However, the tear concentration in epidermal growth factor (EGF), fractalkine, IL-8, IL-1RA, interferon-inducible protein (IP)-10/CXCL10, vascular endothelial growth factor (VEGF) and IL-6, comparing the active and inactive period, was not statistically different. Apart from the tear VEGF levels, the cytokine/chemokine concentration in tears in the active/inactive phase was statistically different (p < 0.05) from the counterpart levels in plasma. In conclusion, no isolated cytokine/chemokine in the tears has been found in a concentration which could be used as a potential biomarker of disease activity and treatment response.Uveitis accounts for up to 20% of blindness in Europe, making the development of new non-invasive biomarkers which could help in its management a field of interest. It has been hypothesised that tear levels of cytokines and chemokines could be used as a potential biomarker in patients with anterior uveitis, and this could be correlated with their concentration in plasma. Therefore, we measured twelve cytokines/chemokines in tear and plasma samples of 22 patients diagnosed with active anterior uveitis. Levels of these molecules in tears and plasma were compared and associated with the degree of activity of the uveitis. It is notable that the percentage of tear interleukin (IL)-6 detection was significantly reduced in the inactive phase ( The term uveitis refers to the inflammation of the uvea tissue, which is composed of the iris, ciliary body and choroid, and it frequently involves the impairment of adjacent structures in the eye such as the cornea, vitreous humour, retina, or optic nerve. It is a severe eye disease, as it has been put forward as the cause of up to 10% of legal blindness in the USA ,3,4,5,6.The correct approach to uveitis patients is usually a challenge to the physician for several reasons. Firstly, it encompasses a great variety of pathologies, either systemic or non-systemic, which may present with a wide range of clinical manifestations, sometimes resulting in a misleading diagnosis and consequently delaying correct treatments. Secondly, a significant percentage of uveitis has a considerable risk for sight-threatening complications, some of which can be successfully treated, such as the formation of cataracts, whereas others result in permanent vision loss ,8,9,10. Although the exact pathophysiological mechanism underlying uveitis has not been completely unveiled, the cytokine and chemokine balance appears to have a critical role in the pathway of this entity. Cytokines and chemokines are secreted proteins mainly derived from mononuclear phagocytic cells and T-lymphocytes’ growth, differentiation and activation functions. They regulate all immune and inflammatory responses, acute and chronic, being a crucial mediator in cytotoxic, humoural, cell-mediated and allergic immunity, and their imbalance could lead to a real impairment in the immune system response . They caWithin this framework, several studies have reported increased levels of cytokines/chemokines in either plasma, vitreous or aqueous humour (Aqh) ,17,18,19In spite of the fact that the outcomes provided by the literature suggest that the analysis of cytokines/chemokines could be of utmost importance in our daily routine, it is also true that the procedures used to obtain these outcomes are not without risk, as they are invasive procedures which could be challenging to apply in the clinical setting. This is why the study of non-invasive tests that could have clinical implications is a field of interest.Being a non-invasive approach, analysis of the cytokines/chemokines concentration in tears could improve our understanding of the ocular immunological changes that take place over the ocular surface in this entity, which could also provide the possibility of more specific and effective treatments according to the stage of the disease ,21,22. PAn observational prospective longitudinal analytical study was performed. The present study was approved by the Institute of Applied Ophthalmobiology (IOBA) institutional review board and the University of Valladolid Clinical Hospital Ethics Committee and followed the tenets of the Declaration of Helsinki. Study participants were selected among patients who attended the Ocular Immunology and Uveitis Unit of both IOBA and University of Valladolid Clinical Hospital with active uveitis between July 2018 and January 2020. Informed consent was obtained from each participant over 18 years old, and parental consent was provided in the case of patients under that age.Clinical evaluation was always performed by authors LC and LG, each one always evaluating the same clinical parameters: slit-lamp anterior chamber cells, anterior chamber flare, vitreous haze grade, intraocular pressure, and estimation of best-corrected visual acuity in logMAR format. All patients, regardless of the type of uveitis, anatomic classification and grade of inflammation, were evaluated according to the criteria proposed by the Standardisation of Uveitis Nomenclature (SUN) Working group . In addiEligible patients had a clinical diagnosis of active anterior uveitis in the first visit and started on dexamethasone 0.1% eye drops six times a day and cyclopentolate 1% three times a day until the suppression of uveitis was achieved, then topical corticosteroids were tapered and cyclopentolate 1% was stopped. Study visits occurred at baseline, and weeks 2, 6, 12 and 24. The presence of anterior chamber cells for more than 1+, a two-step increase in the vitreous haze, or newly formed retinal/choroidal lesions at least three months after the end of corticosteroid treatment were considered as an active uveitis and excluded from the study . TherefoTo ensure as much as possible that the results would not be influenced by the effects of topical medications and/or any other surface disease, exclusion criteria were established. Exclusion criteria were the presence of any other active ocular surface disease at the time of sample collection, such as infectious conjunctivitis, keratitis, allergy or dry eye disease, and the presence of active uveitis during the last visit. In addition, patients with intermediate or posterior uveitis were excluded from the study as well.Single tear samples were obtained from the active-uveitis eye in both the first and the last visit. Following the technique previously described by our group , we usedA venous blood sample was taken from the antecubital vein. The blood samples were immediately placed into individual Eppendorf tubes  and centrifuged at 6000 rpm for five minutes at 4 °C. The sera were stored at −80 °C until used for the assay.β, IL-17A, IL-2, IL-23, IL-6, IL-8/CXCL8, interferon-inducible protein (IP)-10/CXCL10, vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α).The presence and concentration of 12 molecules were determined in both tear and serum samples by a multiplex immunobead-based array ; Millipore, Watford, UK), using a Luminex IS-100 . The following molecules were assayed: epidermal growth factor (EGF), fractalkine, interleukin (IL) 1 receptor antagonist (IL-1RA), IL-1 The samples were analysed as previously described  followinβ = 0.4; IL-17A = 0.25; IL-2 = 0.3; IL-23 = 28.6; IL-6 = 1.1; IL-8/CXCL8 = 0.4; (IP)-10/CXCL10 = 1.14; VEGF = 2.6; TNF-α = 1.1. For statistical analysis, in cases where the assayed molecule was undetectable, a regression-based imputation method for missing values was used, based on the multivariate imputation by chained equations algorithm [The collected data were stored and analysed using the “BEAD VIEW Software” . The minimum detectable concentrations (provided by the assay manufacturer) were (in pg/mL) EGF = 4.47; fractalkine = 4.27; IL-1Ra = 10.97; IL-1 lgorithm . CytokinStatistical analyses were performed by LGO using the R software .t-tests for parametric data or the Wilconxon’s signed rank sum test, the respective nonparametric test. p values less than or equal to 0.05 were considered significant.Results are expressed as mean ± SD unless otherwise indicated. Normality assumptions were checked by the Shapiro–Wilk test. Comparison between samples was performed using either Student’s paired 2-tailed n = 6), active disease in the last follow-up appointment (n = 6), active intermediate or posterior uveitis (n = 4) or concomitant ocular surface condition (n = 2). Eighteen uveitis patients, 10 females (66.6%) and 8 males (44.4%), of age 52.5 ± 12.2 years, were included in the study.Of the 36 patients initially recruited, 18 were excluded for the following reasons: inability to collect tears (Fifteen of the tear and plasma samples (83.3%) came from patients with unilateral uveitis, while three (16.7%) came from patients with bilateral uveitis, in which case only the eye with more severe condition was considered. Clinical data and uveitis characteristics are shown in Fifteen patients (83.3%) were not receiving systemic treatment at the time of sample collection, neither during the first episode nor at the time of final evaluation. Three samples (13.6%) were from patients on systemic antiviral treatment at the time of sample collection either during the first episode or at the time of final evaluation. Two (9.1%) were on a systemic anti-TNF-α agent.p < 0.05)  .Eight out of twelve molecules assessed in the plasma samples were detected in more than 60% of the samples in the active phase: EGF, fractalkine, IL-1RA, IL-6, IL-8, IP-10, TNF-α and VEGF. Eight out of twelve molecules assessed in the plasma sample were detected in more than 60% of the samples in the inactive phase: EGF, fractalkine, IL-1RA, IL-6, IL-8, IP-10, TNF-α and VEGF .α, IL-8, IP-10, VEGF and IL-6 in the tears, comparing the active and inactive periods . However, the difference between the tear and plasma levels of VEGF was not statistically significant (p > 0.05)  .β, IL-17A, IL-1RA, IL-2, IL-23, IL-8, IP-10, TNF-α and IL-6 in patients with uveitis in their active phase were independent of their counterpart levels in plasma, the difference being statistically significant (p < 0.05). However, the difference between the tear and plasma level of VEGF was not statistically significant (p > 0.05)  .One of the cornerstones of the most widely accepted classification system of uveitis, designed by The SUN Working group in 2005, consists of grading the severity of inflammation, which is the most important when assessing the efficacy of different treatment approaches . HoweverRegarding the aetiology, it is vital to consider that the aetiology of the uveitis could vary the cytokine/chemokine profile in tears and plasma. Therefore, sampling patients with different aetiologies could bias our outcomes and should be taken into consideration in future investigations, as the activation of different molecular pathways could lead to anterior uveitis. For instance, Takase et al.  investigIn our study, a panel of 12 inflammatory molecules was analysed in the tears and plasma of the patient throughout the different stages of the uveitis, which led us to investigate the variability of the molecules according to the uveitis activity and the relationship between the plasma and tear levels, as little is known about the origin of the cytokines in the tear. The technological advancements in the analysis of cytokines and chemokines have allowed the investigation of the cytokine profile from a very small sample such as vitreous or aqueous humour, or plasma ,27,28,29For instance, TNF-α was detected in tears in less than 10% of the samples, which suggests there might be no link between the concentration of TNF-α in tears and the intraocular levels, which is of particular interest as TNF-α inhibitors are currently one of the most common treatments in non-infectious uveitis. Hence, adalimumab, a fully human monoclonal antibody, is the only biologic agent approved by the FDA to treat non-anterior non-infectious uveitis . In addiOn the other hand, the percentage of IL-6 detection showed a lower tendency of detection after finishing the treatment. In previous investigations, the aqueous humour level of IL-6 has been correlated with the number of lymphocytes and neutrophils ,37. The The development of new biomarkers has been the focus of multiple investigations as the assessment of disease severity or treatment effect could be a game-changer in managing eye conditions. Recently, studies have suggested that the different cytokines and chemokines levels in aqueous humour, vitreous and serum of uveitis patients could be a potential biomarker ,42,43,44β2 in uveitis patients. However, they could not find significant differences in cytokine tear levels between active and inactive uveitis. To confirm or refute these data, we decided to evaluate the hypothesis that the tear molecules’ concentration could reflect the uveitis activity. Still, our outcomes could not find significant results in concordance with previous studies.Within this framework, Carreño et al.  evaluateTürkçüoğlu et al.  investigOur results are in concordance with these investigations, as no significant changes have been observed in the concentration of the chemokines and cytokines in tears. However, as mentioned above, it is essential to highlight the low tendency in the percentage of detection of IL-6 in the inactive phase, being detected in two-fold more cases in the active phase. In our study, the potential role of IL-10 as a clinical predictor was not assessed as patients who showed any degree of activity during the 6-month time appointment were excluded. Therefore, further investigations trying to unveil possible predictor factors could benefit the clinical practice.On the other hand, other studies have found different protein profiles in the tears of patients with active uveitis. For example, Eidet et al.  analysedIn addition, Kumar et al.  analysedRegarding the potential role of the serum cytokine profile, many investigations have explored the correlation between different uveitis clinical entities and disease activities and serum cytokine levels with contradictory results. Of note, most of these investigations have compared active with inactive uveitis or a healthy control group showing increased serum levels of different cytokines in the active group, such as the IL-8 ,40, IL-6Of note, unveiling new non-invasive biomarkers could be a field of particular interest in patients affected by juvenile idiopathic arthritis (JIA) with eye manifestations, as it commonly manifests as chronic anterior uveitis, which is often clinically silent and can lead to serious visual impairment . AngelesThe cytokine and chemokine tear origin is still controversial, as there is no clear answer as to whether the cytokines in tears are derived from the lacrimal gland, secreted by the conjunctival epithelium, by leakage from the surrounding blood vessels , or in aIn summary, to our knowledge, this is the first study in uveitis investigating whether the cytokine and chemokine concentration in tears could be a potential biomarker for uveitis activity or treatment response without showing significant changes between the active and inactive phases in the molecules studied. In addition, we have tried to unveil whether or not the cytokine and chemokine profiles in the tear samples are related to their concentration in plasma or if they were independent, as there is some controversy about the cytokine and chemokine tear origin, being postulated that it is a reflection of the one in the plasma. However, we have not found any correlation in these levels apart from the VEGF, the only molecule whose tear concentration showed a significant relationship with the plasma concentration. The discrepancy between the TNF-α levels in tear and plasma samples is remarkable since, as mentioned above, one of the main treatments is non-infectious uveitis based on blocking this molecule.The main limitations of this study are the low number of patients included in the study, not enough to permit a proper subanalysis according to the type of uveitis, and the restricted number of molecules studied per sample, limiting a more completed analysis of the tear profile. In addition, we have to consider that uveitis is a rare condition with an estimated prevalence of around 121 cases per 100,000 adults, responsible for 10% of cases of blindness in the United States . MoreoveOne of the limitations of studying small samples is the limited number of molecules evaluated per sample. In this framework, the Luminex method has been used to investigate the potential benefit of the cytokine and chemokine profiles in tears due to its capacity to assess several proteins with one sample. However, new methods such as the quantitative microarray or the TripleTOF 5600 system allow a greater range of molecules to be evaluated per sample, which could be of interest for a more general analysis of the tear profile in uveitis patients according to their degree of activity .Considering everything, our conclusions are tentative, as multiple conditions could interfere with the outcomes. Therefore, further studies are needed to unveil the potential benefit of analysing the tear proteome to monitor treatment response or uveitis activity.


In the present study, gels based on xanthan gum and poloxamer 407 have been developed and characterized in order to convey natural antioxidant molecules included in niosomes. Specifically, the studies were conducted to evaluate how the vesicular systems affect the release of the active ingredient and which formulation is most suitable for cutaneous application. Niosomes, composed of Span 20 or Tween 20, were produced through the direct hydration method, and therefore, borate buffer or a micellar solution of poloxamer 188 was used as the aqueous phase. The niosomes were firstly characterized in terms of morphology, dimensional and encapsulation stability. Afterwards, gels based on poloxamer 407 or xanthan gum were compared in terms of spreadability and adhesiveness. It was found to have greater spreadability for gels based on poloxamer 407 and 100% adhesiveness for those based on xanthan gum. The in vitro diffusion of drugs studied using Franz cells associated with membranes of mixed cellulose esters showed that the use of a poloxamer micellar hydration phase determined a lower release as well as the use of Span 20. The thickened niosomes ensured controlled diffusion of the antioxidant molecules. Lastly, the in vivo irritation test confirmed the safeness of niosomal gels after cutaneous application. Antioxidants molecules (AMs) represent our body’s defense system against damage caused by free radicals: they are chemically defined as reducing agents capable of slowing down or preventing the oxidation chain reactions triggered by free radicals in the presence of oxidative stress.Much of the inputs to the production of radicals occurs as a consequence of the body’s metabolism and respiration, but also psycho-physical stress, environmental factors such as smog and smoke, and UV rays can increase the production of reactive elements inducing a cellular overload (oxidative stress) with altered functionality and consequent cutaneous disorders, e.g., dermatitis, tumors, psoriasis, wrinkles, alopecia, acne and skin aging.For instance, some research has shown that a high concentration of the hydroxyl radical OH· is the cause of the onset of tumors, following an alteration of the genetic information due to the oxidation of the nitrogenous bases . InsteadTherefore, antioxidants are the best allies in keeping the body healthy and for protecting the skin from the harmful effects of external factors. The mechanism of action of AMs in radical inhibition can be summarized in the following three points: (a) neutralization of reactive species, (b) sequestration of transition metal ions (chelating effect) and (c) enzymes inhibition involved in reactive oxygen species production . From a However, it should be kept in mind that many of these substances are particularly insoluble and unstable at different pH and temperature levels and under environmental stress conditions; therefore, the most effective solution is the topical application through delivery systems such as phospholipid-based (liposomes) and niotenside-based (niosomes) vesicular systems  to ensurIn the present study, gallic (GA) and ferulic acid (FA) were considered as example AMs of natural origin due to their activity. Indeed, GA is a potent tyrosinase inhibitor with excellent antioxidant effects, together with antibacterial and anti-inflammatory activity ,5,6. MorFinally, in order to obtain suitable systems for cutaneous application and delivery of AM embedded in niosomes, this research investigated the development and characterization of gels based on poloxamer 407 and xanthan gum.Gallic acid (GA), ferulic acid (FA), cholesterol (CH), Tween 20 (T), Span 20 (S) the copolymers poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) poloxamer 188 (P) and poloxamer 407 (pol) were from Sigma-Aldrich . All other materials and solvents were from Merck Serono S.p.A. .v/v) and subjected to removal under vacuum of the organic solvent residue using a rotary evaporator  obtaining a film on the glass wall that was hydrated using a 2 mg/mL AM aqueous solution, swirled and sonicated, at 60 °C, to give a final concentration of 25 mg/mL in terms of total vesicular components. Borate buffer (B) and Poloxamer 188 (P) solution (2.5% w/w) were alternatively used as a hydration medium. In Niosomes were prepared by thin-layer hydration with slight modifications  10]. In. In10]. 2 at 200 kV and images digitally recorded by CCD camera  using GMS 1.4 software  to process the images. During the visualization, the temperature of the sample was maintained below −175 °C.The morphology of niosomes was investigated using a Cryogenic Transmission Electron Microscope (Cryo-TEM). After vitrifying and transferring each sample to a Zeiss EM922Omega transmission electron microscope using a cryoholder  , specimeQ-range,  extended from 0.003 to 1.6 Å−1. The experiment exploited the mail-in service. Niosome samples were prepared in 2 mm polycarbonate capillaries, loaded into a capillary ladder, and analyzed, at 36 °C. Each image had a 2 min collection time, with a 0.4 mm X-ray beam. Two-dimensional (2D) data were corrected for background, detector efficiency and sample transmission, then radially averaged to derive I(Q) vs. Q curves [Small-angle and wide-angle X-ray scattering (SAXS and WAXS) experiments were performed using the Offline Xeuss 3.0 Diamond-Leeds SAXS facility (DL-SAXS) at Diamond Light Source . A gallium Excillum metaljet (9.4 keV), coupled with a movable beamstop-less Eiger 2 R 1M placed at the two distances of 100 mm and 2000 mm  was used. The final investigated Q curves .Niosomes dimension was measured on aqueous diluted fractions (1:10 by volume) using a Zetasizer Nano S90  equipped with a 5 mW helium neon laser with a wavelength output of 633 nm. Plasticware, cleaned with detergent washed and rinsed with milliQ water, was used for measurements made at 25 °C at 90° angle and run time around 180 s. “CONTIN” method  was usedv/v) the samples were magnetically stirred for 30 min. Afterwards, the amount of AM was quantified by UV, and the encapsulation parameter was calculated using Equation (1).AM is the total AM amount weighed to prepare the formulation. AM quantification was performed by means of a double-ray UV/Vis spectrometer  operating at 260 nm and 318 nm for GA and FA, respectively, in 1 mL quartz cuvettes. To estimate the AM content in the sample, a previously carried out calibration curve for each compound, was used as reference. The evaluation was performed on day 1, 7, 15 and 30 after production.The encapsulation efficiency (EE) in niosomes, expressed as AM content, was determined after 1 day from vesicles production on a ultracentrifuged 300 µl fraction of the formulation using a Microcon centrifugal filter unit YM-10 membrane . Ultracentrifugation was conducted at 8000 rpm for 20 min on a Spectrafuge™ 24D Digital Microcentrifuge . After diluting 100 microliters of retentate with methanol . The receptor compartment contained 5 mL of PBS 1X. This solution was stirred at 500 rpm, with the help of a magnetic bar, and thermostated, at 32 ± 1 °C, during all the experiments [2, were then plotted as a function of time. Diffusion coefficients (Js) were calculated from the linear portion of the accumulation curve and expressed as normalized fluxes (Jn) by dividing by AM concentration in the analyzed form, expressed in mg/mL.AM diffusion was evaluated using Franz cells associated with the mixed cellulose esters membrane. Briefly, dried membranes were immersed in distilled water, at room temperature, for 1 h then mounted on Franz diffusion cells  exposing a surface area of 0.78 cmeriments . Then, 150 values were calculated from the results after applying a previously obtained calibration curve. The obtained values (μg/mL) were the mean ± standard deviation of at least three independent experiments.DPPH radical-scavenging assay was utilized to rapidly evaluate the antioxidant capacity . The DPPescribed . The IC53+) to ferrous ions (Fe2+) under acidic conditions in the presence of 2,4,6-tripyridyl-s-triazine (TPTZ) [3+–TPTZ complex to the ferrous form, leading to an intense blue coloration measured at 593 nm (absorption maximum wavelength). Trolox was employed to perform the calibration curves, and therefore, the FRAP antioxidant activity is given as μmol Trolox equivalent/g of compounds.The quantitative FRAP assay measures the plasma ferric ion reducing capacity as it is based on the reduction of ferric ions (Fee (TPTZ) . Indeed,w/w) was added to niosomal formulation (xg-niosomes) and handily mixed for 10 min up to complete dispersion [w/w) to cold water (5–10 °C) magnetically stirred for 3 h and then kept at 5 °C for 12 h up to complete dispersion of poloxamer [To obtain thickened formulations, xanthan gum and poloxamer 407 were added to niosomes . To be pspersion , while poloxamer .The spreading capacity of niosomal gels was evaluated 24 h from gel preparation, at 37 ± 1 °C, to mimic the body temperature conditions . To be pSpreadability assay was performed three times, and then the mean values ± standard deviations were calculated using Equation (3).diameter .w/w) to a citrate buffer with pH 5.5 and stirred, at 95 °C, until solubilization. Then, the cooled gels agar slides were cut. An amount of 30 mg of gel was placed on the top of an agar slide placed in a Petri plate vertically positioned at 90° on a transparent box wall and maintained, at 37 ± 1 °C. After the gel placement, the time taken by the gel to run along the slide was measured. Gel leakage, expressed as the time traveled by the gel on the plate, was evaluated thrice, and the mean values ± standard deviations were determined.The in vitro leakage of niosomal gels was determined on rectangular agar slides prepared by adding agar  ,22,23. TIt is well known that the low bioavailability and stability of GA and FA hamper the administration on skin and limit the antioxidant potential of these molecules. In order to overcome these limitations, the encapsulation of GA and FA was investigated by the development of a preformulative study ,25. In pThen, the morphology and the inner structure of the produced niosomes were investigated and compared. In particular, the morphological characterization of the different formulations was addressed by Cryo-TEM visualization . Since tVesicular systems with different peculiarities ascribable to the components used in the formulation have been obtained. Indeed, uni- or multilamellar non-ionic surfactant vesicles self-assembled in aqueous medium have been obtained .Notably, both S a,b and TIn order to elucidate the internal structures of the different niosomal formulations, SAXS analysis was performed.Q−4-law, suggesting the presence of very large particles , independent from niosomes composition. However, the vesicles show a different degree of lamellarity and a rather disordered nature. Indeed, the Bragg peak observed at about 0.14 Å−1 in the scattering profiles confirms the lamellar inner structure of the niosomes, but its width and the absence of the characteristic higher-order peaks suggest that the positional correlation between adjacent bilayers is very small and/or that the degree of lamellarity is very low [The results, reported in very low . Interesvery low , seems cAfter production, the AMs-loaded formulations were characterized in terms of size and polydispersity. Afterwards, the dimensional stability was investigated for 30 days, as reported in The size distribution of niosomes after production ranged between 420 and 860 nm; thus, this parameter was strongly influenced by the composition of the vesicles. In particular, the higher dimensional increase was evidenced for niosomes comprising the combination of T and P as a surfactant and hydrating phase. This data agrees with the morphological and SAXS analyses, as well as with the literature , supportMoreover, no significant differences related to the alternative encapsulation of each AM have been evidenced.Concerning dispersity indexes, the presence of heterogeneous populations has been denoted, in particular, one month after production, since PdI values higher than 0.2 indicate a wide size distribution .The percentage of Ams content in the different vesicular systems was evaluated over time, and the chemical stability of the loaded drug was monitored up to 30 days after production. The results reveal that niosomes allowed a quantitative encapsulation of AMs after production, reaching percentages between 90% and 100% independently of the type of formulation.Specifically, in the case of GA, the composition of the formulations did not affect the encapsulation efficiency of the drug, which was maintained stable, reaching content values around 83% one month after production. The stability profile of GA content was similar for all the formulations. Concerning FA, NSB, NSP and NTP retained more than 85% of the drug after one month, while for NTB, the amount of drug decreased, reaching 75% of encapsulation. This behavior suggests that the presence of S, especially in combination with B as a hydration medium, ensured better stability to the system, as observed also in terms of size. Notably, the role of S as a destabilizing agent, when in aqueous solution, facilitates an increase in the entrapment efficiency of drugs, possibly explained by its HLB value being 8.6 .2 and hours, respectively. In The effect of the niosomes composition on AM diffusion has also been investigated by means of Franz-cell experiments. To be precise, to in vitro mimic the physiological conditions of AM diffusion through the skin, mixed cellulose esters membrane  and phosAs expected, from the comparison between each reference solution (crosses) and the corresponding niosomal formulation (dots) in terms of the aqueous phase, a controlled diffusion of AM from the vesicles has been evidenced. This behavior was more pronounced for B as compared to P. However, also for P, the solution ensured a controlled diffusion, ascribable to the chemical nature of the co-polymer.Furthermore, considering the hydration medium, it should be noticed that NSB and NTB displayed a faster drug diffusion profile with respect to those from NSP and NTP for both AMs. Indeed, the use of a micellar solution in the formulation conferred a “matryoshka” configuration to the system , respons50 values and FRAP assay to investigate their total antioxidant potential. Stability, intended as maintenance of the antioxidant effect, was monitored up to 30 days after production, and the results are showed in The use of GA and FA as antioxidants, in particular as radical-scavenging agents, is well demonstrated in the cosmetic and pharmaceutical fields ,32,33,3450 values in the range 2.87–3.77 μg/mL) than for FA .Comparing the different niosomal formulations with the corresponding AM solutions, the data obtained showed that the encapsulation strategy was able to maintain the antioxidant potential of the molecules, confirming the suitability of niosomes as GA and FA delivery systems. Regarding the DPPH assay, as can be seen in 50 values were significantly different; p < 0.0001). Additionally, NSP-GA and NTP-GA showed higher and statistically significant IC50 values than GA/P . In contrast, all FA-containing formulations possessed comparable antioxidant capacity to FA/B .NTB-GA displayed higher antioxidant activity than GA/B solution .In general, the ICp < 0.023 and p < 0.046, respectively). The 30-day stability of FRAP activity was maintained, except for GA/B, NSB-GA and NTP-GA, which showed a statistically significant decrease .Regarding the FRAP assay, the results corroborated what was shown by DPPH, indicating a greater antioxidant effect for GA  than FA (μmol TE/g ranging from 5714.88–6468.45) b. In parBeing niosomes dispersed in liquid form, the administration onto the skin is difficult and onsite permanence limited. Therefore, to obtain formulations with a certain grade of viscosity suitable for topical application, two different gelling agents, namely, xanthan gum and poloxamer 407 have been selected and added to niosome formulations. Indeed, these two biocompatible polymers are able to confer different adhesive properties to niosomes. Xanthan gum, an anionic polysaccharide obtained by bacterial fermentation , is a goAs evidenced, the addition of xanthan gum led to the formation of a stiff gel formulation. In fact, concerning spreadability, the lowest values have been obtained on xg-niosomes, and no running across the agar plate has been observed in leakage tests. This aspect could ensure higher retention on the application site. Conversely, pol-niosomes showed higher values in terms of spreadability and leakage behavior.In general, considering the hydration medium, niosomes composed of P resulted in thicker formulations, independently of the gelling agent used. This result could be related to the above-described role of the P micellar system in creating a rigid and complex structure.Concerning the pH characteristics of niosomal gels, as summarized in The AM diffusion from niosomal gels, obtained after addition of xg or pol, has been investigated and compared. In As depicted, the obtained diffusion profiles showed different behavior for GA and FA. In the case of GA, the passage of the drug through the membrane is mainly influenced by the thickening agent. In fact, the addition of xg provides a higher control on drug diffusion with respect to pol.s) and normalized in function of the experimental AM concentration in each formulation (Jn).Concerning FA, the different behavior seems to be related to the nature of the surfactant employed for the formulation. In the case of NSB and NSP, xg induced a lowest FA passage, while for NTB and NTP no significant differences have been detected with the use of xg or pol. Furthermore, the diffusion kinetics of AMs were expressed as fluxes , extrapoConcerning GA, the pol-made gel led to a faster diffusion of the drug. In fact, the possible solubilization of GA in pol, thanks to the ability of the polymer in creating a micellar network, could be responsible for its increased passage through the membrane.n values of FA diffusion, it was found that when T is used in the composition, the addition of a thickening agent did not affect a controlled passage of the drug with respect to formulations and data are superimposable. This behavior could be ascribed to the interaction between T and FA, and thus, it can be proposed that the diffusion is mainly governed by the vesicular system and less by the hydrophilic nature of the polymers, resulting in less structured gels.Considering the JIn all cases, P as a hydration medium of niosomes plays a crucial role in GA diffusion, corroborating the influence of the “matryoshka” configuration on drug diffusion.Being the topical administration the final target of these formulations, a patch test was performed in order to evaluate the safeness of the produced niosomal gels. Taking into account the results of spreadability and leakage, xg-niosomes have been selected for the patch test. To be precise, the potential irritation caused by applying xg-NSB, xg-NSP, xg-NTB and xg-NTP on skin have been evaluated on 20 healthy volunteers and the results expressed as a percentage of irritative reactions .It was found that the formulations exhibited the same results in terms of safeness; thus, all niosomal gels applied for 48 h under occlusive conditions can be classified as non-irritant. In fact, in 95% of cases, no irritative reactions have been detected, while 5% of cases led to negligible reactions, confirming the suitability of these systems for topical application.The present investigation enabled the design of niosomal gels suitable for AMs loading and subsequent topical delivery. Specifically, this study highlighted the influence of a hydration medium (B or P) and a thickening agent (xg or pol) on the diffusion of AMs.Strikingly, the results of the Franz-cell experiments and patch tests demonstrated that niosomal gel can deliver AMs in different ways depending on the interaction between the loaded drug, the non-ionic surfactant and the thickener agent. Indeed, in the case of FA, the presence of T facilitated an in vitro diffusion mainly governed by the vesicular system, while in the case of GA, an important role is due to the hydrophilic nature of the thickeners, corroborating the influence of the “matryoshka” configuration on drug diffusion. These data are in agreement with the cryo-TEM and SAXS analyses, showing the coexistence of unilamellar, multilamellar and multivesicular systems after hydration with B, or sole multilamellar vesicles, in the case of P hydration. However, an important contribution of the surfactant constituting the niosomes has been detected by means of SAXS analysis. Indeed, T seems to give rise to great hydration of the vesicle’s layers both in the presence of B and of P, while in the case of niosomes prepared with S as the surfactant, the presence of P induces an ordering of the lamellar structure resulting in the enhancement of the mean crystallite size.Moreover, no irritative reactions were detected during patch tests in 95% of cases, confirming the suitability of these systems for topical application.


The identification of non-invasive biomarkers from biological fluids collected by liquid biopsy provides new horizons for individualized therapeutic strategies and improves clinical decision-making in OSCC patients. Circulating microRNAs have emerged as biomarkers that may reflect not only the existence of cancer, but also the dynamic, malignant potential, and drug resistance of tumors. The aim of the systematic review is to evaluate and summarize the results of the published studies regarding the use of microRNAs as biomarkers for OSCC.A literature search was conducted on PubMed, Scopus, Web of Science, and Cochrane databases till November 2020. A total of 34 studies met the inclusion criteria and were therefore subjected to quality assessment. Each study was subjected to data extraction including; patient characteristics, type of fluid sample , molecular analysis method, specific dysregulated microRNA, and microRNA expression pattern. The analysis showed that 57 microRNAs of liquid biopsy samples of four different fluids  were analyzed. The prognostic and therapeutic significance of these microRNAs were suggested by several studies; where 41 microRNAs were upregulated while 16 were downregulated.Scientific evidence supports the interest in the use of microRNAs in the diagnosis and prognosis in OSCC patients; however, further studies in a larger cohort of patients are mandatory to introduce liquid biopsy in the routine clinical practice for the OSCC management. Key words:Biomarkers, liquid biopsy, microRNA, oral squamous cell carcinoma, systematic review. Oral squamous cell carcinoma (OSCC) represents the most common histo-type of head and neck cancers, representing 90% of diagnosed oral malignancies . DespiteThe etiology of OSCC is not still comprehensively understood. It may be multifactorial, where a combination of several genetic alterations and environmental risk factors may play a role, including tobacco and alcohol consumption, human papillomavirus (HPV) infection, and chronic mechanical trauma. OSCC can be evolved from oral potentially malignant disorders, such as oral leukoplakia, erythroplakia, lichen planus, smoking hyperkeratosis, and submucous fibrosis, or from normal epithelium with genetically altered keratinocytes.To date, histopathological analysis of a tissue biopsy is the gold standard for the diagnosis of OSCC. Some limitations can be considered of tissue biopsy; including that it is an invasive, costly, and potentially harmful procedure; furthermore, it is temporally and spatially limited, where it provides a snapshot of a single region of a heterogeneous tumor.Nowadays, cancer research is encouraging to find less invasive methods to provide a more comprehensive view of the cancer profile and to easily monitor its evolution and therapeutic response. In this regard, liquid biopsy has been recently proposed as a method for diagnosis, prognosis, and follow-up of OSCCs.The liquid biopsy involves the study and analysis of body fluids; such as blood, saliva, urine, seminal plasma and cerebrospinal fluid .In this method, include the analysis of circulating tumor cells (CTCs), circulating cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), extra-cellular vehicles (EVs) and microRNAs (miRNAs) .In case of confirmation of the reliability of this method for the diagnosis, prognosis, and follow-up of OSCCs, it may provide the dynamic observation of cancer evolution and for understanding temporal and spatial heterogeneity of tumors. Furthermore, it can also be used to detect early onset of therapy resistance, residual disease and recurrence.miRNAs are a large family of single-stranded non-coding RNA that includes 19-25 nucleotides, regulating the gene expression at the post-transcriptional level.miRNAs play a crucial role in cell cycle regulation, differentiation, apoptosis and migration. During carcinogenesis, some miRNAs are upregulated and some are downregulated, so any change in the expression of miRNAs can cause tumor suppression or act as carcinogens .Circulating miRNAs are extremely sTable under harsh conditions, such as high temperature, extreme pH, and in the presence of RNAs activity, suggesting their potential role as biomarkers for cancer diagnosis .Several studies reported an altered expression of blood and salivary miRNAs in different tumor types, including colon, lung, ovarian and breast cancers -10; howeThe aim of this systematic review was to evaluate and summarize the results of the published studies regarding the use of miRNAs as biomarkers for OSCC.This study was performed according to the PRISMA  guidelines . The proThe focus question was: “Do circulating miRNAs play a role as biomarkers in OSCC?”-Eligibility CriteriaThe inclusion criteria were: 1) studies with significantly dysregulated miRNAs from body fluids between OSCC patients and healthy controls; 2) studies which analyzed the presence of dysregulated miRNA in at least one of the body fluids among blood, serum, plasma, or saliva; 3) studies involving humans; 4) studies published in peer-reviewed journal; 5) and studies in English language.in vitro or in vivo animal studies.The Exclusion criteria were: 1) case-reports, case-series, conference proceedings, letters to the editor, short communications, systematic reviews; 2) -Search StrategyAn electronic search was conducted on PubMed, Scopus, Web of Science, and Cochrane Library databases till 25th November 2020. The following combination of keywords were used with the Boolean term “AND” and “OR”: (“microRNA” OR “miRNA”) AND (“squamous cell carcinoma” OR “squamous cells carcinoma” OR “squamous cell carcinomas” OR “squamous cells carcinomas”) AND . In addition, a manual search of references listed in each included study was performed, to avoid losing potential studies.-Study SelectionThe study selection was carried out in two stages. First, two reviewers (M.C. and F.R.) identified independently the possible eligible studies by screening the titles and abstracts of the resulted studies based on the abovementioned inclusion and exclusion criteria. Then, a full-text read was carried out independently by the same two reviewers for each identified study in the first stage. In case of disagreement between the two reviewers, the final decision for the inclusion of a study was taken by consensus discussion by the inclusion of a third reviewer (G.P.).-Data-Collection and Synthesis ProcessThe two investigators (M.C. and F.R.) independently assessed each eligible article, extracted data using a pre-established form using a Microsoft Excel spreadsheet . Discordant judgments were resolved by consensus discussion with the third investigator (G.P.).The following information were extracted from each study: (i) authors, (ii) country, (iii) year of publication, (iv) sample size, (v) gender, (vi) mean age, (vii) TNM staging, (viii) histological grading, (ix) type of fluid sample , molecular analysis method, (x) specific dysregulated miRNA, and (xi) miRNA expression pattern.In case of missing or incomplete data, an email was sent to the authors to obtain more detailed information; in case of missing answer, the article was excluded.-Reporting Quality and Risk of Bias AssessmentsThe quality assessment of the included studies was carried out using the SIGN “Scottish Intercollegiate Guidelines Network” methodology checklist for case-control studies. The SIGN checklist consisted of 11 statements to evaluate the risk of bias of the internal validity across 6 domains; groups comparability, differentiation, assessor blinding, outcome measures, confounding, statistical analysis. The scores for each statement were; “yes” (low risk) or “no” (high risk). In case of lack of details, the score “can’t say (moderate risk)” was given.After the piloted test of the checklist to confirm its compatibility with this review, the reviewers decided that grouped domains “groups comparability and differentiation”, that consists of more than one statement, would have got the final score “low risk” in case all responses to the statements were “yes”, and it would be “high risk” in case of presence of a negative response (no) or the presence of two or more than of “can’t say” responses. While the final score of “moderate” was given in case of the presence of one response of “can’t say” to one of the statements.Two reviewers (F.R. and F.M.) assessed independently each included study with this checklist. In case of conflicts, a third reviewer (G.P.) was consulted for arbitration. Finally, an overall assessment was provided to each study by the reviewers with the same scores.A total of 2879 studies were resulted from the search of the electronic databases and were distributed as follows; 896 studies from PubMed, 1103 studies from Scopus, 878 studies from Web of Science, and 2 studies from Cochrane database. After the removal of duplicates, a total of 1381 articles were screened. The title and abstract screening resulted in the exclusion of 1321 studies. After the full-text screening, 26 articles were excluded due to the following reasons: out of topic (n=14); insufficient information (n=6); studies considered the same study population of other studies (n=1); studies without a control group (n=5). No disagreement regarding study eligibility was observed between the reviewers. No additional relevant studies were found by checking references and citations of the included studies. A detailed flow chart of the selection process is shown in Figure In the overall assessment of the risk of bias, fourteen studies of a total of 34 studies were rated with a low risk score. The majority (17 studies) of the included studies were awarded a moderate risk score. This was because of the partial or poor identification of the population source with an incomplete demonstration of the baseline similarities and/or differences between the case and control groups. Also, because some studies didn’t provide a complete statistical analysis including the confidence interval (CI). The presence of blinding assessor in all the included studies of this review was unclear. Only 3 studies were rated with a high-risk score, this was mainly due to the incomplete reporting of the participant demographic data mainly that of the control group and the poor presentation of comparison between participants and non-participants to establish their baseline similarities or differences  cont.. FThe characteristics of the included studies are presented in -Group A featuresGroup A involved 25 case-control studies published from 2010 to 2020 and carried out in 10 different countries, where 8 studies in China, 6 studies in Taiwan, 3 studies in Germany, 2 studies in Iran, and 1 study in each of the following countries: Canada, Japan, Brazil, Denmark, and Pakistan. The sample size of the studies ranged from 16 to 170, and comprised a total of 2730 subjects .The percentage of males and females was reported in 19 of the 25 studies. Males represented 62.6% while females were 37.4%. The mean age of OSCC patients was 57 years (20 - 93 years). The percentage of alcohol consumers among OSCC patients was reported in 8 of 25 studies, and 54.1% of the studied patients were alcohol consumers. With regard to smoking, 81.5% of the studied patients were smokers.Histological grading of OSCC was reported in 10 of the 25 studies: 28% of OSCC were well-differentiated, 12% were between well-differentiated and moderately differentiated, 24% were moderately differentiated, 4% were between moderately differentiated and poorly differentiated, and 32% were poorly differentiated. Tumor staging was reported in 12 studies: stage I (10%), stage I-II (23%), stage II (10%), stage I-III (3%), stage III (14%), stage III-IV (23%), and stage IV (17%). Eleven studies reported the presence/absence of lymph node involvement (N), of which 48% was N0, and 52% was N+. Only 4 studies reported the presence/absence of distant metastases (M), of which 15% were M+. With regard to the site of OSCC, 33% of OSCC were on the tongue, 17% on the buccal mucosa, 17% on the gingiva, 25% in other oral sites, and 8% in unspecified sites.The control group consisted of 371 males and 184 females with a mean age of 54 years (15 - 88 years). In this group, 18.3% were smokers, and 12% were alcohol consumers.Expression levels of single miRNA (n=34) were detected by quantitative Polymerase Chain Reaction in real-time. According to the individual miRNA expression levels, 23 miRNAs were up-regulated and 11 were down-regulated .-Group B featuresGroup B involved 10 case-control studies published from 2009 to 2020 and carried out in 8 different countries; 2 studies in Iran, 2 studies in China, and 1 study in each one of the following countries: Taiwan, USA, Turkey, Saudi Arabia, Serbia, and Italy.The sample size of studies included in Group B ranged from 9 to 50 and comprised a total of 372 subjects .The percentage of males and females was reported in 7 of the 10 studies. Males were 65.5% and females were 34.5%. OSCC patients in Group B had a mean age of 48.8 (20 - 78 years).The percentage of alcohol consumers among OSCC patients was reported in 3 out of the 10 studies. Alcohol consumers represented 26% of all the patients. While the percentage of smokers was reported in 5 of the 10 studies, 59% of the studied patients were smokers.Histological grading of OSCC was reported only in 2 studies: 10% of OSCC were well differentiated, 20% were moderately differentiated, 10% were between moderately differentiated and poorly differentiated, 20% were poorly differentiated, 20% were undifferentiated or anaplastic. Tumor staging was reported only in two studies: stage I (10%), stage I-II (10%), stage II (10%), stage III (10%), stage III-IV (10%), and stage IV (10%). Only one study analyzed the lymph node involvement, of which 67% was N0 and 33% was N+. OSCC site has been reported in two studies; where 40% of OSCC was on the tongue, 20% of them were on the buccal mucosa, 20% on the gingiva, and 20% on other oral sites.The control group involved 46 males and 23 females with a mean age of 53.5 (37-75 years). In this group, 30.5% were smokers and 9.5% were alcohol consumers.Expression levels of single miRNA (n=27) were detected by quantitative Polymerase Chain Reaction in real-time. According to the individual miRNA expression levels, 21 miRNAs were upregulated and 6 were downregulated .Recently, many efforts have been made to improve OSCC diagnosis, treatment, and prognosis. However, according to GLOBOCAN, OSCC represents one of the leading causes of morbidity and mortality worldwide .This primarily depends on the delay of the patient being screened and on the diagnostic delay . FurtherIn light of the above, the latest studies have been focused on understanding the molecular basis of OSCC, to develop more precise and personalized methodologies for early detection, prognosis, and establishment of successful therapies.Liquid biopsy with circulating biomarkers could represent a new opportunity to understand the molecular profile and dynamic behavior of the tumor . There iThe aim of the present study was to systematically review the current findings on miRNAs in OSCC patients. The search was carried out in four of the most common databases with a focus on studies with statistically differential expression of one miRNA in one of the body fluids in patients suffering from OSCC compared to healthy control. The year of publication of the included studies ranged from 2013 to 2020. Most of the studies were conducted on plasma, serum, and saliva samples, while whole blood samples were less investigated.et al. demonstrated that OSCC patients affected by early-stage (T:1–2 with N0) had significantly higher levels of miR-31 in their preoperative plasma compared to the healthy controls. Furthermore, the majority of the OSCC patients showed a decrease in plasma miR-31 levels after surgical resection (All the studies showed heterogeneous sampling regarding the stage of the tumor. All the studies showed heterogeneous sampling regarding the stage of the tumor. Of note, the subgroup analysis performed by Liu esection . This woet al. (In addition, the potential role of micro-RNAs in predicting lymph nodal metastasis was evaluated in several studies ,19,22,36et al.  carried et al. (Similarly, Yang et al.  studied et al. (Moreover, in the study by Xu et al. , to explet al. (Pedersen et al. , in a stet al. (et al. (et al. (The prognostic significance of microRNAs as a biomarker was also evaluated. Liu et al. , showed  (et al. , showed  (et al. , demonstet al. (Another important issue was addressed which is the possibility to predict the response to treatment. Karimi et al.  reportedet al. (Of the included studies, only Tachibana et al.  focused Our review presents some limitations: firstly, the absence of meta-analysis because of the heterogeneity of the data. Secondly, studies did not have large sample size and long follow-up, which may raise questions on the reliability of the outcomes. Finally, several studies included were conducted on Asian population with only few studies involved patients from a different ethnic population.The field of liquid biopsy has gained more interest over the last two decades. Due to their stability in a variety of body fluids, miRNAs have been shown to be promising non-invasive biomarkers for the diagnosis, prognosis, and therapeutic goals of OSCC. Further studies in a larger cohort of patients are mandatory to introduce liquid biopsy in the routine clinical practice for the OSCC management.


Breast cancer is still the most common cancer in the female population and is the second leading cause of cancer death in women. Although only 6% of breast cancers have metastatic spread at onset, metastases remain the first cause of death. An artificial intelligence approach could be a valuable noninvasive predictor of the risk of distant metastasis. The purpose of this study is to determine the role of a Deep Learning model approach based on a convolutional neural network in predicting the risk of distant metastasis in patients with breast cancer using dynamic Contrast-Enhanced 3T-MRI images.Background: The incidence of breast cancer metastasis has decreased over the years. However, 20–30% of patients with early breast cancer still die from metastases. The purpose of this study is to evaluate the performance of a Deep Learning Convolutional Neural Networks (CNN) model to predict the risk of distant metastasis using 3T-MRI DCE sequences (Dynamic Contrast-Enhanced). Methods: A total of 157 breast cancer patients who underwent staging 3T-MRI examinations from January 2011 to July 2022 were retrospectively examined. Patient data, tumor histological and MRI characteristics, and clinical and imaging follow-up examinations of up to 7 years were collected. Of the 157 MRI examinations, 39/157 patients (40 lesions) had distant metastases, while 118/157 patients (120 lesions) were negative for distant metastases (control group). We analyzed the role of the Deep Learning technique using a single variable size bounding box (SVB) option and employed a Voxel Based (VB) NET CNN model. The CNN performance was evaluated in terms of accuracy, sensitivity, specificity, and area under the ROC curve (AUC). Results: The VB-NET model obtained a sensitivity, specificity, accuracy, and AUC of 52.50%, 80.51%, 73.42%, and 68.56%, respectively. A significant correlation was found between the risk of distant metastasis and tumor size, and the expression of PgR and HER2. Conclusions: We demonstrated a currently insufficient ability of the Deep Learning approach in predicting a distant metastasis status in patients with BC using CNNs. Currently, patients with early invasive breast cancer (BC) are mainly treated by surgical approaches, with or without radiotherapy, possibly preceded by neoadjuvant chemotherapy and followed by adjuvant systemic therapy to reduce the risk of recurrence and distant metastasis. BC presenting with distant metastasis at onset corresponds to about less than 6% of all invasive BC ,2.Few studies exist that investigate the incidences of metastasis in patients with BC and the relative mean time to metastasis onset and the subsequent survival. A large study from the German cancer registry described that the proportion of patients without metastases at diagnosis, but who later developed metastases within 5 years of initial diagnosis, decreased from 27% in 1978–1984 to 15% in 1995–2003. The same study showed a reduction in the proportion of bone metastases, with a relative increase in metastases to other sites, mainly the liver and brain [The risk of occurrence of distant metastases can be influenced by different factors such as tumor stage, grade, and subtype. A large Australian study demonstrated a difference in the 5-year risk of metastasis between women with node-negative disease (5.3%) and those with node-positive or locally advanced disease at diagnosis (18.1%) .To ensure the best treatment and achieve the best results, accurate BC staging is essential. Magnetic Resonance Imaging (MRI) has demonstrated itself to be a highly sensitive and non-invasive technique that provides both morphologic and functional data. Currently, MRI is used in the diagnosis step, preoperative tumor staging, and evaluation of response to chemotherapy treatment . Most ofIn recent years, image-based Machine Learning (ML) processes have been employed in many oncological fields. Radiomics is a relatively new area of artificial intelligence (AI), first developed by Lambin et al. . It invoAI aims to assist physicians in evaluating lesions beyond the subjective visual interpretation that is possible with currently employed methodologies. Although it is still a developing area of research, the effects of radiomics-derived data analysis on lesion diagnosis, prediction of response to chemotherapy, risk of recurrence, disease-free survival, axillary lymph node status, and the influence of tumor edema in histologic characterization of BC have already been studied in the field of BC ,10,11. HBuilding a ML system requires engineering and expertise to design an extractor that transforms the features into an appropriate representation that the learning subsystem can detect or classify. For this reason, there has been an emergence of the Deep Learning (DL) approach, which improves this process by automatically discovering the representations needed for the specific task from the raw data . A key rDeep Learning has already been applied to BC-MRI in the lesion diagnosis, prediction of tumor molecular subtypes, pathological complete response, and axillary lymph node status ,15,16,17This is a retrospective observational study where only existing information collected from human participants was used, with no identifiers linking individuals to the data/samples. All methods and procedures meet institutional and research committee ethical standards in accordance with the 2013 Declaration of Helsinki.All breast 3T-MRI exams performed at the Radiology Department of our hospital, from January 2011 to July 2022 for pre-operative evaluation, were retrospectively reviewed. The inclusion criteria were as follows: (a) pre-operative breast 3T-MRI with Dynamic DCE-MRI sequences; (b) diagnostic confirmation of invasive breast cancer by histopathological analysis; (c) complete histological analysis including molecular receptor status  and proliferation index Ki-67; (d) patients undergoing clinical and imaging follow-up at our institution for at least seven years or until distant metastases diagnosis.The presence of breast implants, post-chemotherapy follow-up patients, exams for neo-adjuvant treatment evaluation, and images that were not of good diagnostic quality were all considered as exclusion criteria.Written informed consent was obtained for all patients before MRI.Following the mentioned criteria, a total of 157 breast cancer patients with 160 breast lesions were included in the study. Patients clinical data  were collected.T2-weighted axial single-shot fast spin echo sequence with a modified Dixon technique (IDEAL) for intravoxel fat-water separation .Diffusion-weighted axial single-shot echo-planar with fat suppression sequence.b-value of 0, 500, and 1000 s/mm2. with diffusion-sensitizing gradient with a ®, Guerbet S.p.A., Villepinte France, or Claricyclic®, GE Healthcare S.r.l, Chicago, IL, USA) at a rate of 2 mL/sec followed by a bolus of 15 mL saline flush , before, and five to ten times after intravenous contrast medium injection.Dynamic 3D-T1w axial and sagittal gradient echo sequence with fat suppression after injection of 0.1 mmol/kg body weight of Gadoteric acid . Patients were positioned prone and a dedicated eight-channel breast coil (8US TORSOPA) was used. Three orthogonal localizer sequences were employed, then images were acquired following this protocol:Subtracted images were automatically produced in post-processing from the images after contrast medium administration for a more accurate tumor analysis. The largest lesion was considered to be the index lesion for statistical analysis purposes. Reading was performed without access to the original reports and clinical data.The following MRI characteristics were collected for each lesion using DCE sequences as reference images for tumor detection and characterization.Location on the breast quadrant;Margins: regular, irregular, lobulated, speculated, non-mass;Size (mm);Morphology: round, oval, or irregular.For each index lesion, a signal-intensity-to-time curve (SI/T) was automatically generated by placing a region of interest (ROI) within the lesion on a subjectively recognized area of maximal contrast enhancement and evaluating all the acquired DCE series. The kinetics curves were classified as I (progressive wash-in), II (plateau) or III (rapid wash-out), as reported in the current ACR BI-RADS guidelines .ADC values were calculated for quantitative analysis by superimposing the subtracted images on the ADC map. An ROI with a diameter of 3–6 mm was manually drawn on the slice where the lesion reached its greatest diameter. ADC measurements were performed only on the enhanced solid portion to avoid areas of T2 shine-through.Distant metastasis status for each patient was recorded using clinical and imaging follow-up, with definitive histological characterization confirming breast cancer cells as a dichotomous result: positive, if there was at least one organ with metastases, or negative, if there were no metastases. Radiological examinations  and medical records of the Patients were collected up to a maximum of seven years after the initial diagnosis of breast cancer; if distant metastases were found, their organ site was recorded.All breast lesions were characterized on the histological specimen obtained by a core biopsy and on the histological definitive sample after surgery by two pathologists. Histological diagnosis was performed according to the WHO classification. The histopathological grade was evaluated according to the Nottingham Grading System, considering tubule formation, pleomorphism, and mitotic count through a scoring system. The total score ranges from 3 to 9: 3–5 corresponds to grade 1 (G1), 6 or 7 to grade 2 (G2), and 8 or 9 to grade 3 (G3).Immunohistochemical (IHC) analysis was performed to evaluate molecular receptor status  and to calculate the Ki-67 index. Evaluation of ER and PgR status was performed by IHC using Dako monoclonal antibody, 1:100 dilution. The monoclonal antibody Mib-1  was used to assess the Ki-67 index, which was reported as the percentage of immune-reactive cells out of 2000 tumor cells in randomly selected high-power fields surrounding the tumor core. HER2 status was re-evaluated using the Hercep test , following published guidelines. Samples that gave an equivocal IHC result were subjected to fluorescence in situ hybridization (FISH) analysis. A ratio of HER2 gene signals to chromosome 17 signals greater than 2.2 was used as a cut-off value to define HER2 gene amplification. ER and PgR status were considered to be positive if the expression was >1% and negative if the expression was <1%. HER2 expression was classified as 0, 1+, 2+ or 3+; only tumors reaching a score of 3+ were considered to be HER2-positive.Each case was anonymized and identified with a progressive identification number (ID).For the analysis of bilateral tumors, lesions were considered one at a time with different IDs. Image preparation was performed on a personal workstation using 3D Slicer : a freely available open-source software. For each tumor, the second post-contrast subtracted T1w sequence was selected. For each case, a label map was generated. Using manual and thresholding-assisted segmentation techniques, the lesions were drawn manually. Segmentation was initially always performed in the axial projections and subsequently reshaped and optimized in the other projections until an optimal lesion contour was obtained. Multifocal or multicentric lesions were also segmented.To limit the amount of non-tumor tissue to be included, the smallest 3D rectangle circumscribed by the tumor region was considered for each patient in Single Variable-size Box (SVB), as shown in Denoting with dxp × dyp × dzp the size of the bounding rectangle of patient p, a 3D cubic box of size dmp × dmp × dmp is used, where dmp = max, to crop the subject. The cubic box of the SVB option depends on the patient. Therefore, the amount of non-tumor tissue depends on the shape of each patient’s tumor region and the difference between the largest (dmp) and maximum  size, and the difference between the largest (dmp) and smallest (dmp) size.However, in the case of multifocal and multicentric tumors, the parenchyma between lesions was included in the 4D volume extracted. Each voxel was associated with its size information in millimeters (mm). During the acquisition of the DCE sequence for patient p, the Pixel Spacing attribute was the physical distance between the centers of each two-dimensional pixel, specified by two numerical values  that represented the row spacing and vertical spacing, respectively. In addition, the attribute spacing between slices specified by the numerical value ˆzp was the distance between slices measured along the normal to the first image. We exploited CNNs for the prediction of extra-nodal metastasis. The voxel-based (VB)-NET model was used, considering that the size of the selection rectangle varies according to the tumor region of each patient.The proposed networks consist of several reduction layers and two fully connected layers. A reduction layer is a block with a convolutional layer, followed by a normalization layer and the ReLU function. Each convolutional layer reduces the dimensionality of the input feature map and doubles the number of channels, making pooling operations (maximum or average pooling) unnecessary.The architecture of VB-NET is shown in Performance was evaluated in terms of accuracy, sensitivity, specificity, and area under the ROC curve (AUC). The experiment was performed in a 10-fold cross-validation (CV) setting. A patient-based cross-validation was performed to reliably estimate performance, avoiding the use of 3D volumes or slices of the same patient during the training and evaluation phase. The experiment was conducted using Pytorch , while the pre-processing phase, including the different bounding box options, were implemented in MATLAB 2020b.p < 0.05. Spearman’s rank-order correlation was evaluated to assess whether there was a correlation between the presence of distant metastases during follow-up and categorical variables, clinical , MRI , and histologic features . The Kolmogorov–Smirnov test was performed to determine whether age, tumor size, Ki-67 index, and ADC values followed a normal distribution.Descriptive statistics were carried out using the statistical software SPSS© version 25.0. Statistical significance was set at Statistical comparisons between the presence of distant metastases during follow-up and age, tumor size, Ki-67 index, and ADC values were performed using the Kruskal–Wallis H test.-Patients with distant metastases at follow-up ;-Patients negative for distant metastasis .In this study, 157 breast cancer patients with 160 lesions were included. Three patients had bilateral breast cancer, but only one patient with bilateral BC presented distant metastases at follow-up. The mean age of the patients was 55 years (range 30–85 years). The patients were divided into two groups:Metastasis sites are represented as follows: 3 (5%) brain, 17 (31%) lung, 19 (34%) bone, 14 (25%) liver, 1 (2%) gluteal subcutis, and 1 (2%) brachial plexus.Anamnestic and clinical data of the study population were collected. The mean age of patients with metastases at follow-up was 54.3 years (range 30–84 years), while the mean age of the control group was 55.2 years (range 30–85 years). The average time to occurrence of distant metastasis was 22.8 months (range: 1–84 months).The mean diameters of the measured lesions in the study population, the group with distant metastases, and the control group were 22.6 mm (range 6–90 mm), 33.3 mm (range 7–90 mm), 19.7 mm (range 6–80 mm), respectively.The main imaging, histological, and clinical characteristics of the patients are shown in p > 0.5). A significant correlation was found between the presence of distant metastasis at follow-up and the expression of PgR and HER2. The Kruskal–Wallis H test demonstrated a significant correlation between the presence of distant metastasis at follow-up and lesion size . No correlation was found between the presence of distant metastasis at follow-up, patient age, ADC values, and Ki-67 index.Using Spearman’s rank test, no correlation was found between the presence of distant metastasis at follow-up and menopausal status, family history, hormone therapy, lesion margins and morphology, kinetic curve, histologic type, histologic class, grading, and ER expression (The VB-NET model achieved a sensitivity, specificity, accuracy, and AUC of 52.50%, 80.51%, 73.42%, and 68.56%, respectively.Conflicting evidence exists regarding the improved survival of metastatic patients from BC. Some studies have found that the development and spread of adjuvant systemic therapies have resulted in a reduction in the spread of BC metastases, but not an increase in survival, due to the shift from lesions developing in sites with higher survival rates, such as the bone, to more aggressive sites such as the brain and liver . HoweverBC can be characterized by clinical cancer dormancy ,25, and AI has the ability to infer data from the images that are not visible to the subjective eye of the radiologist. Our aim was to test in our sample whether using a 3T-MRI-based Deep Learning model we can non-invasively predict the risk of distant metastasis from BC. We also studied whether clinical, MRI, or histologic features could play a similar role in this task. Ours is the first study that attempts to build a predictive model based on CNN Deep Learning for predicting distant metastasis of BC. However, our results demonstrated low AUC, accuracy, and sensitivity values of 68.56%, 80.51%, and 52.50%, respectively. These results reflect a currently insufficient capacity of the Deep Learning approach with CNN in its ability to predict the development of distant metastasis. In our previous studies, we have shown that both a radiomic and a DL approach can predict with greater than 80% accuracy the lymph node status of patients with BC ,27. We aFurthermore, in this study no correlation was found between the risk of distant metastasis and almost all MRI, histologic, and clinical features considered. Tumor size, and therefore tumor stage, have a very good correlation with the risk of metastasis, as already found in the literature ,29; howeWith a DL approach, which is more complex than conventional radiomics, we did not find a meaningful performance of the model for several possible factors. BC is a heterogeneous disease, characterized by considerable pathophysiologic and histologic variability: the different molecular subtypes each have a different rate of aggressiveness and a different mean period free of recurrence or relapse. In addition, each molecular subtype has preferential sites of metastasis development. In a study by Xiao et al., HR+ BCs were correlated with the risk of bone metastasis, HER2+ subtypes were significantly associated with higher rates of liver, brain, and lung metastasis, and triple-negative tumors had a higher rate of brain metastasis but a significantly lower rate of bone metastasis . These dOur study has some limits: this is a retrospective study, with a relatively small number of patients with highly heterogeneous BC types. In addition, we could not explore the possible differences between the different histologic and molecular subtypes, nor the risks of developing metastases for each organ site, due to the small sample size. Segmentation was performed by one radiologist using the same methodology for all lesions, but with a manual assisted approach, which is a method that is time-consuming as well as not error-free. Only the second post-contrast phase was used as the segmentation mask, which, although it is recognized as the phase with higher contrast resolution for BCs, could cause the loss of additional information available from the other sequences. In particular, by considering only the second post-contrast phase, we lost information on the temporal dimension of the post-contrast tumoral enhancement progression, which was instead included in our previous DL study on the prediction of loco-regional lymph node status, which might explain the higher accuracy obtained .Therefore, several steps are needed to improve the current performance of our model: enrollment of more patients to alleviate histological differences in BCs, sharing of different datasets among multiple institutions, development of prospective and multicenter studies, and validation and comparison of additional AI models. Thus, the study population should be as homogeneous as possible, with equal histological and molecular subtypes, and metastases in the same sites. In this regard, in this study we found a significant correlation between HER2, PgR expression, and metastasis risk, so the first successive step should be to select a cohort of patients with the expression of these two specific molecular receptors.Our study demonstrated a currently insufficient ability of the Deep Learning approach in predicting distant metastasis status in patients with BC using CNNs. Our results suggest that further studies are necessary to investigate the role of AI in this specific task.


Danon disease is a rare X-linked dominant genetic disorder that manifests with a clinical triad of cardiomyopathy, skeletal myopathy, and intellectual disability. It is caused by mutations in the lysosome-associated membrane 2 (LAMP2) gene. We report one case of Danon disease and his family members, characterized by ventricular pre-excitation, ventricular hypertrophy, abnormal muscle enzymes, and aberrant liver function. All the patients were confirmed to have Danon disease through genetic screening. Relevant literature was reviewed as a reference for the diagnosis and treatment of the disease. Danon disease DD; MIM#300257) refers to a rare lysosomal storage disease with various manifestations. Male patients display severe cardiomyopathy, skeletal myopathy, and developmental delay, while females show cardiac insufficiency and arrhythmia . Moreove00257 refThe pathology of DD includes vacuoles containing autophagic substances and the accumulation of massive glycogen in cardiomyocytes and skeletal muscle cells . PatientThe present study reports one case of DD and his family members within three generations  and Atrial Fibrillation: Current Understanding and Treatment Recommendations—2018 , 8. and AtriV5, > 4.0 mV of RV5 + SVl, > 1.5 mV of RI, > 1.2 mV of RaVL, and > 2.0 mV of RaVF, which suggested a hypertrophic remodeling of the left ventricle. Secondary ST-T changes were present in all leads and Delta-waves were detected in II and III leads. The narrow QRS tachycardia indicated atrioventricular reentrant tachycardia (The proband (Pt 1) was a male aged 16 years who was admitted to hospital for palpitation. The electrocardiogram (ECG) showed overt ventricular pre-excitation with 74 ms of PR interval, > 2.5 mV of Rhycardia . The serhycardia . A diffuhycardia . Pt 1 rehycardia . The pathycardia . ThroughPt 1’s mother (Pt 2), aged 38 years, was repeatedly admitted to hospital due to palpitation, chest tightness, short of breath or low endurance to activity. Atrioventricular reentrant tachycardia and hypertrophic cardiomyopathy (HCM) was diagnosed. The electrophysiological examination suggested a concealed accessory pathway in the right anterior interventricular septum. Echocardiography and MRI revealed a thick interventricular septum and posterior wall in the left ventricle , 5. FromPt 3 was the elder sister of Pt 2, aged 40 years, and was hospitalized twice for chest tightness. The coronary angiography showed coronary atherosclerosis and coronary myocardial bridge. The ECG indicated atrial premature contractions, ventricular premature contractions, and poor R wave progression. The echocardiography showed enlargement of both atriums, but normal ventricular wall thickness . Heart MPt 4 was the younger sister of Pt 2, without a medical record. The echocardiography found no anomaly. ECG showed sinus rhythm and aberrant Q wave on the I and aVL leads. The genetic test revealed that she and her two sons held the similar mutation of LAMP2 as in Pt 1. During the follow-up, the patient reported a normal life.Pt 5, aged 8 years, was one of the sons of Pt 4. The genetic test indicated the mutation of LAMP2 as in Pt 1. No clinical manifestation was detected except higher serum concentrations of creatine kinase, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, alanine transferase, and aspartate aminotransferase. The ECG and echocardiograph suggested hypertrophic ventricles. The follow-up found no abnormity in the patient.Family member 5Pt 6, aged 2 years, was another son of Pt 4. The genetic test revealed the similar mutation in LAMP2 as for Pt. 1, and the echocardiograph showed a thickened ventricular wall. The patient remained healthy during the follow-up.Limited studies have shown a 4–6% rate of DD in pediatric patients with HCM, 0.7–4% in adults with HCM, 6–8% in adults with symmetric HCM, 17–30% in LV hypertrophy with pre-excitation, and 33% in HCM with vacuolar cardiomyopathy –11. All Atrioventricular bundle passage could be a sign of DD, and 35–50% of patients with DD suffering from heart block are in need of pacemaker implantation . MoreoveThe left ventricular hypertrophy develops into dilated cardiomyopathy (DCM) in 10–33% of DD cases . This coCardiac embolic stroke and ischemic thromboembolic stroke usually arise in DD  indicatiSkeletal muscle lesions are present in 80–100% of male patients with DD, which in general is mild, but turns severe occasionally and is always accompanied with 3-to-35 times higher serum creatine kinase activity . FemalesAt least 110 types of mutations have been reported in the LAMP2 gene, of which c.926G > A is the commonest. Most mutations were non-functional  or splicing. Missense mutation, recombination, or synonymous substitution are rare . In the As for the treatment of DD, the ultimate strategy should be heart transplantation, especially for the female carriers , 20. BefIn the present study, the DD arose earlier in the males. As seen in Pt 6, the onset age was just 2 years. All the males showed typical clinical manifestations such as LVH, and one developed Wolff-Parkinson-White syndrome. Thus, in a youth with HCM and ventricular pre-excitation, DD should be strongly considered. Liver dysfunction and skeletal muscular impairment were also more common in males in the present study; the females showed diversified cardiac manifestations, and two developed heart failure during follow-up. The definitive gender difference implies that DD occurs later in females than in males, at the age of about 30 years. The two female patients in our study displayed atrial arrhythmias at the beginning, which exacerbated the heart failure significantly. The gender-associated difference in clinical manifestations may be due to estrogen, which promotes autophagy and a lysosome pathway in multiple cells .Regarding diagnosis, a family genetic screen and carrier deduction based on a genetic map should be conducted. Moreover, the cardiac MRI of the three patients in the present study identified delayed enhancement in the myocardium and high T1-mapping, which is valuable for the differential diagnosis of patients with HCM.If clinical manifestations are not present, a close follow-up is necessary. With the advent of Wolff-Parkinson-White syndrome or atrial arrhythmias, medication or RFA should be considered. Malignant ventricular arrhythmia demands the installation of an implantable cardioverter-defibrillator. If the condition has developed into heart failure, active medication treatment should be effective. For patients who are insensitive to medication, an active CRT-D implantation is suggested.It has been mentioned that some DD cases develop abnormalities in cerebral vessels , which, Overall, the auxiliary device implantation at an early stage would improve heart function or defer the exacerbation of cardiac performance in patients of DD. However, all these treatments are only palliative or delay the deterioration of the condition. Without heart transplantation or future gene therapy, most patients will die within the ages of 40–50 years. Unfortunately, although we have recommended patient 2 to make an appointment for HTx, they refused to follow the suggestion for economic reasons or the lack of understanding on severity of the disease.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the People’s Hospital of Fuyang. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.BN: conceptualization, supervision, and writing—review and editing. YW: data curation and writing—original draft. MJ: data curation and formal analysis. YG: formal analysis and supervision. TZ: data curation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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