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Funder: Cancer Research UK Cambridge Cancer Centre, Background: SDHB is one of the major genes predisposing to paraganglioma/pheochromocytoma (PPGL). Identifying pathogenic SDHB variants in patients with PPGL is essential to the management of patients and relatives due to the increased risk of recurrences, metastases and the emergence of non-PPGL tumours. In this context, the ‘NGS and PPGL (NGSnPPGL) Study Group’ initiated an international effort to collect, annotate and classify SDHB variants and to provide an accurate, expert-curated and freely available SDHB variant database. Methods: A total of 223 distinct SDHB variants from 737 patients were collected worldwide. Using multiple criteria, each variant was first classified according to a 5-tier grouping based on American College of Medical Genetics and NGSnPPGL standardised recommendations and was then manually reviewed by a panel of experts in the field. Results: This multistep process resulted in 23 benign/likely benign, 149 pathogenic/likely pathogenic variants and 51 variants of unknown significance (VUS). Expert curation reduced by half the number of variants initially classified as VUS. Variant classifications are publicly accessible via the Leiden Open Variation Database system (https://databases.lovd.nl/shared/genes/SDHB). Conclusion: This international initiative by a panel of experts allowed us to establish a consensus classification for 223 SDHB variants that should be used as a routine tool by geneticists in charge of PPGL laboratory diagnosis. This accurate classification of SDHB genetic variants will help to clarify the diagnosis of hereditary PPGL and to improve the clinical care of patients and relatives with PPGL.

Introduction The purpose of this study was to evaluate the impact of 3-dimensional (3D) digital reconstructions of renal models on the arterial clamping approach during partial nephrectomy (PN). Patients and Methods Fifty-seven patients with T1 renal mass, referred for PN, were prospectively enrolled in 2 groups: Group 1 (n = 32) with revision of both 2-dimensional (2D) computed tomography (CT) imaging and 3D virtual model before surgery; Group 2 (n = 25) with revision of 2D CT imaging. Segmentation of the 3D models from preoperative high-quality CT scan was achieved using D2P software. In a sub-analysis of patients treated with PN with the on-clamp approach (n = 36), the effective intraoperative level of arterial clamping was compared with the preoperative planning. Results In the sub-group of patients referred to PN with the on-clamp approach, the intraoperative selective clamping was performed in 12 (57.1%) patients of Group 1 and in 2 (13.3%) cases of Group 2 (P = .01). The intraoperative management of the renal pedicle was done as preoperatively planned in 61.9% of patients in Group 1 and in 86.6% of cases in Group 2 (P = .1). Conclusion The 3D-guided plan of PN allows to perform selective clamping in higher proportion of patients compared with the standard 2D-guided approach without increasing intraoperative and postoperative complications.

PURPOSE: To perform an external validation of a recently published nomogram aimed to predict positive 68Ga-PSMA-11 PET/CT in patients with biochemical recurrence (BCR) after radical prostatectomy (RP) by Rauscher et al. (Eur Urol 73(5):656-661, 2018). METHODS: Overall, 413 PCa patients with BCR after RP (two consecutive PSA ≥ 0.2 ng/ml) and PSA value between 0.2 and 1 ng/ml were included. A multivariable logistic regression model was produced to assess the predictors of positive 68Ga-PSMA-11 PET/CT results. The performance characteristics of the model were assessed by quantifying the predictive accuracy, according to model calibration. Yuden's index was used to find the best nomogram's cut-off. Finally, decision curve analysis (DCA) was implemented to quantify the nomogram's clinical value. RESULTS: In the external cohort, the overall detection rate of 68Ga-PSMA-11 PET/CT was 44% vs. 64.7% in the original population. At multivariate analysis, PSA at 68Ga-PSMA-11 PET/CT (OR: 7.06, p 35%. CONCLUSION: We assessed similar suboptimal predictive accuracies in the external cohort compared to the original one. PSA and ongoing ADT were confirmed as positive predictors, and the most informative nomogram cut-off resulted 35%.

Background Pheochromocytoma (PHEO) and paraganglioma (PGL) are rare neuroendocrine tumors releasing catecholamines. Metastatic pheochromocytomas/paragangliomas (PPGLs) occur in about 5–26% of cases. To date, the management of patients affected by metastatic disease is a challenge in the absence of guidelines. Aim The aim of this study was to evaluate the overall survival (OS) and the progression-free survival (PFS) in metastatic PPGLs. Methods Clinical data of 20 patients referred to the Careggi University Hospital (Florence, Italy) were retrospectively collected. Follow-up ranged from 1989 to 2019. Site and size of primary tumor, biochemical activity, genetic analysis and employed therapies were considered. Data were analyzed with SPSS version 27. Results Nine PHEOs (45%) and 11 PGLs (55%) were enrolled. Median age at diagnosis was 43.5 years [30–55]. Mean follow-up was 104.6 ± 89.3 months. Catecholamines were released in 70% of cases. An inherited disease was reported in 50% of patients. OS from the initial diagnosis (OSpt) and from the metastatic appearance (OSmtx) were lower in older patients (OSpt p = 0.028; OSmtx p p = 0.007; OSmtx p = 0.041), larger tumors (OSpt p = 0.008; OSmtx p = 0.025) and sporadic disease (OSpt p = 0.013; OSmtx p = 0.008). Conclusion Our data showed that older age at the initial diagnosis, sympathetic extra-adrenal localization, larger tumors and wild-type neoplasms are related to worse prognosis. Notably, the employed therapies do not seem to influence the survival of our patients. At present, effective treatments for metastatic PPGLs are missing and a multidisciplinary approach is indispensably required.

BackgroundLeft hemispatial neglect (LHN) is a neuropsychological syndrome often associated with right hemispheric stroke. Patients with LHN have difficulties in attending, responding, and consciously representing the right side of space. Various rehabilitation protocols have been proposed to reduce clinical symptoms related to LHN, using cognitive treatments, or on non-invasive brain stimulation. However, evidence of their benefit is still lacking; in particular, only a few studies focused on the efficacy of combining different approaches in the same patient.MethodsIn the present study, we present the SMART ATLAS trial (StimolazioneMAgneticaRipetitivaTranscranica nell’ATtenzioneLAteralizzata dopoStroke), a multicenter, randomized, controlled trial with pre-test (baseline), post-test, and 12 weeks follow-up assessments based on a novel rehabilitation protocol based on the combination of brain stimulation and standard cognitive treatment. In particular, we will compare the efficacy of inhibitory repetitive-transcranial magnetic stimulation (r-TMS), applied over the left intact parietal cortex of LHN patients, followed by visual scanning treatment, in comparison with a placebo stimulation (SHAM control) followed by the same visual scanning treatment, on visuospatial symptoms and neurophysiological parameters of LHN in a population of stroke patients.DiscussionOur trial results may provide scientific evidence of a new, relatively low-cost rehabilitation protocol for the treatment of LHN.Trial registrationClinicalTrials.govNCT04080999. Registered on September 2019.

Introduction and Objective: ExactVuTM is a real-time micro-ultrasound system which provides, according to the Prostate Risk Identification Using Micro-Ultrasound protocol (PRI-MUS), a 300% higher resolution compared to conventional transrectal ultrasound. To evaluate the performance of ExactVuTM in the detection of Clinically significant Prostate Cancer (CsPCa). Materials and methods: Patients with Prostate Cancer diagnosed at fusion biopsy were imaged with ExactVuTM. CsPCa was defined as any Gleason Score ≥ 3+4. ExactVuTM examination was considered as positive when PRI-MUS score was ≥ 3. PRI-MUS scoring system was considered as correct when the fusion biopsy was positive for CsPCa. A transrectal fusion biopsy- proven CsPCa was considered as a gold standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the receiver operator characteristic (ROC) curve (AUC) were calculated. Results: 57 patients out of 68 (84%) had a csPCa. PRI-MUS score was correctly assessed in 68% of cases. Regarding the detection of CsPCa, ExactVuTM ’s sensitivity, specificity, PPV, and NPV was 68%, 73%, 93%, and 31%, respectively and the AUC was 0.7 (95% CI 0.5-0-8). For detecting CsPCa in the transition/ anterior zone the sensitivity, specificity, PPV, and NPV was 45%, 66%, 83% and 25% respectively ant the AUC was 0.5 (95% CI 0.2-0.9). Accounting only the CsPCa located in the peripheral zone, sensitivity, specificity, PPV, and NPV raised up to 74%, 75%, 94%, 33%, respectively with AUC 0.75 (95% CI 0.5-0-9). Conclusions: ExactVuTM provides high resolution of the prostatic peripheral zone and could represent a step forward in the detection of CsPCa as a triage tool. Further studies are needed to confirm these promising results.

Background No data are available regarding the impact of time between a previous transrectal prostate biopsy (PB) and holmium laser enucleation of the prostate (HoLEP) on perioperative outcomes. Objective To evaluate the impact of time from PB to HoLEP on perioperative outcomes. Design, setting, and participants A total of 172 consecutive patients treated with HoLEP within 12 mo of a single previous transrectal PB at two tertiary centers were included. Outcome measurements and statistical analysis Patients were stratified into two groups according to the median time from PB to HoLEP (namely, ≤6 and >6 mo). The primary outcome was intraoperative complications. Multivariate logistic regressions were used to identify the predictors of intraoperative complications. Linear regressions were used to test the association between the time from PB to HoLEP and intraoperative complications, enucleation efficiency, and enucleation time. Results and limitations In total, 93 (54%) and 79 (46%) patients had PB ≤ 6 and >6 mo before HoLEP, respectively. Patients in PB ≤ 6 mo group experienced higher rates of intraoperative complications than those in PB > 6 mo group (14% vs 2.6%, p = 0.04). At multivariable analysis, time between PB and HoLEP was an independent predictor of intraoperative complications (odds ratio: 0.74; 95% confidence interval: 0.6–0.9; p = 0.006). Finally, the risk of intraoperative complications reduced by 1.5%, efficiency of enucleation increased by 4.1%, and enucleation time reduced by 1.7 min for each month passed from PB to HoLEP (all p ≤ 0.006). Selection of patients with only one previous PB represents the main limitation. Conclusions The time from PB to HoLEP of ≤6 mo is associated with a higher risk of intraoperative complications, lower enucleation efficacy, and longer enucleation time. Patient summary Patients with a prostate biopsy (PB) ≤6 mo before holmium laser enucleation of the prostate (HoLEP) had significantly worse outcomes than those with a PB > 6 mo before HoLEP.

BACKGROUND: We aimed to assess the detection rate of overall PCa and csPCa, and the clinical impact of MRI/TRUS fusion targeted biopsy (FUSION-TB) compared to TRUS guided systematic biopsy (SB) in patients with different biopsy settings. METHODS: Three hundred and five patients were submitted to FUSION-TB, divided into three groups: biopsy naïve patients, previous negative biopsies and patients under active surveillance (AS). All patients had a single suspicious index lesion at mpMRI. Within these groups, we enrolled men underwent both to FUSION-TB and SB in the same session. Overall detection rate of PCa and csPCa for the two biopsy methods were compared separately between the three groups of patients. RESULTS: No differences were observed between the three groups concerning clinical and radiological characteristics. We found no differences in terms of overall PCa detection (66% vs. 63.8%, P=0.617) and csPCa detection (56.4% vs. 51.1%; P=0.225) concerning biopsy naïve patients. In patients previously submitted to a negative biopsy, FUSION-TB showed higher detection rate of csPCa compared to SB alone (41,3% vs. 27% respectively, P=0.038). In patients under AS, no differences were observed between FUSION-TB and SB in terms of overall PCa (50% vs. 73.1%) and csPCa (30.8% vs. 26.9%, respectively; P=0.705) detection. CONCLUSIONS: Our results suggest that in men with previously negative biopsy, FUSION-TB showed significantly higher diagnostic performance for clinically significant PCa as compared to SB. Combination of FUSION-TB and SB should be recommended in AS population to offer higher chance of csPCa diagnosis.

No studies have carried out an extensive analysis of the possible association between non-syndromic pheochromocytomas and paragangliomas (PPGLs) and other malignancies. To assess the risk of additional malignancy in PPGL, we retrospectively evaluated 741 patients with PPGLs followed-up in twelve referral centers in Italy. Incidence of second malignant tumors was compared between this cohort and Italian patients with two subsequent malignancies. Among our patients, 95 (12.8%) developed a second malignant tumor, which were mainly prostate, colorectal and lung/bronchial cancers in males, breast cancer, differentiated thyroid cancer and melanoma in females. The standardized incidence ratio was 9.59 (95% CI 5.46–15.71) in males and 13.21 (95% CI 7.52–21.63) in females. At multivariable analysis, the risk of developing a second malignant tumor increased with age at diagnosis (HR 2.50, 95% CI 1.15–5.44, p = 0.021 for 50–59 vs. &lt, 50-year category, HR 3.46, 95% CI 1.67–7.15, p &lt, 0.001 for &gt, 60- vs. &lt, 50-year). In patients with available genetic evaluation, a positive genetic test was inversely associated with the risk of developing a second tumor (HR 0.25, 95% CI 0.10–0.63, p = 0.003). In conclusion, PPGLs patients have higher incidence of additional malignant tumors compared to the general population who had a first malignancy, which could have an impact on the surveillance strategy.

Introduction to explore the feasibility of augmented reality (AR) to guide arterial clamping during robot-assisted partial nephrectomy (RAPN). Patients and Methods 15 consecutive patients with T1 renal mass underwent RAPN guided by AR. The 3D virtual model derived by computed tomography was superimposed on the actual view provided by the Da Vinci video stream thought AR technology. Preoperative plan of arterial clamping based on 2D conventional imaging, on 3D model and the effective intraoperative surgical approach guided by AR were compared using the McNeamar test. Results The plan of arterial clamping based on 2D preoperative imaging was recorded as follows: no clamping in 3 (20%), clamping of the main artery in 10 (66.7%) and selective clamping in 1 (6.7%) and super-selective clamping in 1 (6.7%) cases. After revision of the 3D model, the plan of clamping was modified as follows: no clamping in 1 (6.7%), clamping of the main artery in 2 (13.3%), selective clamping in 8 (53.3%) and super-selective clamping in 4 (26.7%) cases (p=0.03). The effective intraoperative clamping approach guided by AR-guidance was performed as planned in 13 (86.7%) patients. Conclusion AR for 3D guided renal surgery is useful to increase the adoption of selective clamping during RAPN.

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis when metastatic and scarce treatment options in the advanced stages. In solid tumors, the chemokine CXCL12/CXCR4 axis is involved in the metastatic process. We demonstrated that the human adrenocortex expressed CXCL12 and its cognate receptors CXCR4 and CXCR7, not only in physiological conditions, but also in ACC, where the receptors’ expression was higher and the CXCL12 expression was lower than in the physiological conditions. In a small pilot cohort of 22 ACC patients, CXCL12 negatively correlated with tumor size, stage, Weiss score, necrosis, and mitotic activity. In a Kaplan–Meier analysis, the CXCL12 tumor expression significantly predicted disease-free, progression-free, and overall survival. In vitro treatment of the primary ACC H295R and of the metastatic MUC-1 cell line with the PPARγ-ligand rosiglitazone (RGZ) dose-dependently reduced proliferation, resulting in a significant increase in CXCL12 and a decrease in its receptors in the H295R cells only, with no effect on the MUC-1 levels. In ACC mouse xenografts, tumor growth was inhibited by the RGZ treatment before tumor development (prevention-setting) and once the tumor had grown (therapeutic-setting), similarly to mitotane (MTT). This inhibition was associated with a significant suppression of the tumor CXCR4/CXCR7 and the stimulation of human CXCL12 expression. Tumor growth correlated inversely with CXCL12 and positively with CXCR4 expression, suggesting that local CXCL12 may impair the primary tumor cell response to the ligand gradient that may contribute to driving the tumor progression. These findings indicate that CXCL12/CXCR4 may constitute a potential target for anti-cancer agents such as rosiglitazone in the treatment of ACC.

BACKGROUND: We aimed at comparing perioperative outcomes in patients submitted to radical cystectomy followed by Fast Track (FT) protocol or standard management, and propose a definition of Trifecta, to improve standardized quality assessment for RC.METHODS: We considered 191 patients submitted to RC between January 2017 and January 2019. Patients followed FT or standard management according to surgeon's preference. Preoperative and intraoperative characteristics, alongside with postoperative outcomes were compared between the two groups. Trifecta was defined as follows: inhospital stay (HS) ≤ 10 days, time to defecation (TtD) below the overall mean and no major (≥ Clavien-Dindo grade III) complications. Finally, Trifecta achievement rates were assessed in both groups.RESULTS: 75 patients (39%) followed the FT protocol and 116 (61%) standard management. The two groups were homogeneous for preoperative, intraoperative and pathological characteristics. Patients in the FT group had shorter TtD (5 vs. 6 days p=0.006), HS (12 vs. 14 days p=0.008) and lower readmission rate (8% vs. 19% p=0.04). Early complication rates and grades were similar, while less late complications were found in FT group (6.7% vs. 21.6% p=0,006). Trifecta achievement rate was higher for FT group (31% vs. 8% p

BACKGROUND: To develop a clinical nomogram aimed to predict the achievement of trifecta in patients treated with open, laparoscopic and robotic partial nephrectomy (PN) for localized renal masses ( < cT2). METHODS: We retrospectively evaluated 482 consecutive patients who underwent PN with open (OPN: 243), laparoscopic (LPN: 156) and robotic (RAPN: 83) approach for T1 renal mass at single tertiary center. Trifecta was defined as follows: warm ischemia time (WIT)

BACKGROUND: We report long-term oncologic outcomes in patients with positive surgical margins (PSMs) at radical prostatectomy (RP) and the oncologic impact of different scenarios of PSMs presentation. METHODS: We selected 494 men with at least 3 years follow-up after surgery. PSMs patterns were recorded as: burden (focal vs multifocal), site (apical-anterior vs posterolateral vs base-bladder neck vs multiple) and side (unilateral vs bilateral). Kaplan-Meier curves depicted the clinical recurrence-free survival (CR-FS) rates at 10-year in the overall population, after biochemical recurrence and according to different PSMs patterns. Multivariate Cox-regression analysis was performed to predict CR. RESULTS: Overall, PSMs sites were apical-anterior, postero-lateral, base-bladder neck and multiple in 19.8%, 23.7%, 3.4% and 43.8%, respectively. Out of 494 patients, 278 (56.3%) had a focal margin, while 216 (43.7%) had a multifocal margin. In 268 (54.3%) and 87 (17.6%) men, PSMs were unilateral and bilateral, respectively. Median follow-up was 93 months. No significant differences were found in CR-FS rates after stratifying according to burden and site of PSMs. Men with unilateral PSMs experienced significant higher CR-FS rates compared to those with bilateral PSMs (87.1% vs. 71,3% at 10 years, p

The presence of amyloid deposits in bladder walls is a rare histological finding. It can be linked to primary (limited to bladder) or secondary (systemic, associated with chronic inflammatory disorders) amyloidosis. Secondary bladder involvement is very uncommon; it usually presents with gross hematuria, which is challenging to manage, due to frail bladder mucosa and/or necrosis. We present a case of 54-year old man with secondary bladder amyloidosis due to Crohn's disease, that caused gross hematuria and severe anemia, which was managed conservatively by endoscopic transurethral resection, diatermocoagulation, clot evacuation and urinary drainage by bilateral percutaneous nephrostomy, with spontaneous resolution. Secondary bladder amyloidosis is a rare condition that presents with severe hematuria, difficult to control with standard management. Owing to chronic nature of the disease, treatment should be aimed to a conservative approach whenever possible. In case of failure, invasive procedures should be considered as salvage therapies.

Background: Minimally-invasive approach is one of the mainstays of Enhanced Recovery After Surgery (ERAS) pathways. Robot-assisted radical cystectomy (RARC) introduction has reduced the surgical burden on patient's recovery. Accordingly, ERAS protocol benefits may be more striking in RARC patients. We evaluated the impact of surgical approach on perioperative outcomes, Fast Track (FT) recovery steps and Trifecta success rates in patients undergoing RC followed by FT protocol. Materials and methods: We considered 147 patients who underwent RC, with open (Open radical cystectomy [ORC]; 47.6%) or robotic (RARC; 52.4%) approach at 2 tertiary centers. Urinary diversions were ileal conduit or orthotopic neobladder. All patients underwent FT protocol. We analyzed perioperative surgical and functional outcomes and Trifecta success rates (namely, defecation 0.05). Conclusion: RARC has more favorable perioperative outcomes compared to ORC, with higher Trifecta success rates. Accordingly, robotic approach should be ideally included in every center where ERAS protocol is applied to RC for maximizing patient's recovery.

Regulatory T Cells (Tregs) are a T-lymphocyte subset involved in the maintenance of immune peripheral tolerance. Despite evidence of the adaptive immune system’s role in Alzheimer’s Disease (AD), the involvement of Tregs is still not clear. We focused on the Flow-Cytometry analysis of the Treg frequencies and phenotypes in the AD. The aim of the study is to analyse similarities and differences in Tregs profile between Alzheimer’s Disease and Multiple Sclerosis. Regulatory T Cells (CD4+/CD25high/CD127low-neg) were identified using an innovative Flow Cytometry method and subtyped as Resting (analysed CD45RApos/CD25dim), Activated (CD45RAneg/CD25bright) and Secreting (CD45RAneg/CD25dim) cells. Our data demonstrate a significant decrease in the total and Resting Tregs in AD patients when compared to healthy subjects. The percentage of the results of the Resting Tregs were also reduced in MS patients together with a parallel frequency increase of Activated Tregs. Our data suggest that altered Treg phenotypes observed in both diseases could play a role in the impairment of the Treg-mediated immunological tolerance, recalling a possible link between the two pathologies. Given that this study was conducted on a restricted population, if confirmed by a further and enlarged study, the implications of the autoimmune mechanisms in AD pathophysiology could open new immunotherapeutic perspectives based on Treg modulation.

Introduction: the Italian Radical Cystectomy Registry (RIC) is an observational prospective study aiming to understand clinical variables and patient characteristics associated with short- and long-term outcomes among bladder cancer (BC) patients undergoing radical cystectomy (RC). Moreover, it compares the effectiveness of three RC techniques - open, robotic, and laparoscopic.Methods: from 2017 to 2020, 1400 patients were enrolled at one of the 28 centers across Italy. Patient characteristics, as well as preoperative, postoperative, and follow-up (3, 6, 12, and 24 months) clinical variables and outcomes were collected. Results: preoperatively, it was found that patients undergoing robotic procedures were younger (pConclusion: the present study summarizes the characteristic of patients included in the RIC. Future results will provide invaluable information about outcomes among BC patients undergoing RC. This will inform physicians about the best techniques and course of care based on patient clinical factors and characteristics.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Background: Mutations in genes encoding one of the subunits of succinate dehydrogenase (SDH) are involved in pheochromocytoma (PHEO) and paraganglioma (PGL) development. Over the last few years, such mutations have also been associated with non-chromaffin tumors. However, immunohistochemistry (IHC) on the tumor tissue and a study on the loss of heterozygosity (LOH) aimed at demonstrating the pathogenic role of SDHx genes have only been employed in a few cases. Case report: We describe the case of a 19-year-old Caucasian man with a germline SDHB mutation, who presented with acne vulgaris resistant to medical treatment. His follow-up for chromaffin tumors was negative, while hormonal tests revealed suppressed gonadotropins with testosterone in the upper range of normality and elevated β-human chorionic gonadotropin (β-hCG). At the whole-body enhanced CT scan, a mediastinal lesion suggestive of a germ cell tumor (GCT) was detected. 18FDG-PET (fluorodeoxyglucose-positron emission tomography) imaging showed low glucose metabolism at the mediastinal site. Surgical removal of the mass was uneventful. Pathology confirmed the diagnosis of GCT consisting of cystic teratoma (95%) and seminoma (5%). IHC for SDHB showed normal protein expression, and genetic analysis of the tumor tissue revealed the absence of SDHB LOH. Normalization of the hormonal tests and acne attenuation were achieved after surgery. Conclusion: We report an incidental association of a germinal SDHB mutation and mediastinal GCT in a young Caucasian man. Our paper highlights the importance of IHC and genetic analysis in confirming the etiologic role of SDHx genes in nonchromaffin tumors, thus excluding incidental associations.

INTRODUCTION AND OBJECTIVE:Due to the low morbidity, local tumor ablation (LTA) like Cryoablation (Cryo) and Radiofrequency ablation (RFA), are gaining popularity as alternative to PN. The aim of o...

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the &ldquo, tip of the iceberg&rdquo, of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV &lt, 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

The genetic approach of pheochromocytomas and paragangliomas has changed in the last two decades. Nowadays, we know that more than 40% of patients have a germline mutation in one of the susceptibility genes identified to date. Our aim is to underline how genetic diagnosis by next-generation sequencing (NGS) can improve the management of patients affected by pheochromocytomas and paragangliomas in our routine diagnostic screening. We reported a case presentation and next-generation sequencing analysis supported by in silico studies and evaluation of mitochondrial status in KIF1Bβ tissue. A 46-year-old male affected by a left secreting pheochromocytoma underwent surgery in 2017. After surgery, the normetanephrine levels decreased very slowly and a suspected abdominal lymph node was detected. We found a novel germline KIF1Bβ gene mutation, c.4052C > T, p. Pro1351Leu associated with tumor loss of heterozygosity, and resulted likely-pathogenetic by in silico studies. This mutation was also associated with an increased number of mitochondria through the electron microscopy compared with wild-type tissues as suggestive for mitochondria neoformation compensatory to the mitochondrial autophagic figures observed. Our results underline the usefulness of next-generation sequencing in the presence of multiple tumor predisposition genes and how, at the same time, its use may result challenging for the clinicians. To date, performing the genetic analysis according to the latest Consensus Statement is mandatory in patients affected by PHEO/PGL.

Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS.

Mutations in one of the five genes encoding the succinate dehydrogenase (SDHx) or mitochondrial complex II cause the corresponding family syndromes characterized by the occurrence of pheochromocytomas (PHEO) and paragangliomas (PGL). Recently, other solid growths, such as gastrointestinal stromal tumors (GISTs), renal cell carcinomas (RCCs) and pituitary adenomas (PAs) have been associated with these syndromes. In the absence of prospective studies assessing their frequency, at present, their occurrence seems too infrequent to suggest systematic screening for SDHx mutation carriers. However, SDHB immunohistochemistry (IHC) on tumor tissues or SDHx genetic testing on blood or tumor samples should be performed in patients affected by GISTs, RCCs or PAs with clinicopathologic phenotypes suggesting an etiologic role of SDHx genes.

Background The SARS-CoV-2 pandemic has led to a remodeling of care activity, including the inflammatory bowel disease (IBD) centers. According to international recommendations, we did not discontinue immunosuppressive or biologic drugs and reorganized biologic drug infusions’ administration to minimize the risk of infection. The aim of this study is to describe the clinical outcome of SARS-CoV-2 infection and the adherence to the scheduled biologic therapies in a single tertiary center including both IBD adult and pediatric patients treated with biological agents during COVID-19 pandemic. Methods Demographic information, clinical data and the adherence to the therapy were collected in all consecutive IBD patients treated with biologic agents from March 2020 to February 2021. Moreover, we reported the clinical outcome of IBD patients infected with SARS-CoV-2. Results A total of 278 subjects (171 male) treated with a biologic agent (34 children) were included. The mean age was 41.6 ± 17.3 years (range 5 – 88), and the mean disease duration was 12.7 ± 10.1 years (range 0.2 – 49). One hundred eleven patients (39.9 %) had ulcerative colitis, and 167 (60.0 %) had Crohn’s disease. About the therapy: 73 patients were receiving infliximab, 93 adalimumab, 20 golimumab, 59 vedolizumab, 29 ustekinumab, and 4 an experimental compound. 31 patient, including 1 child, (11.1 %) were treated with combination therapy. The mean Charlson Comorbidity Index was 1.5 (range 0 – 8). Twenty one patients (2 pediatrics and nineteen adults) (7.5 %) had a confirmed diagnosis of SARS-COV-2 infection. Only one of them (an adult man) was hospitalized but did not require intensive care unit measures. Compared to cumulative SARS-CoV-2 infection in the general population in our Region, the cumulative incidence of SARS-CoV-2 infection in our cohort was significantly higher (7.5% vs 4.0%, p < 0.002). Twelve adult patients (4.3%) postponed biologic therapy for fear of SARS-CoV-2 infection during the first wave of the pandemic, and two of them (16.6%) experienced a clinical relapse. Conclusion In our cohort, the adherence to biologic therapy was high during the pandemic, but the rate of clinical relapse in patients who postponed the scheduled administration of therapy was relevant. Although the incidence of SARS-CoV-2 infection was significantly higher than in general population, the clinical impact was mild as no patients experienced a severe form of COVID-19.

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

Background/aim Germline mutations in any of the succinate dehydrogenase (SDH) genes result in destabilization of the SDH protein complex and loss of SDHB expression at immunohistochemistry. SDHA is lost together with SDHB in SDHA-mutated tumours, but its expression is retained in tumours with other SDH mutations. We investigated whether SDHA/SDHB immunohistochemistry is able to identify SDH-related tumours in a retrospective case series of phaeochromocytomas (PCCs) and paragangliomas (PGLs). Materials and methods SDHA and SDHB immunostaining was performed in 13 SDH gene-mutated tumours (SDHB: n=3; SDHC: n=1; SDHD: n=9) and 16 wild-type tumours. Protein expression by western blot analysis and enzymatic activity were also assessed. Results Tumours harbouring SDH gene mutations demonstrated a significant reduction in enzymatic activity and protein expression when compared to wild-type tumours. SDHB immunostaining detected 76.9% of SDH mutated PCCs/PGLs (3/3 SDHB-mutated samples; 1/1 SDHC-mutated sample; 6/9 SDHD-mutated samples). In three SDHD-related tumours with the same mutation (p.Pro81Leu), positive (n=2) or weakly diffuse (n=1) SDHB staining was observed. All wild-type PCCs/PGLs exhibited SDHB immunoreactivity, while immunostaining for SDHA was positive in 93.8% cases and weakly diffuse in one (6.2%). SDHA protein expression was preserved in all tumours with mutations. Conclusion SDHA and SDHB immunohistochemistry should be interpreted with caution, due to possible false-positive or false-negative results, and ideally in the setting of quality assurance provided by molecular testing. In SDHD mutation, weak non-specific cytoplasmic staining occurs commonly, and this pattern of staining can be difficult to interpret with certainty.

The recent introduction of the “precision medicine” concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326− EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

In the pathophysiology of neurodegenerative disorders, the role of misfolded protein deposition leading to neurodegeneration has been primarily discussed. In the last decade, however, it has been proposed a parallel involvement of innate immune activation, chronic inflammation and adaptive immunity in the neurodegeneration mechanisms triggered by proteinopathies. New insights in the neurodegenerative field strongly suggest a role for the immune system in the pathophysiology of neurodegenerative disorders. Therefore, the hypothesis underlining the modulation of the innate and the adaptive immune system in the events linked to brain deposition of misfolded proteins could open new perspectives in the setting of specific immunotherapeutic strategies for the treatment of neurodegenerative diseases. Therefore, we have reviewed the pathogenic hypothesis in neurodegenerative pathologies, underling the links between the deposition of misfolded protein mechanisms and the immune activation.

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

INTRODUCTION AND OBJECTIVES:Although multiparametric MRI (mpMRI) is the most accurate tool in the detection of clinically significant Prostate Cancer (CsPCa), some limitations have reduced its use ...

Purpose: Metabolic aberrations have been described in neoplasms with mutations in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those mutations. Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. Methods: Using liquid-chromatography-mass-spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multi-gene panel-sequencing was applied to detect driver mutations in cases with indicative metabolite profiles but undetermined genetic drivers. Results: Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline mutations in FH and somatic mutations in IDHx and SDHx, including the first case of a somatic IDH2 mutation in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) mutations. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx, thereby enabling missense-variants associated with SDHx LOF to be distinguished from benign variants. Conclusion: We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with mutations in FH and IDHx.

This paper aims at developing the knowledge on the seismic behavior of dowel beam-to-column connections, typically employed in precast buildings in Europe. Despite the large diffusion of the industrial buildings, a high seismic vulnerability was exhibited by these structures, mostly due to the connection systems deficiencies, during some recent earthquakes (Emilia 2012, Turkey 2011). An experimental campaign was conducted on a typical dowel connection between an external column and a roof beam. In this paper, the performed cyclic shear test is described. According to the experimental results, the seismic response of the system is evaluated in terms of strength, stiffness and failure mechanism. Moreover, the complete damage pattern of the test is described by means of the instrumentations records. The connection failure occurred due to the concrete cover failure in the column (splitting failure). Such a mechanism corresponds to a negligible energy dissipation capacity of the connection, compared to the overall seismic response of the structure. The experimental results are also compared with the results of a similar monotonic shear test, as well as with some literature relationships for predicting the strength of dowel connections under horizontal (seismic) loads.

Phaeochromocytomas and paragangliomas (PPGLs) are neural-crest-derived tumours of the sympathetic or parasympathetic nervous system that are often inherited and are genetically heterogeneous. Genetic testing is recommended for patients with these tumours and for family members of patients with hereditary forms of PPGLs. Due to the large number of susceptibility genes implicated in the diagnosis of inherited PPGLs, next-generation sequencing (NGS) technology is ideally suited for carrying out genetic screening of these individuals. This Consensus Statement, formulated by a study group comprised of experts in the field, proposes specific recommendations for the use of diagnostic NGS in hereditary PPGLs. In brief, the study group recommends target gene panels for screening of germ line DNA, technical adaptations to address different modes of disease transmission, orthogonal validation of NGS findings, standardized classification of variant pathogenicity and uniform reporting of the findings. The use of supplementary assays, to aid in the interpretation of the results, and sequencing of tumour DNA, for identification of somatic mutations, is encouraged. In addition, the study group launches an initiative to develop a gene-centric curated database of PPGL variants, with annual re-evaluation of variants of unknown significance by an expert group for purposes of reclassification and clinical guidance. Funding Agencies|Cancer Prevention and Research Institute of Texas (CPRIT) [RP101202, RP57154]; Department of Defense [CDMRP W81XWH-12-1-0508]; Voelcker Fund; National Institutes of Health (NIH)s National Center for Research Resources; National Center for Advancing Translational Sciences [8UL1TR000149]; INSERM; French National Cancer Institute (INCA); Direction Generale de lOffre de Soins (DGOS); INCA [INCA-DGOS_8663]; European Union [633983]; Brazilian National Council for Scientific and Technological Development (CNPq); Cancer Research for PErsonalized Medicine (CARPEM); ERC Advanced Researcher Award

Heart valve diseases are usually treated by surgical intervention addressed for the replacement of the damaged valve with a biosynthetic or mechanical prosthesis. Although this approach guarantees a good quality of life for patients, it is not free from drawbacks (structural deterioration, nonstructural dysfunction, and reintervention). To overcome these limitations, the heart valve tissue engineering (HVTE) is developing new strategies to synthesize novel types of valve substitutes, by identifying efficient sources of both ideal scaffolds and cells. In particular, a natural matrix, able to interact with cellular components, appears to be a suitable solution. On the other hand, the well-known Wharton's jelly mesenchymal stem cells (WJ-MSCs) plasticity, regenerative abilities, and their immunomodulatory capacities make them highly promising for HVTE applications. In the present study, we investigated the possibility to use porcine valve matrix to regenerate in vitro the valve endothelium by WJ-MSCs differentiated along the endothelial lineage, paralleled with human umbilical vein endothelial cells (HUVECs), used as positive control. Here, we were able to successfully decellularize porcine heart valves, which were then recellularized with both differentiated-WJ-MSCs and HUVECs. Data demonstrated that both cell types were able to reconstitute a cellular monolayer. Cells were able to positively interact with the natural matrix and demonstrated the surface expression of typical endothelial markers. Altogether, these data suggest that the interaction between a biological scaffold and WJ-MSCs allows the regeneration of a morphologically well-structured endothelium, opening new perspectives in the field of HVTE.

Adrenocortical carcinoma (ACC), a rare and aggressive neoplasia, presents poor prognosis when metastatic at diagnosis and limited therapies are available. Specific and sensitive markers for early diagnosis and a monitoring system of therapy and tumor evolution are urgently needed. The liquid biopsy represents a source of tumor material within a minimally invasive blood draw that allows the recovery of circulating tumor cells (CTCs). CTCs have been recently shown to be detectable in ACC. In the present paper, we evaluated the prognostic value of CTCs obtained by size-filtration in a small pilot cohort of 19 ACC patients. We found CTCs in 68% of pre-surgery and in 38% of post-surgery blood samples. In addition, CTC clusters (CTMs) and cancer associated macrophages (CAMLs) were detectable in some ACC patients. The median number of CTCs significantly decreased after the mass removal. Finally, stratifying patients in high and low pre-surgery CTC number groups, assuming the 75th percentile CTC value as cut-off, CTCs significantly predicted patients&rsquo, overall survival (log rank = 0.005), also in a multivariate analysis adjusted for age and tumor stage. In conclusion, though preliminary and performed in a small cohort of patients, our study suggests that CTC number may represent a promising marker for prognosis and disease monitoring in ACC.