Mechanisms of glioblastoma replication: Ca2+ flares and Cl- currents.

Glioblastoma (GBM) is amongst the deadliest types of cancers, with no resolutive cure currently available. GBM cell proliferation in the patient's brain is a complex phenomenon controlled by multiple mechanisms. The aim of this study was to determine whether the ionic fluxes controlling cell duplication could represent a target for GBM therapy. In this work, we combined multi-channel Ca2+ and Cl- imaging, optical tweezers, electrophysiology and immunohistochemistry to describe the role of ion fluxes in mediating the cell volume changes that accompany mitosis of U87 GBM cells. We identified three main steps: (i) in round GBM cells undergoing mitosis, during the transition from anaphase to telophase and cytokinesis, large Ca2+ flares occur, reaching values of 0.5-1 µM; (ii) these Ca2+ flares activate Ca2+-dependent Cl- channels, allowing the entry of Cl- ions; (iii) to maintain osmotic balance, GBM cells swell to complete mitosis. This sequence of steps was validated by electrophysiological experiments showing that Cl- channels are activated either directly or indirectly by Ca2+, and by additional live-cell imaging experiments. Cl- channel blockers with different molecular structures, such as niflumic acid and carbenoxolone, blocked GBM replication by arresting GBM cells in a round configuration. These results describe the central role of Ca2+ flares and Cl- fluxes during mitosis and show that inhibition of Ca2+-activated Cl- channels blocks GBM replication, opening the way to new approaches for the clinical treatment of GBM. Implications: Our work identifies ionic fluxes occurring during cell division as targets for devising novel therapies for the glioblastoma treatment.

Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

Extracellular vesicle features are associated with COVID-19 severity.

COVID-19 is heterogeneous; therefore, it is crucial to identify early biomarkers for adverse outcomes. Extracellular vesicles (EV) are involved in the pathophysiology of COVID-19 and have both negative and positive effects. The objective of this study was to identify the potential role of EV in the prognostic stratification of COVID-19 patients. A total of 146 patients with severe or critical COVID-19 were enrolled. Demographic and comorbidity characteristics were collected, together with routine haematology, blood chemistry and lymphocyte subpopulation data. Flow cytometric characterization of the dimensional and antigenic properties of COVID-19 patients' plasma EVs was conducted. Elastic net logistic regression with cross-validation was employed to identify the best model for classifying critically ill patients. Features of smaller EVs (i.e. the fraction of EVs smaller than 200 nm expressing either cluster of differentiation [CD] 31, CD 140b or CD 42b), albuminemia and the percentage of monocytes expressing human leukocyte antigen DR (HLA-DR) were associated with a better outcome. Conversely, the proportion of larger EVs expressing N-cadherin, CD 34, CD 56, CD31 or CD 45, interleukin 6, red cell width distribution (RDW), N-terminal pro-brain natriuretic peptide (NT-proBNP), age, procalcitonin, Charlson Comorbidity Index and pro-adrenomedullin were associated with disease severity. Therefore, the simultaneous assessment of EV dimensions and their antigenic properties complements laboratory workup and helps in patient stratification.

Potential role of IgM-enriched immunoglobulin as adjuvant treatment in severe SARS-CoV-2 infection.

BACKGROUND: Severe COVID-19 patients are characterized by a dysregulated host response to an infection, with uncontrolled pro- and anti- inflammatory pathway activation. Consistent proportion of patients require admission in intensive care units and are at risk of progression to severe forms of disease. These patients are generally admitted during later stages of the disease, when effective antiviral and monoclonal antibody are not indicated. We aimed to assess the potential role of IgM-enriched intra venous immunoglobulins (IGAM) preparations in this setting. METHODS: This retrospective, observational case-controlled study was conducted at a single-center University Hospital of Udine in the Friuli Venezia Giulia Region of Italy. Patients referring to the center between March 2020 and April 2021 was included. During the study period, patient who received Pentaglobin® IGAM treatment (N.=56), administered as compassionate use, was compared with a control group (N.=169) to assess, by propensity score analysis, clinical outcome. RESULTS: Untreated controls required, respect to patient treated with IGAM therapy, longer time to hospitalization with no significant differences in death and orotracheal intubation requirement. Significant differences in the two cohort were in: SOFA was higher in treated, while D-dimer and P/F ratio was better in the treatment cohort. Multivariate logistic regression analysis performed on the "matched sample," obtained by a weighting propensity score approach, identify, as significant protective factor for death outcome, the Pentaglobin® treatment (0.820 [0.698-0.963], P=0.016) and low C-reactive protein (1.001 [1.000-1.002], P=0.031) value while the delay of onset hospitalization is associate with a worst outcome (0.983 [0.967-0.999], P=0.041). CONCLUSIONS: The present study offers a significant insight concerning the use of IgM-enriched immunoglobulin preparations in patients with SARS-CoV-2 severe infection and also could identifying the specific immunological and biochemical profile of the patient who can more benefit from this therapeutic option.

Machine learning to improve interpretability of clinical, radiological and panel-based genomic data of glioma grade 4 patients undergoing surgical resection.

BACKGROUND: Glioma grade 4 (GG4) tumors, including astrocytoma IDH-mutant grade 4 and the astrocytoma IDH wt are the most common and aggressive primary tumors of the central nervous system. Surgery followed by Stupp protocol still remains the first-line treatment in GG4 tumors. Although Stupp combination can prolong survival, prognosis of treated adult patients with GG4 still remains unfavorable. The introduction of innovative multi-parametric prognostic models may allow refinement of prognosis of these patients. Here, Machine Learning (ML) was applied to investigate the contribution in predicting overall survival (OS) of different available data (e.g. clinical data, radiological data, or panel-based sequencing data such as presence of somatic mutations and amplification) in a mono-institutional GG4 cohort. METHODS: By next-generation sequencing, using a panel of 523 genes, we performed analysis of copy number variations and of types and distribution of nonsynonymous mutations in 102 cases including 39 carmustine wafer (CW) treated cases. We also calculated tumor mutational burden (TMB). ML was applied using eXtreme Gradient Boosting for survival (XGBoost-Surv) to integrate clinical and radiological information with genomic data. RESULTS: By ML modeling (concordance (c)- index = 0.682 for the best model), the role of predicting OS of radiological parameters including extent of resection, preoperative volume and residual volume was confirmed. An association between CW application and longer OS was also showed. Regarding gene mutations, a role in predicting OS was defined for mutations of BRAF and of other genes involved in the PI3K-AKT-mTOR signaling pathway. Moreover, an association between high TMB and shorter OS was suggested. Consistently, when a cutoff of 1.7 mutations/megabase was applied, cases with higher TMB showed significantly shorter OS than cases with lower TMB. CONCLUSIONS: The contribution of tumor volumetric data, somatic gene mutations and TBM in predicting OS of GG4 patients was defined by ML modeling.

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

Background: Metastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC. Methods: A cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita's overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher's, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method. Results: Stratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03). Conclusion: These data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.

Nanotechnologies in Obstetrics and Cancer during Pregnancy: A Narrative Review.

Nanotechnology, the art of engineering structures on a molecular level, offers the opportunity to implement new strategies for the diagnosis and management of pregnancy-related disorders. This review aims to summarize the current state of nanotechnology in obstetrics and cancer in pregnancy, focusing on existing and potential applications, and provides insights on safety and future directions. A systematic and comprehensive literature assessment was performed, querying the following databases: PubMed/Medline, Scopus, and Endbase. The databases were searched from their inception to 22 March 2022. Five independent reviewers screened the items and extracted those which were more pertinent within the scope of this review. Although nanotechnology has been on the bench for many years, most of the studies in obstetrics are preclinical. Ongoing research spans from the development of diagnostic tools, including optimized strategies to selectively confine contrast agents in the maternal bloodstream and approaches to improve diagnostics tests to be used in obstetrics, to the synthesis of innovative delivery nanosystems for therapeutic interventions. Using nanotechnology to achieve spatial and temporal control over the delivery of therapeutic agents (e.g., commonly used drugs, more recently defined formulations, or gene therapy-based approaches) offers significant advantages, including the possibility to target specific cells/tissues of interest (e.g., the maternal bloodstream, uterus wall, or fetal compartment). This characteristic of nanotechnology-driven therapy reduces side effects and the amount of therapeutic agent used. However, nanotoxicology appears to be a significant obstacle to adopting these technologies in clinical therapeutic praxis. Further research is needed in order to improve these techniques, as they have tremendous potential to improve the accuracy of the tests applied in clinical praxis. This review showed the increasing interest in nanotechnology applications in obstetrics disorders and pregnancy-related pathologies to improve the diagnostic algorithms, monitor pregnancy-related diseases, and implement new treatment strategies.

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

Carmustine Wafers Implantation in Patients With Newly Diagnosed High Grade Glioma: Is It Still an Option?

Background: The implantation protocol for Carmustine Wafers (CWs) in high grade glioma (HGG) was developed to offer a bridge between surgical resection and adjuvant treatments, such as radio- and chemotherapy. In the last years, however, a widespread use of CWs has been limited due to uncertainties regarding efficacy, in addition to increased risk of infection and elevated costs of treatment. Objective: The aims of our study were to investigate the epidemiology of patients that underwent surgery for HGG with CW implantation, in addition to the assessment of related complications, long-term overall survival (OS), and associated prognostic factors. Methods: Three different medical databases were screened for conducting a systematic review of the literature, according to the PRISMA statement guidelines, evaluating the role of BCNU wafer implantation in patients with newly diagnosed HGG. The search query was based on a combination of medical subject headings (MeSH): "high grade glioma" [MeSH] AND "Carmustine" [MeSH] and free text terms: "surgery" OR "BCNU wafer" OR "Gliadel" OR "systemic treatment options" OR "overall survival." Results: The analysis of the meta-data demonstrated that there was a significant advantage in using CWs in newly diagnosed GBM in terms of OS, and a very low heterogeneity among the included studies [mean difference 2.64 (95% CI 0.85, 4.44); p = 0.004; I2149 = 0%]. Conversely, no significant difference between the two treatment groups in terms of PFS wad detected (p = 0.55). The analysis of complications showed a relatively higher rate in Carmustine implanted patients, although this difference was not significant (p = 0.53). Conclusions: This meta-analysis seems to suggest that CWs implantation plays a significant role in improving the OS, when used in patients with newly diagnosed HGG. To minimize the risk of side effects, however, a carful patient selection based mainly on patient age and tumor volume should be desirable.

Identification of a Prognostic Microenvironment-Related Gene Signature in Glioblastoma Patients Treated with Carmustine Wafers.

Despite the state-of-the-art treatment, patients diagnosed with glioblastoma (GBM) have a median overall survival (OS) of 14 months. The insertion of carmustine wafers (CWs) into the resection cavity as adjuvant treatment represents a promising option, although its use has been limited due to contrasting clinical results. Our retrospective evaluation of CW efficacy showed a significant improvement in terms of OS in a subgroup of patients. Given the crucial role of the tumor microenvironment (TME) in GBM progression and response to therapy, we hypothesized that the TME of patients who benefited from CW could have different properties compared to that of patients who did not show any advantage. Using an in vitro model of the glioma microenvironment, represented by glioma-associated-stem cells (GASC), we performed a transcriptomic analysis of GASC isolated from tumors of patients responsive and not responsive to CW to identify differentially expressed genes. We found different transcriptomic profiles, and we identified four genes, specifically down-regulated in GASC isolated from long-term survivors, correlated with clinical data deposited in the TCGA-GBM dataset. Our results highlight that studying the in vitro properties of patient-specific glioma microenvironments can help to identify molecular determinants potentially prognostic for patients treated with CW.

Triple negative breast cancer-derived small extracellular vesicles as modulator of biomechanics in target cells.

Extracellular vesicle (EV) mediated communication has recently been proposed as one of the pivotal routes in the development of cancer metastasis. EVs are nano-sized vesicles swapped between cells, carrying a biologically active content that can promote tumor-induced immune suppression, metastasis and angiogenesis. Thus, EVs constitute a potential target in cancer therapy. However, their role in triggering the premetastatic niche and in tumor spreading is still unclear. Here, we focused on the EV ability to modulate the biomechanical properties of target cells, known to play a crucial role in metastatic spreading. To this purpose, we isolated and thoroughly characterized triple-negative breast cancer (TNBC)-derived small EVs. We then evaluated variations in the mechanical properties (cell stiffness, cytoskeleton/nuclear/morphology and Yap activity rearrangements) of non-metastatic breast cancer MCF7 cells upon EV treatment. Our results suggest that TNBC-derived small EVs are able to directly modify MCF7 cells by inducing a decrease in cell stiffness, rearrangements in cytoskeleton, focal adhesions and nuclear/cellular morphology, and an increase in Yap downstream gene expression. Testing the biomechanical response of cells after EV addition might represent a new functional assay in metastatic cancer framework that can be exploited for future application both in diagnosis and in therapy.

Definition of the Prognostic Role of MGMT Promoter Methylation Value by Pyrosequencing in Newly Diagnosed IDH Wild-Type Glioblastoma Patients Treated with Radiochemotherapy: A Large Multicenter Study.

Background. O6-methylguanine (O6-MeG)-DNA methyltransferase (MGMT) methylation status is a predictive factor for alkylating treatment efficacy in glioblastoma patients, but its prognostic role is still unclear. We performed a large, multicenter study to evaluate the association between MGMT methylation value and survival. Methods. We evaluated glioblastoma patients with an assessment of MGMT methylation status by pyrosequencing from nine Italian centers. The inclusion criteria were histological diagnosis of IDH wild-type glioblastoma, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) ≤2, and radio-chemotherapy treatment with temozolomide. The relationship between OS and MGMT was investigated with a time-dependent Receiver Operating Characteristics (ROC) curve and Cox regression models. Results. In total, 591 newly diagnosed glioblastoma patients were analyzed. The median OS was 16.2 months. The ROC analysis suggested a cut-off of 15% for MGMT methylation. The 2-year Overall Survival (OS) was 18.3% and 51.8% for MGMT methylation <15% and ≥15% (p < 0.0001). In the multivariable analysis, MGMT methylation <15% was associated with impaired survival (p < 0.00001). However, we also found a non-linear association between MGMT methylation and OS (p = 0.002): median OS was 14.8 months for MGMT in 0−4%, 18.9 months for MGMT in 4−40%, and 29.9 months for MGMT in 40−100%. Conclusions. Our findings suggested a non-linear relationship between OS and MGMT promoter methylation, which implies a varying magnitude of prognostic effect across values of MGMT promoter methylation by pyrosequencing in newly diagnosed IDH wild-type glioblastoma patients treated with chemoradiotherapy.

Reinfection of Transplanted Livers in HCV- and HCV/HIV-Infected Patients Is Characterized by a Different MicroRNA Expression Profile.

BACKGROUND: After liver transplantation, HCV/HIV co-infected patients present, compared to the HCV mono-infected ones, increased HCV viral load, rapid progression to liver fibrosis and higher mortality. Liver biopsies (LB), obtained routinely 6 months after transplantation, represent a unique model to assess the early events related to graft re-infection. Here, we used miRNA sequencing of LB obtained from both HCV-and HCV/HIV-infected recipients, to identify transcriptional profiles able to explain the more severe outcome of these latter. METHODS: miRNAs of 3 healthy livers, 3 HCV-LB and 3 HCV/HIV-LB were sequenced by Illumina HiSeq2500 platform. The DIANA-miRPath v3.0 webserver and DIANA-microT-CDS algorithm (v5.0) were used to characterize the functions of differentially expressed (DE-) miRNAs, querying the KEGG and Gene Ontology-Biological Process databases. RESULTS: LB obtained from infected patients were characterized, with respect to controls, by a miRNA profile related to viral infection, immune system signaling and DNA damage in HCV-induced carcinogenesis. Instead, HCV-LB and HCV/HIV-LB differed in the expression of miRNAs involved in immunological and apoptotic processes and in extracellular matrix remodeling. CONCLUSIONS: liver reinfection processes are associated with early miRNA changes. Further studies are necessary to establish their prognostic role and possible actionability.

Immunoregulatory effects of glioma-associated stem cells on the glioblastoma peritumoral microenvironment: a differential PD-L1 expression from core to periphery?

OBJECTIVE: Glioma-associated stem cells (GASCs) have been indicated as possible players in supporting growth and recurrence in glioblastoma. However, their role in modulating immune response in the peritumoral area has not yet been described. In this study, the authors aimed to investigate programmed death-ligand 1 (PD-L1) differential expression at the protein level in GASCs derived from different tumor areas (core, periphery, and surrounding healthy brain). METHODS: Tumor tissue samples were collected from patients who underwent surgery for a histopathologically confirmed diagnosis of glioblastoma. Sampling sites were confirmed via neuronavigation and categorized on 5-aminolevulinic acid (5-ALA) fluorescence as bright (ALA+), pale (ALA PALE), or negative (ALA-), which corresponds to the tumor mass, infiltrated peritumoral area, and healthy brain, respectively, during surgery. GASCs were first isolated from the 3 regions and analyzed; then Western blot analysis was used to evaluate the level of PD-L1 expression in the GASCs. RESULTS: Overall, 7 patients were included in the study. For all patients, the mean values ± SD of PD-L1 expression in GASCs for ALA+, ALA PALE, and ALA- were 1.12 ± 1.14, 0.89 ± 0.63, and 0.57 ± 0.18, respectively. The differentially expressed values of PD-L1 in GASCs sampled from the 3 areas were found to be significant (p < 0.05) for 3 of the 7 patients: patient S470 (ALA+ vs ALA- and ALA PALE vs ALA-), patient S473 (ALA+ vs ALA PALE and ALA PALE vs ALA-), and patient S509 (ALA+ vs ALA-). CONCLUSIONS: This analysis showed, for the first time, that GASCs expressed a constitutive level of PD-L1 and that PD-L1 expression in GASCs was not uniform among patients or within the same patient. GASC analysis combined with 5-ALA-guided sampling (from core to periphery) made it possible to highlight the role of the tumor microenvironment at the infiltrating margin, which might cause clinical resistance, opening interesting perspectives for the future.

Image Analysis of Circulating Tumor Cells and Leukocytes Predicts Survival and Metastatic Pattern in Breast Cancer Patients.

BACKGROUND: The purpose of the present work was to test whether quantitative image analysis of circulating cells can provide useful clinical information targeting bone metastasis (BM) and overall survival (OS >30 months) in metastatic breast cancer (MBC). METHODS: Starting from cell images of epithelial circulating tumor cells (eCTC) and leukocytes (CD45pos) obtained with DEPArray, we identified the most significant features and applied single-variable and multi-variable methods, screening all combinations of four machine-learning approaches (Naïve Bayes, Logistic regression, Decision Trees, Random Forest). RESULTS: Best predictive features were circularity (OS) and diameter (BM), in both eCTC and CD45pos. Median difference in OS was 15 vs. 43 (months), p = 0.03 for eCTC and 19 vs. 36, p = 0.16 for CD45pos. Prediction for BM showed low accuracy (64%, 53%) but strong positive predictive value PPV (79%, 91%) for eCTC and CD45, respectively. Best machine learning model was Naïve Bayes, showing 46 vs 11 (months), p <0.0001 for eCTC; 12.5 vs. 45, p = 0.0004 for CD45pos and 11 vs. 45, p = 0.0003 for eCTC + CD45pos. BM prediction reached 91% accuracy with eCTC, 84% with CD45pos and 91% with combined model. CONCLUSIONS: Quantitative image analysis and machine learning models were effective methods to predict survival and metastatic pattern, with both eCTC and CD45pos containing significant and complementary information.

The Longevity-Associated Variant of BPIFB4 Reduces Senescence in Glioma Cells and in Patients' Lymphocytes Favoring Chemotherapy Efficacy.

Glioblastoma (GBM) is the most common primary brain cancer with the median age at diagnosis around 64 years, thus pointing to aging as an important risk factor. Indeed, aging, by increasing the senescence burden, is configured as a negative prognostic factor for GBM stage. Furthermore, several anti-GBM therapies exist, such as temozolomide (TMZ) and etoposide (ETP), that unfortunately trigger senescence and the secretion of proinflammatory senescence-associated secretory phenotype (SASP) factors that are responsible for the improper burst of (i) tumorigenesis, (ii) cancer metastasis, (iii) immunosuppression, and (iv) tissue dysfunction. Thus, adjuvant therapies that limit senescence are urgently needed. The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) gene previously demonstrated a modulatory activity in restoring age-related immune dysfunction and in balancing the low-grade inflammatory status of elderly people. Based on the above findings, we tested LAV-BPIFB4 senotherapeutic effects on senescent glioblastoma U87-MG cells and on T cells from GBM patients. We interrogated SA-ß-gal and HLA-E senescence markers, SASP factors, and proliferation and apoptosis assays. The results highlighted a LAV-BPIFB4 remodeling of the senescent phenotype of GBM cells, enhancement of their sensitivity to temozolomide and a selective reduction of the T cells' senescence from GBM patients. Overall, these findings candidate LAV-BPIFB4 as an adjuvant therapy for GBM.

Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. METHODS: In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. RESULTS: The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa = 0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. CONCLUSIONS: RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

TFEB, SIRT1, CARM1, Beclin-1 expression and PITX2 methylation in breast cancer chemoresistance: a retrospective study.

BACKGROUND: Breast cancer chemoresistance is attributed to a wide variety of mechanisms, including autophagy. Transcription factor EB (TFEB) has been recently identified and characterized as one major regulator of autophagy and lysosomal genesis. OBJECTIVE: This study aims to evaluate the prognostic impact of TFEB and its pathway in breast cancer chemoresistance. METHODS: This retrospective study analyzes the expression of TFEB, CARM1, SIRT1, and Beclin-1 and the methylation of PITX2 in breast carcinoma. A group of breast cancer patients treated with chemotherapy, who relapsed within 12 months from treatment initiation, were compared to a sub-cohort of chemo-treated patients who did not recur within 12 months of follow-up. The expression of TFEB, CARM1, SIRT1, and Belcin-1 was analyzed using immunohistochemistry or RT-PCR on formalin-fixed paraffin-embedded samples. PITX2 methylation was tested with the diagnostic CE-marked kit Therascreen PITX2 RGQ PCR. In the final model, 136 cases of chemo-treated breast cancer were included. RESULTS: A higher TFEB and Beclin-1 expression correlate with shorter survival in patients with chemo-treated invasive breast cancer (respectively HR 3.46, CI.95 1.27-9.47, p < 0.05 and 7.11, CI.95 2.54-19.9). TFEB, CARM1, and SIRT1 are positively correlated with Beclin-1. The protein expression of SIRT1 is significantly associated with TFEB and CARM1 so that a very low SIRT1 expression (lower than the first quartile of the H-score distribution) correlates with a low expression of TFEB and CARM1 and with longer survival. SIRT1 seems to have a lower H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. Beclin-1 and TFEB seem to have a higher H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. PITX2 methylation analysis was feasible only in 65% of the selected samples, but no significant differences between cases and controls were found, and there was also no correlation with the expression of the TFEB pathway. CONCLUSIONS: TFEB, SIRT1, and Beclin-1 seem to have a potential prognostic significance in patients with chemo-treated breast cancer, likely because of their role in the regulation of autophagy. In addition, no correlation between TFEB and PITX2 methylation was found, likely because they perform two different roles within the autophagy process.

A Rare Cause of Respiratory Insufficiency in a 30-Year-Old Transgender Woman.

CASE PRESENTATION: A 30-year-old transgender woman who was HIV positive presented to the ED with progressive severe dyspnea and hemoptysis that started 1 day earlier. The patient was undergoing antiretroviral therapy with emtricitabine-rilpivirine-tenofovir with good compliance and feminizing hormone therapy with cyproterone acetate. She was otherwise healthy and was not taking any other medications.