The Pathogenic RET Val804Met Variant in Acromegaly: A New Clinical Phenotype?

Several genetic investigations were conducted to identify germline and somatic mutations in somatotropinomas, a subtype of pituitary tumors. To our knowledge, we report the first acromegaly patient carrying a RET pathogenic variant: c.2410G>A (rs79658334), p.Val804Met. Alongside the fact that the patient's father and daughter carried the same variant, we investigated the clinical significance of this variant in the context of somatotropinomas and other endocrine tumors, reviewing the RET mutations' oncogenic mechanisms. The aim was to search for new targets to precisely manage and treat acromegaly. Our case describes a new phenotype associated with the RET pathogenic variant, represented by aggressive acromegaly, and suggests consideration for RET mutation screening if NGS for well-established PitNET-associated gene mutations renders negative.

Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South-Central Italian Cohort.

The incidence of cystic fibrosis (CF) and the spectrum of cystic fibrosis transmembrane conductance regulator (CFTR) gene variants differ among geographic regions. Differences in CF carrier distribution are also reported among Italian regions. We described the spectrum of the CFTR variants observed in a large group of subjects belonging from central-southern Italy. We also provide a predictive evaluation of the novel variants identified. CFTR screening was performed in a south-central Italian cohort of 770 subjects. We adopted a next-generation sequencing (NGS) approach using the Devyser CFTR NGS kit on the Illumina MiSeq System coupled with Amplicon Suite data analysis. Bioinformatics evaluation of the impact of novel variants was described. Overall, the presence of at least one alternative allele in the CFTR gene was recorded for 23% of the subjects, with a carrier frequency of CF pathogenic variants of 1:12. The largest sub-group corresponded to the heterozygous carriers of a variant with a conflicting interpretation of pathogenicity. The common CFTR p.(Phe508del) pathogenic variants were identified in 37% of mutated subjects. Bioinformatics prediction supported a potential damaging effect for the three novel CFTR variants identified: p.(Leu1187Phe), p.(Pro22Thr), and c.744-3C > G. NGS applied to CF screening had the benefit of: effectively identifying asymptomatic carriers. It lies in a wide overview of CFTR variants and gives a comprehensive picture of the carrier prevalence. The identification of a high number of unclassified variants may represent a challenge whilst at the same time being of interest and relevance for clinicians.

Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family.

Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

A novel low-density lipoprotein receptor variant in a Ukrainian patient: a case report and overview of the disease-causing low-density lipoprotein receptor variants associated to familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is characterized by high low-density lipoprotein-cholesterol levels and it is primarily caused by pathogenic/likely pathogenic variants (P/LPVs) in LDLR, APOB or PCSK9 genes. Next generation sequencing (NGS) technology allows the evaluation of more genes simultaneously, rising the diagnostic throughput of genomics laboratories. MATERIALS AND METHODS: We report a Ukrainian 37-year-old woman hypercholesterolemic since 2010. Despite a suggestive family history, FH was suspected only when the patient referred to the Endocrine and Metabolic Diseases Center of the Fondazione Policlinico Universitario A. Gemelli IRCCS in Rome. After specialist advice, genetic testing was offered to the patient at our Molecular and Genomic Diagnostics Unit. RESULTS: A targeted NGS-based pipeline highlighted a novel out-of-frame deletion in the LDLR gene. This variant has a clear deleterious effect on the LDLR protein and it can be classified as PV. CONCLUSIONS: The ideal model of care for FH is an evidence-based system aimed to provide the highest-quality health services to all FH patients. In fact, this study reports that the integrated care pathway adopted in our hospital for FH patients led successfully to the discovery of a novel LDLR PV in an Ukrainian patient. The finding of this LDLR variant allowed the clinical FH diagnosis in this patient and in her family, expanding the knowledge of FH-related genetic variants in the Ukrainian population.

Mutational status of plasma exosomal KRAS predicts outcome in patients with metastatic colorectal cancer.

Liquid biopsy has become a useful alternative in metastatic colorectal cancer (mCRC) patients when tissue biopsy of metastatic sites is not feasible. In this study we aimed to investigate the clinical utility of circulating exosomes DNA in the management of mCRC patients. Exosomes level and KRAS mutational status in exosomal DNA was assesed in 70 mCRC patients and 29 CRC primary tumor and were analysed at different disease steps evaluating serial blood samples (240 blood samples). There was a significant correlation between the extension of disease and exosomes level and the resection of primary localized tumor was correlated with a decrease of KRAS G12V/ D copies and fractional abundance in metastatic disease. CEA expression and liver metastasis correlated with a higher number of KRAS G12V/D copies/ml and a higher fractional abundance; in the subgroup of mCRC patients eligible for surgery, the size of tumor and the radiological response were related to exosomes level but only the size was related to the number of KRAS WT copies; both KRAS wild-type and mutated levels were identified as a prognostic factor related to OS. Finally, we found that 91% of mutated mCRC patients became wild type after the first line chemotherapy but this status reverted in mutated one at progression in 80% of cases. In a prospective cohort of mCRC patients, we show how longitudinal monitoring using exosome-based liquid biopsy provides clinical information relevant to therapeutic stratification.

Glucose-6-phosphate dehydrogenase deficiency and hydroxychloroquine in the COVID-19 era: a mini review.

The coronavirus disease 2019 (COVID-19) is until today a global health emergency. In an immense effort, effective drugs against COVID-19 are searched and intensive researches on possible repurposing of antiviral agents are performed. Since chloroquine (CQ) and hydroxychloroquine (HCQ) have shown in vitro anti- COVID-19 activities, the potential effect of CQ/HCQ to treat and/or prevent COVID-19 infection has caused global attention. However, concern regarding possible hemolysis in G6PD-deficient COVID-19 patients exists and for this reason, the association between HCQ and G6PD deficiency (G6PDD) is back in the limelight. This study aims to answer the question raised by Mastroianni et al. "Hydroxychloroquine: Culprit or Innocent Bystander in G6PD-Deficient Patients with COVID-19?", reporting all cases of HCQ in G6PD deficient COVID-19 patients published on PubMed (pubmed.ncbi.nlm.nih.gov), in addition to the Mastroianni's patient. In our opinion, after an accurate revision of these cases and responding the question raised by Mastroianni et al., we believe that it is difficult to reach a final verdict about the definitive role of HCQ in these patients. The COVID-19 pandemic has reopened attention on HCQ use and G6PDD. G6PD status is extremely important in modulating the level of reactive oxygen species and many cellular immune responses such as enhanced production of the pro-inflammatory cytokine and inflammasome activation. Since these processes are involved in COVID-19 infection, acute hemolytic anemia, a severe complication of the G6PDD, can occur in these patients. In this context, the role of HCQ, usually effective, safe, and well tolerated in G6PD deficient patients, must be redefined in these patients with COVID-19.As consequence, answering the question: "Hydroxychloroquine: Culprit or Innocent Bystander in G6PD-Deficient Patients with COVID-19?", we state that it is risky to believe that HCQ may be an "innocent bystander" in G6PD-deficient COVID-19 patients.

Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

Extracellular Vesicles and Cancer: A Focus on Metabolism, Cytokines, and Immunity.

A better understanding of the mechanisms of cell communication between cancer cells and the tumor microenvironment is crucial to develop personalized therapies. It has been known for a while that cancer cells are metabolically distinct from other non-transformed cells. This metabolic phenotype is not peculiar to cancer cells but reflects the characteristics of the tumor microenvironment. Recently, it has been shown that extracellular vesicles are involved in the metabolic switch occurring in cancer and tumor-stroma cells. Moreover, in an immune system, the metabolic programs of different cell subsets are distinctly associated with their immunological function, and extracellular vesicles could be a key factor in the shift of cell fate modulating cancer immunity. Indeed, during tumor progression, tumor-associated immune cells and fibroblasts acquire a tumor-supportive and anti-inflammatory phenotype due to their interaction with tumor cells and several findings suggest a role of extracellular vesicles in this phenomenon. This review aims to collect all the available evidence so far obtained on the role of extracellular vesicles in the modulation of cell metabolism and immunity. Moreover, we discuss the possibility for extracellular vesicles of being involved in drug resistance mechanisms, cancer progression and metastasis by inducing immune-metabolic effects on surrounding cells.

Low-intensity pulsed ultrasound affects growth, differentiation, migration, and epithelial-to-mesenchymal transition of colorectal cancer cells.

Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.

CD147 Promotes Cell Small Extracellular Vesicles Release during Colon Cancer Stem Cells Differentiation and Triggers Cellular Changes in Recipient Cells.

Cancer cells secrete small extracellular vesicles (sEVs) that are involved in the remodeling of tumor microenvironment (TME) and can promote tumor progression. The role of sEVs and their molecular key players in colon cancer stem cells differentiation are poorly understood. This study aimed to analyze the role and content of sEVs released during the differentiation of colorectal cancer stem cells. Here we show that sEVs secretion during colon cancer stem cells differentiation is partially controlled by CD147, a well-known player involved in colon cancer tumorigenesis. CD147 + sEVs activate a signaling cascade in recipient cells inducing molecular invasive features in colon cancer cells. CD147 knockdown as well as anti-CD147 antibodies impaired sEVs release and downstream effects on recipient cells and blocking multivesicular body maturation prevented sEVs release during the differentiation. Our findings reveal a functional role of CD147 in promoting sEVs release during the differentiation of colon cancer stem cells and in triggering cellular changes in recipient cells.