow to evaluate the dynamic changes during cancer evolution and treatment.

Simple Summary Many bilateral breast cancer patients with increased hereditary susceptibility to breast cancer result negative for BRCA1 or BRCA2 pathogenic variants and, thus, need a further genetic testing through a broader gene panel. Some patients with negative test result for BRCA1/2 pathogenic variants may harbor pathogenic variants in other breast cancer susceptibility genes, including ATM, CHEK2, PALB2, PTEN, TP53. Of course, the use of a multi-gene panel provides clinicians more information through a single test. Therefore, we focused on potential clinical impact of a NGS-based multi-gene panel testing in bilateral breast cancer patients, in order to evaluate the utility of perform a most comprehensive genetic analysis in these subjects, regardless the criteria concerning personal and family history of cancer established by the current guidelines. Our study revealed that the use of a NGS-based multiple-gene panel testing could increase the detection rates of germline alterations in bilateral breast cancer patients. Abstract Patients with unilateral breast cancer (UBC) have an increased risk of developing bilateral breast cancer (BBC). The annual risk of contralateral BC is about 0.5%, but increases by up to 3% in BRCA1 or BRCA2 pathogenic variant (PV) carriers. Our study was aimed to evaluate whether all BBC patients should be offered multi-gene panel testing, regardless their cancer family history and age at diagnosis. We retrospectively collected all clinical information of 139 BBC patients genetically tested for germline PVs in different cancer susceptibility genes by NGS-based multi-gene panel testing. Our investigation revealed that 52 (37.4%) out of 139 BBC patients harbored germline PVs in high- and intermediate-penetrance breast cancer (BC) susceptibility genes including BRCA1, BRCA2, PTEN, PALB2, CHEK2, ATM, RAD51C. Nineteen out of 53 positively tested patients harbored a PV in a known BC susceptibility gene (no-BRCA). Interestingly, in the absence of an analysis performed via multi-gene panel, a significant proportion (14.4%) of PVs would have been lost. Therefore, offering a NGS-based multi-gene panel testing to all BBC patients may significantly increase the detection rates of germline PVs in other cancer susceptibility genes beyond BRCA1/2, avoiding underestimation of the number of individuals affected by a hereditary tumor syndrome.

Background: Several available data suggest the association between specific molecular subtypes and BRCA1/2 mutational status. Previous investigations showed the association between BRCA1/2 pathogenic variants (PVs) in specific genomic regions and phenotypic variations of cancer relative risk, while the role of PV type and location in determining the breast cancer (BC) phenotypic features remains still unclear. The aim of this research was to describe the germline BRCA1/2 PVs in triple-negative breast cancer (TNBC) versus luminal-like BC and their potential leverage on BC phenotype. Patients & methods: We retrospectively collected and analyzed all clinical information of 531 patients with BC genetically tested for germline BRCA1/2 PVs by Next-Generation Sequencing analysis at University Hospital Policlinico “P. Giaccone” of Palermo (Sicily) from January 2016 to February 2020. Results: Our results corroborate the evidence that BRCA1-related tumors often have a profile which resembles the TNBC subtype, whereas BRCA2-associated tumors have a profile that resembles luminal-like BC, especially the Luminal B subtype. Interestingly, our findings suggest that the PVs identified in TNBC were not largely overlapping with those in luminal-like tumors. Differences in the frequency of two PVs potentially associated with different molecular tumor subtypes were observed. BRCA1-633delC was detected with relatively higher prevalence in patients with TNBC, whereas BRCA2-1466delT was found mainly in Luminal B tumors, but in no TNBC patient. Conclusion: Future studies examining the type and location of BRCA1/2 PVs within different molecular subtypes are required to verify our hypothesis and could provide an interesting insight into the complex topic of genotype–phenotype correlations. Additionally, a more in-depth understanding of the potential correlations between BRCA PVs and clinical and phenotypic features of hereditary BC syndrome patients could be the key to develop better strategies of prevention and surveillance in BRCA-positive carriers without disease.

Over the past 15 years, gastrointestinal stromal tumors (GISTs) have emerged from a poorly understood neoplasm to a well-defined tumor entity. Starting from 2000, the discovery of gain-of-function mutations involving KIT or PDGFRα (platelet-derived growth factor-α) genes and the development of tyrosine kinase inhibitors (TKIs), such as imatinib, revolutionized dramatically the management of GISTs. Due to the almost continual emergence of new data about biological complexity of GISTs and more sophisticated whole-genome technologies, to date, the role of molecular biology is clinically important to drive therapeutic decision making. The possibility of using liquid biopsy in GISTs was reported for the first time at the 2013 ASCO Annual Meeting. Detection of circulating tumor DNA (ctDNA) offers a wide spectrum of applications in GIST management. CtDNA correlates with the tumor burden, thus, after surgery may indicate the presence of minimal residual disease and patients with high risk of recurrence. In addition, during the clinical treatment, this approach could be used to identify early biomarkers of response and assess variations in whole genome, early identifying the development of secondary resistance.

Introduction/Aim: Chemotherapy or BCG given intravesically to prevent recurrence after transurethral resection (TUR) of non-muscle invasive bladder cancer (NMI-BC) cause frequent, sometime severe, local toxicity. As a consequence, many patients do not complete the planned treatment (1). A major challenge for the urologists is to identify an early biomarker of urothelial damage to recognize and prevent local toxicity improving patient’s compliance. The purpose of our research was to investigate the relation between urothelial injury by intravesical treatment and the expression of potential biomarkers in urine and/or in barbotage solution. The urinary HB-EGF expression in interstitial cystitis has been analyzed by a few studies (2, 3). As a preliminary step, the variations of Fibronectin (FN), Epidermal Growth Factor- Receptor (EGF-R) and Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) during intravesical therapy and after the administration of a solution with the potential role of urothelial repairing (hyaluronic acid and chondroitin sulphate) were investigated. Patients and Methods: the toxicity of intravesical therapy with mitomycin, epirubicin or BCG was classified in 3 grades (absent, moderate, severe). Urine and bladder washing solution during intravesical therapy in 55 patients after NMI-BC TUR and in 10 healthy controls for a total of 200 samples were collected. Total cellular RNA was isolated from the cell pellet using miRNeasy Mini Kit (Qiagen®). FN and EGF-R gene expression by Real Time quantitative PCR were analyzed. The expression of HB-EGF was measured in urine samples by ELISA (Abcam®). Results: In barbotage samples the FN gene expression and the EGF-R levels in our patients were respectively increased a median of 4.7 fold and decreased of 0.9 fold compared to controls. Before intravesical therapy and in absence of local toxicity, gene expression increased 1.9 fold for FN and 1.1 fold for EGF-R. In contrast, in patients with local toxicity due to intravesical therapy, the FN gene expression levels increased to a median of 5.82 fold, while EGF-R remained unchanged. The administration of hyaluronic acid and chondroitin sulphate solution decreased the mean FN gene expression from 3 to 0.6 fold, with concomitant symptomatic relief. HB-EGF protein median urine levels were 25.7 pg/ml in 13 patients before intravesical therapy and 18.9 pg/ml in 5 healthy controls. No significant variations in relation to the local toxicity. During therapy median HB-EGF protein levels in urine varied from 21.6 pg/ml in absence of toxicity to 25.7 pg/ml in case of severe toxicity to 18.5 pg/ml after hyaluronic acid and chondroitin sulphate solution. Preliminarily, the observed variations of HB-EGF, increasing no more than 1.2 fold compared to healthy controls, do not seem possible marker of urothelial damage. Discussion and Conclusion: EGF-R gene and HBEGF expressions do not seem to vary significantly in relation to local toxicity due to intravesical therapy. FN gene is overexpressed in presence of urothelial damage significantly reduced by intravesical hyaluronic acid and chondroitin sulphate solution administration, according with symptoms relief.

Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression, angiogenesis and metastasis. Thrombospondin-1 (TSP-1) has been shown to contrast angiogenesis in vivo. TSP-1 expression levels are inversely correlated with tumor vascularity and metastasis in colon cancer. Bio-informatic statistical analysis indicated that TSP-1 is hypothetical target of miR-21, miR-182, overexpressed in CRC, and let-7i which expression is down-regulated in this tumor. In this work we investigated whether TSP-1 expression could be regulated by miR-21, mir-182 and let-7i in HT29 colon cancer cell line. Methods: To investigated whether miR-21, mir-182 and let-7i directly modulates TSP-1 expression, we transfected HT29 cell line with pre-mir21, pre-mir182 and pre-let7i by using siPortNeo FX tranfection agent and after 48h we evaluated TSP-1 mRNA, using Quantitative Real Time-PCR, and intracellular and secreted protein level performed by Western blotting and ELISA. To confirm the modulation of TSP-1 by miRNAs we transfected HT29 cell line with anti-mir to target the mature form of miR-21, miR182 and let-7i. Results: Using Real-Time PCR we did not find any variation of TSP-1 mRNA expression levels after transfection with pre-mir21 in HT29 cell line, but we observed a down-regulation of cytosolic and secreted protein by Western blot and ELISA. In cells transfected with pre-mir182 we did not observe any down-regulation both TSP-1 mRNA and cytosolic and secreted protein. Finally, we did not find any variation of TSP-1 level in cells transfected with let- 7i. Results were confirmed by transfection with anti-mir21, anti- mir182 and anti-let7i and, using the same method, we evaluated TSP-1 expression. Conclusions: Data suggest that mir-182 induces degradation of TSP-1 mRNA in HT29 cell line, whereas mir-21 affects probably by blockage of TSP-1 translation. Let-7i does not seem involved in regulation of TSP-1 expression in HT29 cells. Understanding the molecular mechanism by which miRNAs regulate TSP-1 expression could be used to restore TSP-1 expression to contrast angiogenic events in colon cancer.