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The natural polyphenol resveratrol (RSV) might counteract the skeletal muscle age-related loss of muscle mass and strength/function partly acting on mitochondria. This work analysed the effects of a six-week administration of RSV (50 mg/kg/day) in the oxidative Soleus (Sol) skeletal muscle of old rats (27 months old). RSV effects on key mitochondrial biogenesis proteins led to un unchanged amount of SIRT1 protein and a marked decrease (60 %) in PGC-1α protein. In addition, Peroxyredoxin 3 (PRXIII) protein decreased by 50 %, which on overall suggested the absence of induction of mitochondrial biogenesis by RSV in old Sol. A novel direct correlation between PGC-1α and PRXIII proteins was demonstrated by correlation analysis in RSV and ad-libitum (AL) rats, supporting the reciprocally coordinated expression of the proteins. RSV supplementation led to an unexpected 50 % increase in the frequency of the oxidized base OH8dG in mtDNA. Furthermore, RSV supplementation induced a 50 % increase in the DRP1 protein of mitochondrial dynamics. In both rat groups an inverse correlation between PGC-1α and the frequency of OH8dG as well as an inverse correlation between PRXIII and the frequency of OH8dG were also found, suggestive of a relationship between oxidative damage to mtDNA and mitochondrial biogenesis activity. Such results may indicate that the antioxidant activity of RSV in aged Sol impinged on the oxidative fiber-specific, ROS-mediated, retrograde communication, thereby affecting the expression of SIRT1, PGC-1α and PRXIII, reducing the compensatory responses to the age-related mitochondrial oxidative stress and decline.

Abstract Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ischemic end organ injury due to microvascular platelet-rich thrombi. iTTP pathophysiology is based on a severe ADAMTS13 deficiency, the specific von Willebrand factor (vWF)-cleaving protease, due to anti-ADAMTS13 autoantibodies. Early diagnosis and treatment reduce the mortality. Front-line therapy includes daily plasma exchange (PEX) with fresh frozen plasma replacement and immunosuppression with corticosteroids. Caplacizumab is recently added to the front-line therapy. Caplacizumab is a nanobody that binds to the A1 domain of vWF, blocking the interaction of ultra-large vWF multimers with the platelet, and thereby preventing the formation of platelet-rich thrombi. Caplacizumab reduces mortality due to ischemic events, refractoriness and exacerbations after PEX discontinuation. Until now, the criteria for response to treatment mainly took into account the normalization of platelet count and discontinuation of PEX, now with the use of caplacizumab, leading to rapid normalization of platelet count, it has been necessary to redefine the response criteria, taking into account also the underlying autoimmune disease. Monitoring of ADAMTS13 activity is important to identify cases with low value of activity (

Abstract Background The current management of lung cancer patients has reached a high level of complexity. Indeed, besides the traditional clinical variables (e.g., age, sex, TNM stage), new omics data have recently been introduced in clinical practice, thereby making more complex the decision-making process. With the advent of Artificial intelligence (AI) techniques, various omics datasets may be used to create more accurate predictive models paving the way for a better care in lung cancer patients. Methods The LANTERN study is a multi-center observational clinical trial involving a multidisciplinary consortium of five institutions from different European countries. The aim of this trial is to develop accurate several predictive models for lung cancer patients, through the creation of Digital Human Avatars (DHA), defined as digital representations of patients using various omics-based variables and integrating well-established clinical factors with genomic data, quantitative imaging data etc. A total of 600 lung cancer patients will be prospectively enrolled by the recruiting centers and multi-omics data will be collected. Data will then be modelled and parameterized in an experimental context of cutting-edge big data analysis. All data variables will be recorded according to a shared common ontology based on variable-specific domains in order to enhance their direct actionability. An exploratory analysis will then initiate the biomarker identification process. The second phase of the project will focus on creating multiple multivariate models trained though advanced machine learning (ML) and AI techniques for the specific areas of interest. Finally, the developed models will be validated in order to test their robustness, transferability and generalizability, leading to the development of the DHA. All the potential clinical and scientific stakeholders will be involved in the DHA development process. The main goals aim of LANTERN project are: i) To develop predictive models for lung cancer diagnosis and histological characterization; (ii) to set up personalized predictive models for individual-specific treatments; iii) to enable feedback data loops for preventive healthcare strategies and quality of life management. Discussion The LANTERN project will develop a predictive platform based on integration of multi-omics data. This will enhance the generation of important and valuable information assets, in order to identify new biomarkers that can be used for early detection, improved tumor diagnosis and personalization of treatment protocols. Ethics Committee approval number 5420 − 0002485/23 from Fondazione Policlinico Universitario Agostino Gemelli IRCCS – Università Cattolica del Sacro Cuore Ethics Committee. Trial registration clinicaltrial.gov - NCT05802771.

Cavitating lung tumors occur in approximately 10-15% of the patients, are more commonly associated with squamous histology, and are typically located in the lung parenchyma. Herein we describe an exceedingly rare series of 5 patients, 4 of whom treatment-naïve, whose tumor caused the disruption of the normal airway anatomy at the level of lobar or segmental bronchi, leading to the formation of an endoscopically-visible cavity which ended up in the lung parenchyma or even into the pleural space. Sex (3 males, 2 females), smoking habit (2 never smokers, 2 former smokers, 1 current smoker), and histology (3 adenocarcinoma, 2 squamous cell carcinoma) were heterogeneous, but the 4 patients treatment-naïve presented with metastatic disease, poor ECOG performance status, similar clinical complaints of long duration, and lack of actionable mutations. The only patient who exhibited a meaningful response to treatment had the lowest symptoms’ duration, the smallest size of the cavitated mass, and the best performance status at the time of diagnosis. This series provides the first comprehensive description of a rare presentation of lung cancer characterized by similar clinical complaints, delayed diagnosis and poor prognosis.

Alessandra Micozzi,1 Clara Minotti,2 Saveria Capria,2 Claudio Cartoni,2 Silvia Maria Trisolini,2 Giovanni Manfredi Assanto,1 Walter Barberi,2 Maria Luisa Moleti,2 Stefania Santilli,3 Maurizio Martelli,1 Giuseppe Gentile1 1Haematology, Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy; 2Department of Haematology, Oncology and Dermatology, Azienda Policlinico Umberto I, Rome, Italy; 3Department of Diagnostics, Azienda Policlinico Umberto I, Rome, ItalyCorrespondence: Alessandra Micozzi, Haematology, Department of Translational and Precision Medicine, Sapienza University of Rome, Via Benevento 6, Rome, 00161, Italy, Tel +39 6 857951, Fax +39 6 44241984, Email alessandra.micozzi@uniroma1.itPurpose: To evaluate the benefits and safety of the empiric antibiotic treatment (EAT) active against KPC-K. pneumoniae in febrile neutropenic patients with acute leukaemia (AL) who are colonised by KPC-K. pneumoniae.Patients and Methods: A 7-year (2013– 2019) retrospective observational cohort study was conducted at the Haematology, Sapienza Rome University (Italy) on 94 febrile neutropenia episodes (FNE) in AL patients KPC-K. pneumoniae carriers treated with active EAT.Results: Eighty-two (87%) FNE were empirically treated with antibiotic combinations [38 colistin-based and 44 ceftazidime-avibactam (CAZAVI)-based], 12 with CAZAVI monotherapy. Successful outcomes were observed in 88/94 (94%) FNE, 46/49 (94%) microbiologically documented infections, and 24/27 (89%) gram-negative bloodstream infections (GNB-BSI). Mortality due to infective causes was 4.2% (2.1% within 1 week). KPC-K. pneumoniae infections caused 28/94 FNE (30%) and KPC-K. pneumoniae-BSI was documented in 22 FNE (23.4%) (85% of GNB-BSI), in all cases patients received active EAT, and 21 survived. KPC-K.pneumoniae-BSI mortality rate was 4.5%. CAZAVI-based EAT showed better results than colistin-based EAT (55/56 vs 33/38, p = 0.037), overall and without EAT modification (41/56 vs 20/38, p = 0.02). Empirical combinations including CAZAVI were successful in 98% of cases (43/44 vs 33/38 for colistin-based EAT, p = 0.01), without modifications in 82% (36/44 vs 20/28, p = 0.02). All deaths occurred in patients treated with colistin-based EAT (4/38 vs 0/56, p = 0.02). CAZAVI-containing EAT was the only independent factor for an overall successful response (HR 0.058, CI 0.013– 1.072, p = 0.058). Nephrotoxicity occurred in 3(8%) patients undergoing colistin-based EAT (none in those undergoing CAZAVI-based EAT, p = 0.02).Conclusion: KPC-K. pneumoniae infections are frequent in colonised AL patients with FNE. EAT with active antibiotics, mainly CAZAVI-based combinations, was effective, safe, and associated with low overall and KPC-K. pneumoniae-BSI-related mortality.Keywords: ceftazidime-avibactam, colistin, haematological malignancies, KPC-K. pneumoniae-BSI mortality rate

Abstract Background Achieving complete remission (CR) is the main goal in AML treatment and a prerequisite for successful autologous stem cell transplantation (ACT). Methods Comparing results of peripheral blood ACT in patients with AML in CR1 attained following 1 versus 2 chemotherapy courses transplanted in 2000–2019. Results Patients 1532 (84%) with one and 293 (16%) patients with two induction chemotherapies courses (a total of 1825 patients) were included in the study. Follow‐up was 7.9 (95% CI: 7.4–8.4) and 7.7 (95% CI: 7.0–8.6) years (p = 0.8). Time from diagnosis to ACT was 4.7 (range, 3.9–5.8) versus 5.7 (range, 4.7–7.1) months (p

Supercritical fluid extraction (SFE) was used to extract bioactive compounds from apple (Malus domestica) peel waste from three different Italian cultivars. The bioactive fractions were extracted applying a temperature of 60 °C and a pressure of 250 bar for 15 min with 20% ethanol as co-solvent, at a flow rate of 2 mL/min. The total polyphenol (TP), anthocyanin (TA), ascorbic acid (AA), and antioxidant activity contents (TACs) were measured, while chromatographic analyses were performed to highlight the differences between the extracts. The Stark cultivar had the highest levels of polyphenols, anthocyanins, and ascorbic acid, while the Royal Gala cultivar showed the highest total antioxidant activity. SFE extracts were then tested for their effect on the mitochondrial NADH–ubiquinone oxidoreductase (Complex I) activity on mitochondria isolated from human embryonic kidney cells (HEK239). The Stark extract showed the most positive response in terms of NADH oxidation. The results obtained in this work highlight the potential of apple peel waste as a source of functional phytocompounds and suggest that Stark cultivar extracts may be exploited for pharmacological applications. This study supports the circular bioeconomy by promoting the use of waste products as a valuable resource.

Mozzarella is one of the most popular unripened Apulian cheeses. Knowledge about microbial composition and variability of artisanal mozzarella and its production chain is increasingly growing. In this study, microbial communities from natural whey starters to end products from four renowned Apulian artisanal dairy factories have been explored by means of 16S metagenomics. The chemical properties of mozzarella samples were also detected and analyzed. Lactobacillus is the core acidifying component of the used starters, while some psychrophilic or contaminants bacteria appear in site-specific products. Biodiversity was found to be quite similar between the whey and mozzarella sample pools, while a significant variability among production sites (factories) has been detected. Furthermore, mozzarella microbial diversity seems to be in positive correlation with its lactic acid content. Targeted metagenomics would then be a powerful and relatively quick technique to characterize the microbiological variability of traditional milk-based foods.

Abstract Pleomorphic xanthoastrocytoma (PXA) in its classic manifestation exhibits distinct morphological features and is assigned to CNS WHO grade 2 or grade 3. Distinction from glioblastoma variants and lower grade glial and glioneuronal tumors is a common diagnostic challenge. We compared a morphologically defined set of PXA (histPXA) with an independent set, defined by DNA methylation analysis (mcPXA). HistPXA encompassed 144 tumors all subjected to DNA methylation array analysis. Sixty-two histPXA matched to the methylation class mcPXA. These were combined with the cases that showed the mcPXA signature but had received a histopathological diagnosis other than PXA. This cohort constituted a set of 220 mcPXA. Molecular and clinical parameters were analyzed in these groups. Morphological parameters were analyzed in a subset of tumors with FFPE tissue available. HistPXA revealed considerable heterogeneity in regard to methylation classes, with methylation classes glioblastoma and ganglioglioma being the most frequent mismatches. Similarly, the mcPXA cohort contained tumors of diverse histological diagnoses, with glioblastoma constituting the most frequent mismatch. Subsequent analyses demonstrated the presence of canonical pTERT mutations to be associated with unfavorable prognosis among mcPXA. Based on these data, we consider the tumor type PXA to be histologically more varied than previously assumed. Histological approach to diagnosis will predominantly identify cases with the established archetypical morphology. DNA methylation analysis includes additional tumors in the tumor class PXA that share similar DNA methylation profile but lack the typical morphology of a PXA. DNA methylation analysis also assist in separating other tumor types with morphologic overlap to PXA. Our data suggest the presence of canonical pTERT mutations as a robust indicator for poor prognosis in methylation class PXA.

Introduction Mechanical thrombectomy (MT) is the gold standard for acute ischemic stroke (AIS) due to large vessel occlusion (LVO). Although >70% of patients in the randomized clinical trials (RCTs) assessing the efficacy of MT for AIS‐LVO had successful recanalization, only up to 27% were free of disability according to the 90‐day modified Rankin scale (mRS) score. It is suggested that microcirculation disruption distal to the LVO might contribute to futile recanalization. Hence, combining intra‐arterial (IA) tissue plasminogen activator (tPA) with MT was investigated in a few studies in an attempt to reduce the burden of distal microthrombi and microcirculation disruption. We conducted this meta‐analysis to provide collective evidence in this regard. Methods We performed this meta‐analysis following the Preferred Reporting Items for Systematic Review and Meta‐Analyses statement recommendations. We aimed to include all original studies investigating the benefits of IA tPA in AIS patients undergoing MT.All screening and extraction stages were conducted by two authors, with a third author resolving any conflicts. Using R software, we calculated pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CI). A fixed‐effect model was adopted to pool all data due to the absence of significant heterogeneity among the included studies. Heterogeneity was assessed using Q statistics and the I2test, where I2> 50% or P‐value < 0.05 were considered significant. Results Following the database search, a manual search of references, and different screening phases, we included 5 studies (2,686 patients) satisfying the predefined inclusion criteria. Successful recanalization rates were comparable between both groups. The 90‐day functional independence was reported in 4 studies (2,474 patients), with comparable rates between both groups (OR = 1.25; 95%CI = 0.92‐1.70; P‐value = 0.154). Mortality was reported in 3 studies (368 patients), with comparable rates between both groups (OR = 0.73; 95%CI = 0.44‐1.23; P‐value = 0.240). Symptomatic intracranial hemorrhage (sICH) was reported in 5 studies (2,678 patients) with no significant difference between the two groups (OR = 0.66; 95%CI = 0.34‐1.26; P‐value = 0.304). Conclusions The current evidence does not show any significant differences between MT alone versus MT plus IA tPA in terms of 90‐day functional independence, mortality, and sICH. With the limited number of studies and included patients, more studies are needed to investigate the benefits and safety of the combined MT and IA tPA.

Abstract Background KPC-K.pneumoniae bloodstream infection (KPC-KpBSI) mortality rate in patients with hematological malignancies is reported about 60%. The initial treatment active against KPC-K.pneumoniae is crucial for survival and KPC-K.pneumoniae rectal colonization usually precedes KPC-KpBSI. We evaluated the impact on KPC-KpBSI mortality of the preemptive use of antibiotics active against KPC-K.pneumoniae, as opposed to inactive or standard empiric antibiotics, for the empiric treatment of febrile neutropenia episodes in patients with hematological malignancy identified as KPC-K.pneumoniae intestinal carriers. Methods We compared the outcomes of KPC-KpBSIs occurring in high-risk hematological patients known to be colonized with KPC-K.pneumoniae, during two time periods: March2012-December2013 (Period 1, initial approach to KPC-K.pneumoniae spread) and January2017-October2018 (Period 2, full application of the preemptive strategy). The relative importance of the various prognostic factors that could influence death rates were assessed by forward stepwise logistic regression models. Results KPC-KpBSI-related mortality in hematological patients identified as KPC-K.pneumoniae carriers dropped from 50% in Period 1 to 6% in Period 2 (p

COVID-19 disease has a strong impact on hematological patients; those receiving autologous hematopoietic stem cell transplantation (aHSCT) represent a particularly vulnerable group, in which the effectiveness of vaccination is very variable. Chiarucci et al. showed that patients affected by non-Hodgkin lymphoma (NHL) and treated with rituximab experienced a lower rate of immunization against SARS-CoV-2 (54%), as well as significantly lower IgG antibody titers. In our multicenter retrospective observational study, we included 82 patients who underwent aHSCT, divided into two groups: 58 patients vaccinated after aHSCT (group A) and 24 vaccinated before getting transplantation (group B). In group A, 39 (67%) patients had positive serology, and the rate of positivity increased with time after aHSCT. In the subgroup of patients with NHL, the administration of rituximab predicted negative serology, particularly when administered in the 6 months before vaccination (13% response rate). Patients affected by plasma cells had a higher rate of positivity (83% overall), independently of the time to aHSCT. In group B, no patient who initially showed positive serology became negative after transplantation, so the aHSCT did not affect the response to the vaccination. Our study confirmed the role of rituximab as a negative predictor of response to SARS-CoV-2 vaccination, whereas the conditioning and transplantation procedure itself seemed to be less important.

Introduction: The majority of the money spent on possible new medications’ clinical trials is accounted for by the innovative pharmaceutical sector, which also stimulates the economy of a nation. The objective of this study was to evaluate the impact of pharmaceutical industry-sponsored clinical trials (ISCTs) in inflammatory bowel diseases (IBDs) towards the national health service (NHS) in terms of avoided costs and leverage effect.Methodology: The research was conducted at National Institute of Gastroenterology, “Saverio De Bellis”, Castellana Grotte (Apulia, Italy) collecting data from profit ISCTs of pharmaceutical products conducted over the time period 2018-2020 with focus on inflammatory bowel diseases. After the quantification of health services and drug costs from the latter studies, avoided costs and leverage effects were then estimated.Results: The results on the avoided costs for healthcare facilities deriving from the conduct of clinical studies show that, in relation to the sample of five drug companies participating in our 2018-2020 analysis, out of a total of 235,102.46 €, identified as direct investment, 628,158.21 € of avoided costs for the NHS were measured, with an additional saving (leverage effect) for the NHS of 3.67 € for each € invested by the companies promoting clinical trials.Conclusion: Conducting profit clinical trials has practical benefits and a favourable macroeconomic impact that, by completing its limited resources, helps to sustain one country NHS thanks to the avoided costs while also contributing to locational and industrial policy while guaranteeing novel therapeutics and health services for the patients enrolled.

Natural killer (NK) cells are emerging as unique players in the immune response against cancer; however, only limited data are available on tumor infiltrating NK cells in head and neck squamous cell carcinoma (HNSCC), one of the most common cancer. Occurrence of HNSCC is closely related to the immune microenvironment, and immunotherapy is increasingly being applied to this setting. However, the limited success of this type of treatment in this tumor calls for further investigation in the field.Surgical HNSSC specimens of 32 consecutive patients were mechanically and enzymatically dissociated. Tumor cells were separated from infiltrating cells by short centrifugation and infiltrating NK cells were phenotypically and functionally characterized by multiple antibody staining and flow cytometry. Tumor infiltrating NK cells in HNSCC showed a peculiar phenotype predominantly characterized by increased NKG2A and reduced Siglec-7, NKG2D, NKp30 and CD16 expression. This phenotype was associated with a decreased ability to perform antibody-dependent cellular cytotoxicity (ADCC). However, NK, CD4 and CD8 shared an increment of glucocorticoid-induced tumor necrosis factor-related (GITR) costimulatory receptor which could be exploited for immunotherapy with agonistic anti-GITR antibodies combined with checkpoint inhibitors.

Echobronchoscope-guided transbronchial needle aspiration (EBUS-TBNA) is mainly used as the transbronchial approach to hilar/mediastinal lymph nodes or lesions, for diagnostic or staging purposes. Moreover, the role of linear EBUS-TBNA as a diagnostic tool for central intrapulmonary lesions adjacent to the trachea or the major bronchi is also well established. However, since the tip of the ultrasound probe at the distal end of the echobronchoscope is very thin, it can be wedged through smaller peripheral bronchi, reaching the distal parenchyma and allowing for peripheral pulmonary lesion sampling. The main aim of this retrospective study was to evaluate the diagnostic yield and the safety of EBUS-TBNA in the diagnosis of pulmonary peripheral nodules. The database of the Interventional Pulmonology Unit of Azienda Ospedaliero-Universitaria delle Marche (Ancona, Italy) was evaluated to identify peripheral pulmonary nodules approached by EBUS-TBNA. Thirty patients with a single peripheral pulmonary nodule located peripherally to the subsegmental bronchi of the lower lobes and adjacent to a small bronchus greater than 3 mm in diameter were included in this study. The nodule was visible using endoscopic ultrasound in 28 patients and the diagnosis was obtained via EBUS-TBNA in 26 cases (12 adenocarcinoma, 5 typical carcinoid tumors, 4 hamartoma and 5 metastatic lesions). The diagnostic yield was 86.6% for all 30 patients and 92.8% if only the 28 patients in which the lesion was visualized via echobronchoscopy were considered. No relevant adverse events were observed. We conclude that EBUS-TBNA may be an effective and safe option to sample pulmonary peripheral nodules in selected patients with lower lobe peripheral pulmonary lesions adjacent to small bronchi greater than 3 mm in diameter and reachable with the EBUS-TBNA probe.

The continuously growing number of objects orbiting around the Earth is expected to be accompanied by an increasing frequency of objects re-entering the Earth’s atmosphere. Many of these re-entries will be uncontrolled, making their prediction challenging and subject to several uncertainties. Traditionally, re-entry predictions are based on the propagation of the object’s dynamics using state-of-the-art modelling techniques for the forces acting on the object. However, modelling errors, particularly related to the prediction of atmospheric drag, may result in poor prediction accuracies. In this context, we explored the possibility of performing a paradigm shift, from a physics-based approach to a data-driven approach. To this aim, we present the development of a deep learning model for the re-entry prediction of uncontrolled objects in Low Earth Orbit (LEO). The model is based on a modified version of the Sequence-to-Sequence architecture and is trained on the average altitude profile as derived from a set of Two-Line Element (TLE) data of over 400 bodies. The novelty of the work consists in introducing in the deep learning model, alongside the average altitude, and three new input features: a drag-like coefficient (B*), the average solar index, and the area-to-mass ratio of the object. The developed model was tested on a set of objects studied in the Inter-Agency Space Debris Coordination Committee (IADC) campaigns. The results show that the best performances are obtained on bodies characterised by the same drag-like coefficient and eccentricity distribution as the training set.

Lung cancer is still the leading cause of cancer-related death worldwide. Interest is growing towards early detection and advances in liquid biopsy to isolate circulating tumor cells (CTCs). This pilot study aimed to detect epithelial CTCs in the peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients. We used Smart BioSurface® (SBS) slide, a nanoparticle-coated slide able to immobilize viable nucleated cellular fraction without pre-selection and preserve cell integrity. Forty patients undergoing lung resection for NSCLC were included; they were divided into two groups according to CTC value, with a cut-off of three CTCs/mL. All patients were positive for CTCs. The mean CTC value was 4.7(± 5.8 S.D.) per ml/blood. In one patient, next generation sequencing (NGS) analysis of CTCs revealed v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation, which has also been identified in tissue biopsy. CTCs count affected neither overall survival (OS, p = 0.74) nor progression-free survival (p = 0.829). Multivariable analysis confirmed age (p = 0.020) and pNodal-stage (p = 0.028) as negative predictors of OS. Preliminary results of this pilot study suggest the capability of this method in detecting CTCs in all early-stage NSCLC patients. NGS on single cell, identified as CTC by immunofluorescence staining, is a powerful tool for investigating the molecular landscape of cancer, with the aim of personalized therapies.