Noonan Syndrome Growth Charts and Genotypes: 15-Year Longitudinal Single-Centre Study.

INTRODUCTION: Growth patterns in Noonan syndrome (NS) remain relatively unknown. The objective of this study was to provide growth reference curves for patients with NS and identify correlations between their growth, genotype, and clinical features. METHODS: This was a 15-year-long, monocentric, observational, retrospective, non-interventional study. Children with NS followed up between 2005 and 2022 at "Bambino Gesù" Children's Hospital, Italy, were included and excluded if they had received growth hormone treatment. Comparison of growth curves of participants with NS versus the general Italian population and further genotypic analyses were performed. RESULTS: Overall, 190 eligible participants with NS were identified, with median (interquartile range) age of 14.01 (9.05-19.25) years (55.8% male). Cardiovascular anomalies were present in 85.3% of participants, most commonly pulmonary stenosis (52.6%) and atrial septal defects (36.8%); 48.1% of male participants had cryptorchidism. The most frequently detected mutations were in PTPN11 (66.3%) and SOS1 (13.9%). NS sex-specific centile curves for height, weight, body mass index, and height velocity were produced. For both sexes, the 50th percentile of height and weight for participants with NS overlapped with the 3rd percentile for the general Italian population. Both sexes with a PTPN11 mutation had a significantly lower height and weight than those with "other mutations" at 5 years old. No significant associations were observed between cardiac anomalies and PTPN11 mutation status. CONCLUSION: We present longitudinal data describing growth curves and trends, the natural history, and genotypes of the NS population, which provide a useful tool for clinicians in the management of NS.

MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: Expression signatures and in vitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain­containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed.

Comparison of the clinical usefulness of different urinary tests for the initial detection of bladder cancer: a systematic review.

OBJECTIVES: The standard initial approach in patients with hematuria or other symptoms suggestive of bladder cancer (BC) is a combination of cystoscopy and urine cytology (UC); however, UC has low sensitivity particularly in low-grade tumors. The aim of the present review was to critically analyze and compare results in the literature of promising molecular urinary tests for the initial diagnosis of BC. METHODS: We searched in the Medline and Cochrane Library databases for literature from January 2009 to January 2019, following the PRISMAguidelines. RESULTS: In terms of sensitivity, ImmunoCyt showed the highest mean and median value, higher than UC. All tests analyses showed higher mean and median sensitivity when compared with UC. In terms of specificity, only UroVysion and Microsatellite analyses showed mean and median values similar to those of UC, whereas for all other tests, the specificity was lower than UC. It is evident that the sensitivity of UC is particularly low in low grade BC. Urinary tests mainly had improved sensitivity when compared to UC, and ImmunoCyt and UroVysion had the highest improvement in low grade tumors. CONCLUSIONS: Most of the proposed molecular markers were able to improve the sensitivity with similar or lower specificity when compared to UC. However, variability of results among the different studies was strong. Thus, as of now, none of these markers presented evidences so as to be accepted by international guidelines for diagnosis of BC.

Alopecia areata treated with dimethyl fumarate: A case series.

Alopecia areata is an autoimmune condition leading to non-scarring hair loss. Clinically, several presentations ranging from single or few small patches to complete hair loss are documented. The management of alopecia areata is challenging and all available treatments do not ensure a long-term remission to assess the safety and efficacy of systemic dimethyl fumarate in alopecia areata patients not responding to other systemic treatments. After obtaining informed consent, we administered off-label dimethyl fumarate to 10 adult patients with alopecia areata, for a period ranging from 4 to 37 weeks. Medical information for each patient and the severity of alopecia tool (SALT) score before and after dimethyl fumarate administration were recorded. During the treatment, 50% of patients (5 patients out of 10) had a slight improvement of hair regrowth; it was mainly as partial hair regrowth (ranging from 8% to 32%) and only one patient (10%) achieved > 50% terminal hair regrowth. Authors conclude that dimethyl fumarate is not advisable as a treatment of alopecia areata, also considering the risk of fumaric acid esters toxicity.

Predictive value of MCM5 (ADXBLADDER) analysis in urine of men evaluated for the initial diagnosis of bladder cancer: A comparative prospective study.

BACKGROUND: To compare predictive value of MCM5 to urinary cytology (UC) for the primary diagnosis of bladder cancer (BCa). METHODS: We prospectively enrolled 91 patients who presented macroscopic hematuria or persistent lower urinary tract symptoms at our institution. Single voided mid-stream urine specimens were collected for UC and MCM5 (ADXBLADDER; Arquer Diagnostics Ltd, Sunderland, United Kingdom) assessment. Cystoscopy was used as confirmatory test, and positive cases underwent transurethral resection of bladder tumor with histopathological evaluation. RESULTS: Forty cases (43.9%) showed a positive cystoscopy for BCa. Histology was obtained in 37 cases: 16 (43.2%) high-grade (HG) and 21 (56.8%) low-grade (LG) transitional cell carcinoma (TCC). UC had a sensitivity of 62.5%, specificity of 86.3%, PPV of 78.1% and NPV of 74.6%. Sensitivity, specificity, PPV and NPV of MCM5 were 60.0%, 88.2%, 80.0% and 73.8%, respectively. According to tumor grade, MCM5 and UC showed a sensitivity of 47.6% and 52.4% in LG, and 87.5% and 75.0%, respectively, in HG TCC. False-positive rates were 11.8% and 13.7% of negative cases for BCa with MCM5 and UC test, whereas false-negative results were found in 40.0% and 37.5% of BCa cases, respectively. The combination of the two tests showed a sensitivity of 71.4% in LG, and 93.8% in HG TCC. CONCLUSION: In the present analysis, MCM5 showed lower sensitivity than UC in predicting BCa primary diagnosis. According to tumor grade, MCM5 showed a higher sensitivity in the detection of HG BCa compared to UC, although values were not significantly different.

Correction to: HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3ß kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3ß at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3ß and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

Predictors of Hospitalization After Ureteroscopy Plus Elective Double-J Stent as an Outpatient Procedure.

PURPOSE: To evaluate the safety and feasibility of ureteroscopy plus elective double-J stent as an outpatient procedure in an unselected population with regard to the treatment for ureteral calculi and to present a multivariate analysis of factors predict hospitalization. MATERIALS AND METHODS: Ureteroscopy was performed as an outpatient procedure on 308 consecutive patients with ureteral stones. Contraindication for day case surgery was the only exclusion criteria from the study. All causes that led to immediate hospitalization were recorded; at the same time, all causes of hospitalization that occurred within 72 h from the procedure were also recorded and included in the final analysis. RESULTS: The overall stone-free rate and the rate of hospitalization were 94.5 and 9.7% respectively. Intraoperative complications were observed in 16 patients (5.1%). In terms of the variables related to hospitalization, the univariate analysis showed a statistical significant association between the American Society of Anesthesiologists (ASA) score (p < 0.001) and operative time (p = 0.018). At multivariate analysis, the only independent factor predictor of hospitalization was the ASA score (p < 0.001). CONCLUSIONS: In our experience, semirigid ureteroscopy is a safe and effective treatment that is independent of intraoperative local conditions or stone size. Elective Double-J stenting avoids major complications as the first reason for hospitalization. We suggest that ASA score > 2 should be taken into account when ureterorenoscopy is planning as an outpatient procedure.

MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment.

Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.

MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells. RESULTS: Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells. CONCLUSIONS: Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

BACKGROUND: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts. METHODS: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo. RESULTS: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo. CONCLUSIONS: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.