During the recent outbreak of COVID-19 (coronavirus disease 2019), Lombardy was the most affected region in Italy, with 87,000 patients and 15,876 deaths up to May 26, 2020. Since February 22, 2020, well before the Government declared a state of emergency, there was a huge reduction in the number of emergency surgeries performed at hospitals in Lombardy. A general decrease in attendance at emergency departments (EDs) was also observed. The aim of our study is to report the experience of the ED of a third-level hospital in downtown Milan, Lombardy, and provide possible explanations for the observed phenomena.This retrospective, observational study assessed the volume of emergency surgeries and attendance at an ED during the course of the pandemic, i.e. immediately before, during and after a progressive community lockdown in response to the COVID-19 pandemic. These data were compared with data from the same time periods in 2019. The results are presented as means, standard error (SE), and 95% studentized confidence intervals (CI). The Wilcoxon rank signed test at a 0.05 significance level was used to assess differences in per-day ED access distributions.p < 0.002) and in ED admittance  was observed in 2020. In particular, there were significant decreases in medical (40%), surgical (74%), specialist  (92%), and psychiatric (60%) cases. ED admittance due to domestic violence (59%) and individuals who left the ED without being seen (76%) also decreased. Conversely, the number of deaths increased by 196%.Compared to 2019, a significant overall drop in emergency surgeries (60%, During the COVID-19 outbreak the volume of urgent surgeries and patients accessing our ED dropped. Currently, it is not known if mortality of people who did not seek care increased during the pandemic. Further studies are needed to understand if such reductions during the COVID-19 pandemic will result in a rebound of patients left untreated or in unwanted consequences for population health.The online version contains supplementary material available at 10.1186/s12873-021-00445-z. The outbreak of COVID-19 started in Wuhan, China, in December 2019 and quickly spread beyond the borders of the People’s Republic of China. The World Health Organization (WHO) declared COVID-19 a worldwide pandemic on March 11, 2020 when more than 118,000 people were affected by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) throughout the world. In Italy, the first patient who tested positive for SARS-CoV-2 was admitted to the intensive care unit (ICU) of Codogno Hospital, located in a town near Lodi (Lombardy), on February 20, 2020. On February 22, the Government of Lombardy instituted a task force to address the emergency and established containment measures by quarantining several towns near Codogno , where several COVID-19 clusters had emerged. Within fourteen days, ICU admissions exceeded 550 and hospital admissions totalled 2217 [Of note, on March 8 the Lombardy Government established the “Hub and Spoke” model. Accordingly, certain hospitals were designated as hubs for healthcare time-dependent diseases, i.e. polytrauma, and cardiovascular and neurological emergencies . Our hosSince February 22, the Policlinico Hospital emergency department has prepared for COVID-19 patients with logistic measures and new triage rules. All elective surgeries were cancelled, and surgeons were reallocated to ED and COVID-19 wards to provide care for less critical patients. At the same time, a drop in ED attendance for non-COVID-19 diseases, especially patients with surgical complaints, was observed. This reduction inversely followed an increase in COVID-19 patients.The aim of this paper is to quantify the extent of these observed reductions, to assess the characteristics of the potentially surgical patients who did not access to ED, and to analyze how surgical emergencies could have been managed without ED access.° Period 1 , when the first COVID-19 patient was admitted to Codogno Hospital and ten small towns near Milan were quarantined.° Period 2 , start of the lockdown in Lombardy, as well as in 14 provinces in Piemonte, Veneto, Emilia Romagna, and Marche.° Period 3 , when lockdown was expanded to include the entire Italian nation. All non-necessary businesses and industries were shut down.° Period 4 , when ED attendance numbers for COVID-19 patients decreased and it was clear that the national lockdown would have been attenuated, as it was effectively announced on April 26 .A retrospective, observational study was performed to assess all the emergency surgeries performed and the attendance number at the ED of a level III university hospital immediately before, during and after a progressive community lockdown in response to the COVID-19 pandemic. By using an anonymous hospital-based administrative database, automatically generated with data from ED software , the data on ED attendances were collected and analyzed along four time periods:For each of these four periods, attendances at the ED were stratified according to specialties: general medicine, surgery, specialist examinations , psychiatric examinations and attendances for domestic violence. Number of deaths in the ED and numbers of patients who left the ED without being seen by a physician (LWBS) were also collected. All of these data were compared with the same time periods in 2019.Similarly, emergency surgical interventions during the same time periods in 2020 and in 2019 were compared.Data are presented as means, standard errors (SE), and 95% studentized confidence intervals (CI). All the 95% studentized confidence intervals were computed with 2500 bootstrap iterations.The Wilcoxon rank signed test at a 0.05 significance level was used to assess the differences in the per-day access distribution according to the following groups: medicine, surgery, specialist examinations, psychiatry, domestic violence, LWBS, and deaths.2020) with respect to the number of events in 2019 (x2019) were computed as follows: drop% = abs(x2019-x2020)/ x2020.For each category, drop percentages of the number of events in 2020  was used to perform data analysis and to generate plots.Compared to the same periods in 2019, a 48% reduction in overall ED attendances was observed for non-COVID-19 diseases starting February 22, 2020 (Period 1) (3108 vs 1607). Figure (Table 1).This trend continued through second and third periods, with a 61% drop in total admittances  Admittance for surgery also had mean decreases of 56, 85 and 88% through Periods 1, 2, and 3, respectively. Compared with 2019, a 74% reduction was observed over all the four periods in 2020 (2313 vs 609).p < 0.001). Categorizing the surgeries performed according to indications and applying the Wilcoxon sided test, significant differences in the distributions of appendicitis  and incarcerated hernia  were observed .In the fourth period, the drop showed a moderate reduction for medicine (40%) and surgery (63%), but it was still significant Fig. .Fig. 2DAttendances for domestic violence exhibited a mean decrease of 86 and 85% in Periods 2 and 3, respectively, and only a 10% decrease in the fourth period compared to 2019.Emergency psychiatric examinations decreased by 50 and 74% in the second and third period, and the trend went on in the fourth period (65%).In 2020, deaths in ED increased by 50, 280 and 238% through Periods 1–3, and slightly reduced to 167% in Period 4, as compared to 2019.The percentage of patients who left the ED without being seen by a physician, or during the diagnostic process (LWBS), with respect to all the ED attendances, showed a significant drop in Period 2 (2.8% vs 9.6%), and in Period 3 (5.1% vs 7.8%), while in the first period (7.2% vs 8%), and in the fourth period (6.2% vs 7.8%) the drops, though statistically significant, were less evident  has been suggested in recent literature for uncomplicated appendicitis, selected cholecystitis patients and colonic diverticulitis –21. In tBowel and gastro-duodenal perforations halved in 2020, with 4 interventions in 2020 vs 8 interventions in 2019. Surgery for incarcerated umbilical, inguinal and incisional hernia dropped to only one case in 2020, compared to 10 cases in 2019. We suppose that home-confinement, accompanied by reductions in physical activity and hard work, may have contributed to the reduced surgeries for incarcerated hernia. However, in Spain, an increase in surgery for incarcerated hernia was reported , and we For gastro-intestinal perforations, we cannot identify a convincing and plausible explanation. Covered duodenal perforations, which can be treated conservatively with nasogastric suction and antibiotics, usually represent a minority of observed cases, and it seems inconsistent that such patients could stay at home and be cured only by fasting. The same for colon perforation, often caused by a complicated cancer. Obstruction, another frequent complication of colon cancer, was an uncommon finding during COVID-19 outbreak. In one case endoscopic placement of an endoprosthesis allowed surgery to be postponed for 2 weeks.nilper os and intravenous fluids, was the treatment of choice for no more than 72 h, as suggested by literature [The number of surgeries for small bowel obstruction did not significantly differ between 2019 and 2020. Conservative treatment, with naso-gastric suction, terature , and it Severe COVID-19 has been associated with a marked inflammatory and prothrombotic state . HoweverOverall, the results of this study confirm a decrease in ED attendances for all medical and surgical complaints during the COVID-19 pandemic. The decrease in surgical patients was impressive and this led to a significant reduction in emergency surgeries. It is possible that some diseases were managed conservatively. However, if our data are confirmed in other settings, it is important to determine what consequences were incurred for the patients who did not seek medical attention, as well as the possible future rebound on the health care system.We must acknowledge some limitations of the present work. The study did not analytically analyzed the single diagnoses of ED access to assess which diseases were missing with respect to the previous year.Moreover, no updated data regarding surgeries performed at other hospitals in the metropolitan area of Milan are available; however, informal communications between general and emergency surgeons confirm that a drop in emergency surgeries was observed.Finally, patients may have died at home due to complications of untreated surgical urgencies. However, it is currently difficult to assess possible changes in mortality rates for non-COVID-19 diseases from national and regional death registries.During the COVID-19 outbreak, indications for emergency surgery did not change with respect to previous periods. However, the volume of patients accessing to ED, and of surgeries for urgent and emergent diseases, did change. Actually, it is not known if the mortality rate of people who did not seek care for fear of contracting COVID-19 has risen during the pandemic. It is also unclear if, in the near future, the impressive drop in the volume of ED attendances in the COVID period will result in a rebound of more severe diseases left untreated in their early onset. Further studies, at both regional and national levels are needed to understand if drops in ED attendances and emergency surgeries during the COVID-19 pandemic will result in an increased mortality rate and unwanted complications in the population.Additional file 1. Time-series plots (with dashed trend lines) of daily ED accesses for specialities, and deaths during the same four periods in 2020 (red) and 2019 (blue).


The recovery of scandium (Sc) from wastes and various resources using solvent extraction (SX) was discussed in detail. Moreover, the metallurgical extractive procedures for Sc recovery were presented. Acidic and neutral organophosphorus (OPCs) extractants are the most extensively used in industrial activities, considering that they provide the highest extraction efficiency of any of the valuable components. Due to the chemical and physical similarities of the rare earth metals, the separation and purification processes of Sc are difficult tasks. Sc has also been extracted from acidic solutions using carboxylic acids, amines, and acidic β-diketone, among other solvents and chemicals. For improving the extraction efficiencies, the development of mixed extractants or synergistic systems for the SX of Sc has been carried out in recent years. Different operational parameters play an important role in the extraction process, such as the type of the aqueous phase and its acidity, the aqueous (A) to organic (O) and solid (S) to liquid (L) phase ratios, as well as the type of the diluents. Sc recovery is now implemented in industrial production using a combination of hydrometallurgical and pyrometallurgical techniques, such as ore pre-treatment, leaching, SX, precipitation, and calcination. The hydrometallurgical methods (acid leaching and SX) were effective for Sc recovery. Furthermore, the OPCs bis(2-ethylhexyl) phosphoric acid (D2EHPA/P204) and tributyl phosphate (TBP) showed interesting potential taking into consideration some co-extracted metals such as Fe(III) and Ti(IV). After thermal treatment, an oxide product was produced with 65.5% Sc, 0.6% Mg, and 0.4% Fe. Scandium can also be recovered from scraps of aluminum and iron alloys. The considerable amount of scandium present in the scraps formed during the Sc-alloy manufacturing cannot be considered as a resource worthy for valorization. Furthermore, there is currently no reliable information about this industrial processes suitable for this purpose.To remove the co-extracted Fe from the organic phase, HCl solutions with a low concentration can be utilized. To obtain the Sc(OH)2O3 leaching rate of 82%. Wang et al. 3− and Sc(OH)3 using 2 mol/L NaOH at an O/A ratio of 1:1.Scandium was then recovered from the strip liquid and purified as mixture of 3−, which was then diluted with water to precipitate scandium as Sc(OH)3. To achieve pure Sc2O3, additional purification processes such as the dissolution of Sc(OH)3 with hydrochloric or sulfuric acid, precipitation as scandium oxalate, and calcination could be used.Wang et al.  investig4. In this context, different parameters such as temperature, time, pH, and MgSO4 concentration were tested to see how they affected the scandium extraction efficiency. They observed that Sc can be extracted in excess of 63% at pH 4, 80 °C, after 1 h, whereas a higher content could be extracted at pH 2. Furthermore, increasing the pH from 2 to 4 reduced the iron extraction from 7.7% to 0.03%.Shoppert et al.  investig4Sc(SO4)2, which effectively separates scandium from impurities such as Al, Fe, Zr, Ti, and Ca as shown in Pasechnik et al.  developeRecently, Shoppert et al.  studied In order to achieve an environmentally friendly separation, Jumadilov et al.  investigThis enables the development of novel sorption technologies and processes. An interesting study by Rout et al.  looked iScandium availability is limited because of its natural deficiency and high production costs in Asia and Russia, while it is not produced in Europe. As a result, the extraction of scandium from other resources, such as secondary resources in Europe, is a significant cause of concern. Among the most crucial scandium recovery challenges, the co-dissolution and co-extraction of metal impurities such as iron, titanium, zirconium, uranium, thorium and rare earth, which interfere with the scandium extraction ,45, are This work explored the most recent applied protocols for scandium recovery by solvent extraction technique using different extractants from various sources. We reviewed the most important previous published papers related to the recovery of scandium from various sources (ores and waste) for the period from 1969 to 2022. We hope that this review could be useful for all which are interested in scandium recovery at industrial scale from all existing scandium sources, providing a solid information background for such applications.To separate and purify scandium from a variety of solutions, solvent extraction is the most extensively applied technique. In addition to being found in ores of other metals, scandium can also be recovered from the wastes and tailings of other metals. When the comparatively high scandium content and availability are taken into consideration, red mud can be considered a valuable and potential scandium resource rather than as a solid waste. Scandium recovery from red mud should be considered, because of its rarity and increasing market demand, as a primary goal rather than a secondary by-product in the design process of the entire flow-chart. One of the most popular approaches for scandium recovery from red mud applies hydrometallurgical processes, such as acid digestion, SX, and IX. Industrial-scale operations for the extraction of scandium from bauxite wastes and nickel laterites are currently being considered. Due to their high energy consumption, pyrometallurgical techniques are not appropriate for scandium recovery from wastes and sludge. The most extensively used processes for the scandium recovery are hydrometallurgical. Contaminants in high concentrations could be removed, regardless of the cost increase, using strong mineral acids during the digestion process.Several unwanted metals, such as Fe, Al, Zr and REEs, could interfere with the scandium extraction if they were simultaneously dissolved together or extracted at the same time. Some of OPCs extractants have several disadvantages, the most notable of which are the difficulty in recovering of Sc and the insufficiency of selectivity.In order to improve the extraction selectivity and efficiency, new techniques or optimizations to current processes must be developed in the area of digestion methods and SX. Red mud and nickel laterite ores are identified as the most interesting and promising scandium resources for future valorization.235) or diethyl ether, from the red mud leachate (8 mol/L HCl). The solvent extraction by diethyl ether was preferred due to its high efficiency and better economy. In order to remove the Ti(III) content from the leachate, the scrubbing process by 3 mol/L of HCl or H2SO4 was proposed, depending on the leachate.Based on our findings during scandium recovery from red mud, the hydrometallurgical methods (acid leaching and solvent extraction) were effective. Moreover, the organo-phosphorus compounds (TBP and D2EHPA) showed interesting potential taking into consideration some co-extracted metals such as Fe(III) and Ti(III). In the case of iron, it was found that the removal of iron content before scandium recovery is the optimum choice, by using solvent extraction with trinoctylamine (N


To observe the changes in retinal and choroidal microstructures in patients with different stages of diabetic retinopathy (DR) and to evaluate the vascular perfusion of retina and choroid retinal thickness, retinal and choroidal vessel density by the swept-source optical coherence tomography angiography (SS-OCTA).Subjects were divided into three groups: healthy control group , non-proliferative diabetic retinopathy  and proliferative diabetic retinopathy . The area of the foveal avascular zone (FAZ), retinal and choroidal vascular perfusion, and the deep vascular complexes, including the intermediate capillary plexus (ICP) and deep capillary plexus (DCP) within the radius of 3, 6, 9, and 12 mm around the fovea were measured by SS-OCTA.Compared with the healthy control group, DR patients presented significantly increased fovea avascular area, while vessel density (VD) in the ICP and DCP, vascular perfusion rate, and the retinal thickness were considerably decreased. There were significant differences in the retinal thickness, ICP, and DCP vessel densities between the control and NPDR groups, or control and PDR groups, or PDR and NPDR groups. The deep vascular perfusion rate also significantly differed between the control and PDR groups, but there was no significant difference between the PDR and NPDR groups. The choroidal perfusion exhibited significant differences across different areas and amongst the three groups. Furthermore, the choroidal thickness showed a significant difference between the PDR and NPDR groups.Our results showed significant differences in the area of the avascular fovea and the VD of deep vascular complexes between DR patients and healthy control subjects. Moreover, there were significant differences in retinal VD, especially in the deep-retinalregions, choroidal perfusion, and the volume of large vessel choroid in DR patients with different degrees of disease severity. Notably, SS-OCTA provided in-depth information for detecting the potential VD damage in DR patients caused by a multitudeof factors. Diabetic retinopathy (DR) is one of the most detrimentallong-term diabetes complications that damage fundus retinal microvasculature complications in DM patients . AlthougThe ultra-wide-angle imaging of the swept-source optical coherence tomography angiography (SS-OCTA) has multiple advantages like faster scanning speed, higher signal intensity, and deeper penetration depth. This technique is also used to detect early phases of retinal microvascular damages in DM patients, It did not lead to obvious visual impairment –11. SincSS-OCTA can also present clearer choroidal images, automatically detect and analyze choroidal thickness, reduce manual measurement errors, and improve the efficiency of choroidal observation and diagnosis. With increasing scan depth, the possibility of signal attenuation will be further reduced, which is suitable for imaging larger fields and distinct observation of the vitreous body, retina, choroid, and sclera for detailed study of the fundus in DM patients . Such unIn this study, the SS-OCTA technique was applied to observe and quantitatively analyze the retinal and choroidal microvascular parameters like VD, capillary network, and morphological alterations and compare the foveal avascular zone (FAZ) in different macular regions between healthy subjects and patients with NPDR or PDR. Therefore, this study would provide critical diagnostic guidelines for DM patients with different degrees of retinal damage.This study recruited 73 patients who were Type 2 diabetes mellitus . Among them 31 PDR patients including 24 males (40 eyes) and 7 females (13 eyes), 42 NPDR patients including 29 males (51 eyes) and 13 females (20 eyes). And 30 healthy control subjects without a history of diabetes, including 12 males (17 eyes) and 18 females (34 eyes). All subjects involved in our study have been recruited from the Department of Fundus Medicine of Shandong Eye Hospital in 2020–2021.The inclusion criteria were as follows: (1) the symptoms of the patients met the diagnostic criteria of DR with T2DM ; (2) excAll the subjects were examined by SS-OCTA  , with thFurthermore, SS-OCTA also may not show leakage. However, these artifacts do not affect the analysis of non-perfused regions. At the same time, these artifacts are also subtle. We try to avoid these problems in methods and eliminate artifacts as much as possible. We manual segmentation when machine had segmentation errors.t-test to analyze the data. The detection level threshold was set at α = 0.05 and calculated as mean ± Standard Deviation (mean ± SD). Results with p < 0.05 were considered a statistically significant difference.The data obtained were statistically analyzed by SPSS 22.0 software. Our data included the basic demographic information of the subjects, the lesion area, perimeter, acircularity index (AI) and VD of FAZ. We also evaluated the changes of retinal and choroidal VD, vascular perfusion, and retinal thickness of the deep vascular complex in the circular radii of 3, 6, 9, and 12 mm around the fovea. We used an independent sample x2 = 0.628, p = 0.596). Concurrently, for age, there were no significant differences between the healthy control group  and PDR  or NPDR  group, and there was also no significant difference between PDR and NPDR (p = 0.139) group. The BCVA of healthy controls was 0.01 ± 0.03 LogMAR. The BCVA of NPDR patients was 0.3 ± 0.24 LogMAR, and PDR patients was 0.6 ± 0.50 LogMAR, our results suggested that the vision of PDR was worse than that of NPDR group (p < 0.05). The average intraocular pressure (IOP) of patients was also decreased to 16 ± 1.3 mmHg , there was no significant difference in IOP between NPDR and PDR groups.In this study, we investigated 30 cases (51 eyes) healthy control subjects and 73 cases DR patients (124 eyes), which included two groups, namely NPDR (71 eyes) and PDR group (53 eyes). And 73 DR patients also suffered from T2DM. There were no significant differences in gender between patients with NPDR and PDR . The perimeter, AI, and VD of NPDR subjects were also increased significantly, compared to that of the healthy control group . However, these indices were not significantly different between the NPDR and PDR groups. Hence, these results suggest that the rate of changing of FAZ may be related to the advancement of DR pathology, as observed as differential FAZ pathobiology between NPDR and PDR patients, compared to the healthy control subjects.We compared the area, perimeter, AI, and VD of the fovea FAZ between DR patients and healthy controls, as shown in Next, we compared the VD, perfusion density (PD), and retinal thickness (RT) of deep vascular complexes in the circular radius at the center of the fovea of DR patients and healthy controls, as shown in 2 and 9–6 mm2 captures of the PDR were l± ± over than that of control subjects. Moreover, the mean values of VD in 3 mm (3 × 3 mm), 6 mm (6 × 6 mm), 9 mm (9 × 9 mm), 12 mm (12 × 12 mm), and 9–6 mm2 captures of the PDR lesions were also lower than that of the NPDR group. There were significant differences in mean values of PD between NPDR or PDR and control subjects in all captures, with the lower mean PD in DR patients compared to control subjects , 6 mm (6 × 6 mm), 9 mm (9 × 9 mm), and 12 mm (12 × 12 mm) captures between the healthy control and NPDR group or PDR group . And thesubjects . But theThese data indicated that VD, PD, and RT in NPDR and PDR patients have different degrees of damage, especially in VD and RT. Notably, RT and VD had reciprocal correlations between the PDR and NPDR groups, suggesting that PDR might be characterized by hyperplasia. Moreover, the mean retinal PD was significantly lower in DR patients (in both NPDR and PDR patients) compared to control subjects.(CVI), choroidal vascularity volume (CVV), and choroidal thickness (CT) in the circular radius at the center of the fovea of DR patients and healthy controls. The results showed that the CT was significantly increased in NPDR and PDR patients than in control subjects (2) captures of CT between the PDR and NPDR groups.subjects . There wAdditionally, the mean CP values were significantly decreased in both NPDR and PDR groups, compared to that of control subjects. Importantly, the rate of CP was lower in the PDR group than that in the NPDR group in terms of captures in 3 mm (3 ×3 mm), 6 mm (6 ×6 mm), 9 mm (9 × 9 mm). Thus, the rate of CP was significantly decreased in the diabetic eyes of PDR and NPDR patients, and the intensity of reduction in the rate of CP was directly linked to the progression of DR pathology from NPDR to PDR stage.2 compared to NPDR group. However, there was only difference in the 3 mm (3 × 3 mm) captures of CVV between the control subjects and PDR patients .Subsequently, the PDR group exhibited a significantly different mean value of CVI at 9–6 mmPrevious study has shown that patients with T2DM are more likely to have enlarged FAZ compared to their age-matched non-diabetic control subjects . In diabCompared with OCTA, the SS-OCTA provides superior angiogram montages covering larger areas with distinct retinal capillaries to the mid-periphery , 26. It Furthermore, we observed that both NPDR and PDR patients exhibited considerable ischemic alterations in the macular regions, in addition to the decreased retinal VD and PD around the central fovea. This observation indicated that the change of retinal ischemic area in DR patients might not be limited on the macular area. This is similar to previous studies . AdditioNotably, the difference in the ischemic damage of macular area between the DR patients and control subjects was found to be directly associated with the area measured in the annular region with the fovea at the center. While, based on these findings, it was obvious that there would be a significant difference in macular lesion area between NPDR and PDR patients. On the other hand, a comparatively larger lesion area might be helpful in providing a more accurate diagnosis in the case of PDR than in NPDR. Subsequently, we identified differential damage intensities in peripheral retinal perfusion rates between the PDR and NPDR patients, as well as with the healthy control group, which might provide a new paradigm shift in our understanding of the mechanism of the progression of DR. Together, these findings demonstrated the potential benefits of SS-OCTA evaluation for diagnostic stratification between different stages of retinal injury.Although SS-OCTA devices provide the possibility of wide-field imaging, there are still some limitations. For example the artifacts caused by the eye movement or blinking, the segmentation errors, and so on. And the leakage could not be detected by SS-OCTA. Additionally, a controlled prospective study with larger sample size was needed to further study.In summary, identification of the differences in retinal VD and RT between NPDR and PDR patients could indicate possible mechanistic of DR progression. SS-OCTA examination has promising benefits in the early diagnosis of DR pathology as well as in the differentiation between various stages of disease progression.The original contributionspresented in the study are included in the article/The Human Research was approved by the Institutional Ethics Committee of Shandongeye hospital  on November 11, 2019. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.TiL, QZ, and WLdesigned and conducted clinical examinations, wrote, and revised the manuscript. GS, WW, MF, XX, and ToL prepared the figures and tables and interpreted the results. All authors contributed to the revision steps and approved the final version of the manuscript for submission.This studywas supported by a grant from the Natural Key Research and Development Project (2016YFC1305500), Bethune Langmu Young Scholars Research Fund (BJ-LM2021007J).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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasma cell leukemia (PCL) is a very aggressive plasma cell disorder with a dismal prognosis, despite the therapeutic progress made in the last few years. The implementation of genomic high-throughput technologies in the clinical setting has revealed new insights into the genomic landscape of PCL, some of which may have an impact on the development of novel therapeutic approaches. The purpose of this review is to provide a comprehensive overview and update of the genomic studies carried out in PCL.Plasma cell leukemia (PCL) is a rare and highly aggressive plasma cell dyscrasia characterized by the presence of clonal circulating plasma cells in peripheral blood. PCL accounts for approximately 2–4% of all multiple myeloma (MM) cases. PCL can be classified in primary PCL (pPCL) when it appears de novo and in secondary PCL (sPCL) when it arises from a pre-existing relapsed/refractory MM. Despite the improvement in treatment modalities, the prognosis remains very poor. There is growing evidence that pPCL is a different clinicopathological entity as compared to MM, although the mechanisms underlying its pathogenesis are not fully elucidated. The development of new high-throughput technologies, such as microarrays and new generation sequencing (NGS), has contributed to a better understanding of the peculiar biological and clinical features of this disease. Relevant information is now available on cytogenetic alterations, genetic variants, transcriptome, methylation patterns, and non-coding RNA profiles. Additionally, attempts have been made to integrate genomic alterations with gene expression data. However, given the low frequency of PCL, most of the genetic information comes from retrospective studies with a small number of patients, sometimes leading to inconsistent results. Plasma cell leukemia (PCL) is an uncommon plasma cell dyscrasia with an aggressive course and poor prognosis. PCL represents less than 3% of all plasma cells neoplasms, and its incidence has been estimated at 0.04 cases per 100,000 persons/year .9/L of PCs in peripheral blood [Historically, PCL has been defined by the presence of more than 20% of circulating plasma cells (PCs) and an absolute number of ≥2 × 10al blood . Howeveral blood .PCL is classified as primary PCL (pPCL) when it occurs de novo so that the patient has no evidence of previous multiple myeloma (MM), and as secondary PCL (sPCL) when leukemic progression occurs in the context of pre-existing refractory or relapsing MM ,6. pPCL The clinical presentation of PCL is more aggressive than that observed in MM, including more severe cytopenias, hypercalcemia, and renal insufficiency. Higher tumor burden and proliferation activity of PCL are manifested by greater levels of B2-microglobulin and lactate dehydrogenase (LDH). Extramedullary involvement  at diagnosis is more common in pPCL and sPCL than in MM, but osteolytic lesions are more frequent in sPCL and MM than in pPCL ,11,12,13Various studies have analyzed the immunophenotype of PCL. The two common PCs markers, CD38 and CD138 antigens, are similarly expressed in MM and PCL. However, PCL displays a more immature phenotype than MM, expressing more frequently CD20, CD23, CD28, CD44, and CD45, and less frequently CD9, CD56, CD71, CD117, and HLA-DR antigens ,15,16.BCL2 inhibitors and the new immunotherapeutic approaches, such as chimeric antigen receptor T-cells (CAR-T cells) and monoclonal and bispecific antibodies, opens up new opportunities in the treatment of PCL patients.PCL patients are characterized by short remissions and early relapses. The 5-year survival rate from the diagnosis of PCL does not exceed 10%. Survival of sPCL patients is consistently shorter than pPCL . The incEarly cytogenetic studies performed in PCL had already revealed some differences between this entity and MM. Later on, the widespread use of fluorescence in situ hybridization (FISH) has increased our knowledge of genetic alterations. In recent years, the development of high-throughput genomic analysis tools has helped to better understand the genetic particularities of PCL. However, the robustness of the results is undermined by the limited number of patients included in the studies because of the low incidence of this disease. In this review, we mainly focus on the genomic characteristics of pPCL, although some data concerning sPCL are provided when considered of interest. A summary of the most relevant results provided by the main genomic studies carried out in PCL is shown in Early cytogenetic and DNA content studies carried out in PCL revealed that there was a predominance of non-hyperdiploid cases (more than 50% of pPCL) compared to that observed in MM ,14,18. TMonosomy and deletions of chromosome 13 (del(13q)) have been observed in approximately 85% of pPCL ,35. AbnoTaken together, all these results showing the increasing frequency of the aforementioned chromosome imbalances from MM to PCL support the multistep transformation model from monoclonal gammopathy of undetermined significance (MGUS) through smoldering multiple myeloma (SMM) and MM to PCL that leads to progressive accumulation of secondary genetic alterations.IGH translocations is significantly higher in pPCL than in MM. Several studies show that t leading to CCND1 dysregulation are significantly more frequent in pPCL than in MM, reaching percentages as high as 45–70% in some series [The incidence of e series ,27,40,41e series ,23,36,41e series .The t has largely been demonstrated to be a neutral prognostic factor for MM survival . AlthougMYC rearrangements have also been found in PCL, although the reported incidence varies between 13% and 40% [MYC rearrangement and shorter overall survival of pPCL patients has been shown [ and 40% ,40,48. MOther chromosomal abnormalities have been identified in pPCL, especially the loss of chromosome 16 (80%) ,23,49, 7RAS oncogenes (NRAS and KRAS), the two most prevalent mutated genes in MM, and of the tumor suppressor TP53, had been explored in pPCL using traditional DNA sequencing methodologies. Two studies demonstrated a high incidence of NRAS and KRAS activating mutations: one of them reported these mutations at codons 12, 13, or 61 in 27% of pPCL and 15% of sPCL cases [NRAS and/or KRAS mutations were found in 50% of pPCL cases and in 55% of MM [TP53 is one of the most frequently mutated genes in pPCL in all the published series, reaching frequencies of 25% [TP53 is also greater in pPCL than in MM (17–35% vs. 3–4%) [TP53 coding mutations involving 5–8 exons were found, predicting all of them a non-functional p53 protein [Before the availability of next-generation sequencing (NGS) technologies, the mutational status of CL cases , and in 5% of MM . Strikins of 25% ,23,27. Ts. 3–4%) ,27. TP53CIDEC), RNA binding and degradation , and cell-matrix adhesion and membrane organization  were considered as potential cancer driver genes in pPCL. Other studies have confirmed that the number of nonsynonymous mutations per sample is higher in pPCL than in MM [The first whole-exome sequencing (WES) analysis of pPCL revealed a highly heterogeneous mutational profile . Almost an in MM .N/KRAS mutations have been identified in pPCL using WES methodologies, although the proportions were significantly unequal between the two of the studies. The first study reported mutations of KRAS and NRAS only in two distinct samples (<10% of the pPCL). This study highlighted that KRAS and NRAS were three-fold less frequently mutated in pPCL compared to that observed in MM [KRAS was the most frequently mutated gene in pPCL samples (around 39%), and mutations of NRAS were present in 13% of pPCL [KRAS mutations were detected in 17% of pPCL, 18% of sPCL, and 33% of MM, and NRAS mutations in 4% of pPCL, 36% of sPCL, and 27% of MM [MEK/ERK signaling pathway was less affected by mutation events in pPCL than in sPCL and MM [As in MM, activating ed in MM . On the  of pPCL . Using t7% of MM . ApparenL and MM .BRAF gene have also been detected in pPCL samples. A low frequency and even absence of BRAF mutations in pPCL patients have been described using WES [BRAF mutations detected in pPCL was higher (21% in pPCL and 9% in sPCL). It is worth highlighting the role that the different coverage levels among NGS studies and the small number of patients analyzed may be playing in the conflicting results.Mutations of the sing WES ,36. HoweTP53 gene has also been analyzed by NGS in pPCL [TP53 mutations in pPCL. Interestingly, the presence of TP53 mutations has been associated with significantly shorter survival in the study, including the largest number of patients with pPCL to date [IRF4 mutations have recently been shown to be significantly more frequent in pPCL than in MM patients (11% vs. 4%) [DIS3 and PRMD1 mutations in 5% and 13% of patients with pPCL, respectively, while Cifola et al. identified DIS3 mutations in 25% of cases and no variants in the PRMD1 gene. Both studies have described a similar incidence of FAM46C mutation (10–12%) in pPCL patients [ in pPCL ,36,41, cpatients ,36.IL6 pathways in one study [NF-kB pathway, FAS signaling, structural organization of the cell and migration processes in the other study [Several studies have explored the gene expression profile (GEP) analyzed by microarrays in pPCL. All of them have identified a transcriptome signature characteristic of pPCL and different from that of MM. The first two reports identified a gene-specific signature that distinguished pPCL from MM cases, although the number of overlapped genes between both datasets containing the differentially expressed genes was only around 15%. The functional annotation analysis identified dysregulation of lipid metabolism, glucocorticoid receptor, and ne study , and alter study . A transer study .PHF19 and TAGLN2, and underexpression of the adhesion molecules VCAM1 and CD163, which are highly expressed in MM and have been correlated with poor survival [More recently, the GEP of 41 pPCL patients has been compared to that of more than 700 newly diagnosed MM . In pPCLsurvival ,52.MYC target genes and lower expression of genes involved in p53 pathway, hypoxia, and TNF alpha signaling via NF-κB [CDKN2A, CCND3, and CCND1 genes, using quantitative RT-PCR, has been reported in PCL compared to MM samples, indicating a marked cell cycle dysregulation in the transition from MM to PCL [RNA-seq analysis of pPCL has also shown a specific transcriptional landscape of pPCL, as previously demonstrated by GEP using microarrays. Compared to MM, pPCL showed significantly higher expression of genes involved in G2M checkpoint and ia NF-κB . In thisA comprehensive molecular analysis of pPCL integrating data from FISH, SNP-arrays, and GEP has revealed a strong correlation between chromosomal imbalances and transcriptional modulation. The gene dosage effect was particularly observed in those genes mapping 1q chromosome . In addiDKK1, KIT, NCAM1, and FRZB  and a similar cytogenetic background . This apand FRZB . InteresIL2-STAT5 signaling but negatively associated with the regulation of cell and cell adhesion pathways. In any case, the most relevant finding of this study was that pPCL showed a different expression pattern of the BCL2 family genes and of the B-cell-associated genes, despite the presence of t in both PCL and MM samples. These results suggest that the efficacy of venetoclax in pPCL and MM patients with t may be associated with different molecular programs.A similar approach has been used to ascertain the differences in the transcriptome between pPCL and MM samples harboring t . In lineNon-coding RNAs (ncRNAs) are classified as short (<200 nucleotides) and long (>200 nucleotides). The miRNAs are short ncRNAs of 19–22 nucleotides that regulate gene expression at the post-transcriptional level. Since their discovery, numerous studies have attributed a wide variety of functions for ncRNAs in the pathogenic mechanisms of MM ,55,56.IGH translocation. Three miRNAs  were overexpressed in PCL patients with t; three  underexpressed in PCL with t; and the miR-342-3p was underexpressed in PCL with t. Notably, four miRNAs, miR-22, miR-146a, miR-92a, and miR-330-3p, were found to have an impact on the survival of pPCL patients. The overexpression of miR-146a, which was associated with shorter progression-free survival (PFS) in pPCL cases, and miR-22, which was associated with longer PFS, showed a pro- and anti-survival effect, respectively, in myeloma cell lines [There is only one study analyzing the expression pattern of miRNAs in pPCL . The anall lines . Accordill lines   are a group of very heterogeneous non-coding RNAs with a length of more than 200 nucleotides. They have a similar structure to mRNAs but are not translated to functional proteins. LncRNAs represent more than 50% of the non-coding RNAs, and their functions are related to the regulation of transcription, genome integrity, cell differentiation, X-chromosome inactivation, and development, among others .LY86-AS1) and VIM antisense RNA 1 (VIM-AS1) was observed in PCL compared to MM and further validated by qRT-PCR. However, their functions in MM to PCL progression remain unknown.LncRNAs expression profile has also been investigated in a large cohort of PC dyscrasias, including samples from MGUS, SMM, MM, and PCL together with NPC . DiffereSNHG6, whose overexpression was associated with significantly inferior overall survival in MM patients from the CoMMpass dataset, was found to be upregulated in the pPCL patients.Differential expression of lncRNAs has also been detected between pPCL and MM samples with t . In partDIS3 gene mutations, t, and t. On the contrary, Walker et al. [The analysis of global methylation patterns in pPCL using high-density arrays has identified a global hypomethylation profile in pPCL samples  Figure . The comr et al.  had prevChromosomal, genetic, and genomic alterations found in pPCL are sufficiently different from those observed in MM to consider it a distinct clinicopathological entity and not merely a more aggressive form of MM. However, the low incidence of this disease makes it extremely difficult to gather enough pPCL cases to carry out genomic studies that provide consistent results. On the other hand, the paucity of clinical trials specifically designed for this disease precludes prospective studies. In this regard, proposals aimed at collecting hundreds of pPCL samples involving numerous centers in order to conduct biological studies could represent a breakthrough in identifying dysregulations of signaling pathways that could be therapeutically targeted.


V600EBRAF mutated ATC. A new generation of TKIs, specifically for single altered oncogenes, is under evaluation in phase 2 and 3 clinical trials. The aim of this review was to provide an overview of the current and future treatments of thyroid cancer with regards to the advanced and progressive cases that require systemic therapies that are becoming more and more targeted on the molecular identity of the tumor.Differentiated thyroid cancers (DTC) are commonly and successfully treated with total thyroidectomy plus/minus radioiodine therapy (RAI). Medullary thyroid cancer (MTC) is only treated with surgery but only intrathyroidal tumors are cured. The worst prognosis is for anaplastic (ATC) and poorly differentiated thyroid cancer (PDTC). Whenever a local or metastatic advanced disease is present, other treatments are required, varying from local to systemic therapies. In the last decade, the efficacy of the targeted therapies and, in particular, tyrosine kinase inhibitors (TKIs) has been demonstrated. They can prolong the disease progression-free survival and represent the most important therapeutic option for the treatment of advanced and progressive thyroid cancer. Currently, lenvatinib and sorafenib are the approved drugs for the treatment of RAI-refractory DTC and PDTC while advanced MTC can be treated with either cabozantinib or vandetanib. Dabrafenib plus trametinib is the only approved treatment by FDA for  Thyroid cancer (TC) is the most common endocrine neoplasia and represents approximately 2.9% of all new cancer cases in the United States each year . In the 131I (RAI) and decrease of OS rate (less than 10% at 10 years) [Differentiated thyroid cancer (DTC) is the most frequent tumor type, representing > 90% of all TC . It orig0 years) . As well0 years) , an excePoorly differentiated thyroid cancer (PDTC) is a heterogeneous group that includes those TCs that, according to the Turin classification, lose the papillary nuclear features and have a solid, insular, or trabecular growth pattern with an increased number of mitoses and necrosis ,8. PDTC 131I therapy and thyroid stimulating hormone (TSH) suppressive therapy [According to the most recent guidelines , the gol therapy .2 every 3 weeks) remains the single most effective and approved cytotoxic chemotherapy for the treatment of RAI-refractory DTC patients with metastatic, rapidly progressive, symptomatic, and/or imminently threatening disease but it should be used only when cases are not manageable with other approaches. Nowadays, as described in following paragraphs, targeted therapies able to inhibit abnormal activated tyrosine kinases (TKI) represent the systemic therapies that should be used as first line in metastatic TCs.For patients with metastatic DTC that progresses despite standard therapies, systemic cytotoxic chemotherapy has been evaluated both in phase 2 and retrospective studies but, to date, there is no role for its routinary use. Doxorubicin , its application in DTC/PDTC patients remains controversial and, according to the literature, there is no role for routine adjuvant EBRT to the neck in DTC patients, especially when an initial complete surgical removal of the tumor is achieved. However, EBRT should be considered for those patients undergoing multiple and frequent neck re-operations for palliation of locoregional recurrences and, in some cases of bone metastases especially to control the pain that is frequently associated with these lesions. ,13.Recently, several studies have reported the efficacy of other localized treatments with thermal (radiofrequency or cryo-) ablation ,15, etha131I and with no chance of cure [Anaplastic thyroid carcinoma (ATC) represents about 1% of all TC and is the most aggressive thyroid tumor, accounting for the majority of all TC death . The ATC of cure ,20. It i of cure . Median  of cure ,21. Due > 60 Gy on primary tumor) and adjuvant chemotherapy. Standard cytotoxic chemotherapy includes taxane, platinum-based drugs, and doxorubicin [The initial management of ATC should include the evaluation of airways’ stability to establish whether an immediate intervention is necessary, and the evaluation of full resectability (R0) of primary tumor, since the only debulking of the cancer does not improve the patient’s outcome. Therefore, surgery should be of first choice when a full resection of tumor can be obtained and when no distant metastases are identified. About 2 to 4 weeks after surgery, chemoradiation should be offered to the patient, while unresectable ATC should be treated directly with radiation therapy  should also be offered to those patients with small-volume metastases while systemic therapies with or without palliative radiation therapy should be considered in patients with large-volume metastases. Nowadays, only specific TKI can be used in restricted subgroups of ATC with specific molecular alterations such as Medullary thyroid cancer (MTC) originates from neural crest C cells and represents 4% of all TC. The biological behavior of MTC is more severe than that of DTC: the 10-years survival rate is approximately 50% and can be lower in patients with advanced disease at diagnosis . DistantNowadays, the current systemic options for the treatment of advanced and progressive MTC are both multi-targeted and specific TKI as we will describe thereafter.p53, epigenetic alteration, and infiltration of immune cells, lead to the onset of ATC  show different patterns mostly affecting two signaling pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways ,32,33 F. When a t of ATC .BRAF, RAS, PTEN, AKT, and PI3KCA  activity, leading to RAI refractoriness [ and HTC ,36, and  and HTC . BRAFV60toriness .RAS mutations are reported in 30–40% of FTC, in 30–45% of follicular variant PTC , in 15% of HTC, in 20–40% of PDTC and ATC, and in 10–30% of MTC [% of MTC ,40,41,42PTEN, AKT, and PIK3CA are the most common mutated mediators in PI3K pathways. Mutations, deletions, and epigenetic changes of oncosuppressor PTEN were described in DTC, PDTC, ATC, and HTC [AKT1, AKT2, and AKT3 were reported in very few cases of TC and some AKT1 mutations were reported in ATC and in FTC [AKT1, PIK3CA amplifications, and less commonly mutations, were observed late in TC progression and more frequently in ATC than DTC [Although their frequency is low , PTEN, A and HTC ,35,42. Ad in FTC  and in ad in FTC . As wellthan DTC ,46.RET proto-oncogene is one of the most common altered oncogene coding for a TKR in TC [RET, this oncogene became constitutively activated with a subsequent activation of MAPK and PI3K pathways [RET alterations are RET/PTC rearrangements. In particular, a RET fusion was found in about 50% of PTC diagnosed in atomic bomb survivors after a high radiation dose exposure and was very common, especially RET/PTC1 and RET/PTC3, in childhood TC diagnosed after the Chernobyl accident [KR in TC . As wellpathways . In PTC,accident ,49.RET oncogene gain of function point mutations, both in sporadic MTC cases, in which a somatic RET mutation is found in about 40–70% of patients, and in familiar/hereditary MTC cases, in which a germline mutation is present in 95% of kindred [RET point mutation detected in multiple endocrine neoplasia syndrome (MEN) type 2A occurs on codon 634, while in 95% of MEN 2B and in 75–95% of sporadic cases the most frequent alteration is RET mutation M918T [RET mutations produce distinct phenotypes that can differ in terms of age of onset of MTC, aggressiveness of MTC, and association with other endocrine malignancies [In MTC, the most common genetic alterations are the  kindred . The moson M918T ,52. Diffgnancies .NTRK1, NTRK2, and NTRK3 genes and their rearrangements were reported in PTC, PDTC, and ATC [NTRK, lead to constitutive activation of MAPK, PI3K, and phospholipase C-signaling pathways [Neurothrophic receptor tyrosine kinase (NTRK) are a group of tyrosine kinase (TK) receptors encoded by the  and ATC ,53,54. DALK) were also reported, mainly in PDTC and ATC and less frequently in PTC [Mutations and gene fusions involving anaplastic lymphoma kinase , ve genes ,61.TP53 are described. The same alteration can also be found in the most aggressive FTC and PTC [β-catenin gene and the loss of function mutation of Axin1 gene [In 50–80% of ATC and in up to 35% of PDTC, mutations of oncosuppressor  and PTC ,62. Othein1 gene ,64.TERT promoter. The same promoter alterations can also be found in the aggressive forms of HTC, PTC, and FTC [Another mutation frequently found in ATC (40–70%) and in PDTC (40%) involves  and FTC ,65.+CD25+T cells were demonstrated to be more expressed in PTC than in benign thyroid nodules and were associated with PTC invasiveness [The immune system is the major determinant of tumor microenvironment. Many studies have shown that the escape from immune response called “tumor immunoediting” has a role in thyroid tumor progression and that immune cells, both from adaptive (T cells) and innate system (macrophage), secreted soluble mediators, and immune checkpoints are the principal mediators in this scenario . More insiveness . Cytotoxsiveness . In PTC,siveness . Tumor-asiveness ,71. In Tsiveness .V600EBRAF mutation and a lower degree of differentiation [Immune checkpoints are regulators of T cells immune response through co-stimulatory or inhibitory molecules: cytotoxic T lymphocyte antigen4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death protein 1 ligand (PD-L1) inhibit the T cells mediated control on tumor cells proliferation. In TC, PDL-1 was differently expressed according to the hystotype, with higher expression levels in ATC (822.25) with respect to FTC (7.6%) and PTC (6.1%), with even higher expression in advanced TC . In PTC,ntiation . On the ntiation . Based oOver the last decades, several small molecular agents able to inhibit TK and TKR with different mechanisms have been generated and have been demonstrated to be effective anti-solid tumor and anti-leukemic agents with the name of TKIs. This first generation of TKIs are able to inhibit specific oncogene alterations but they also act against several other TKRs and for this reason they are also recognized as multikinase inhibitors (MKI). Sometimes, the activity of these MKI against these other receptors is stronger than the activity they have against the driver oncogene alteration of TC, and several adverse events (AEs) are due to these “off-targeted” activities.Four MKI, lenvatinib and sorafenib for the treatment of advanced RAI-refractory DTC, and vandetanib and cabozantinib for the treatment of MTC, have lastly been approved by both the Food and Drug Administration (FDA) and the European Medical Agency (EMA).Recently, a second generation of TKIs has been developed which are specific for a specific altered oncogene. The high specificity of these drugs is the reason for a greater tolerability because of a much smaller number of AEs having very few “off-targeted” activities.So far, four of them, larotrectinib, entrectinib, selpercatinib, and pralsetinib have been approved by FDA and are under evaluation of EMA.p < 0.0001) with a response rate of 12.2% in the first group and of 0.5% in the second one. OS was not different in the two groups, but this was probably due to the crossover from placebo to sorafenib. Sorafenib is a small oral molecule with a strong anti-angiogenic activity. Sorafenib is able to inhibit VEGF1–3, PDGF, FGF, KIT, RET receptors and, weakly, RAF . After t131I refractory DTC.Initially approved for renal and hepatocellular carcinoma, DECISION study led to the approval of sorafenib for the treatment of Since the response rate appeared to be low (12.2%) when sorafenib was used as a single-agent, a phase 2 study  was starBRAF oncogene is mutated to its active form in up to 24% of ATC cases. Twenty ATC patients were enrolled and sorafenib was administered at 400 mg twice daily. The evaluation of the disease by RECIST revealed 10% of PR and 25% of stable disease (SD). The duration of response in the two responders was 10 and 27 months, respectively. For the patients with SD, the median duration was 4 months (range 3–11 months). The overall median PFS was 1.9 months with a median and a 1-year survival of 3.9 months and 20%, respectively. Furthermore, in this study, toxicity was manageable and as previously described for sorafenib, included hypertension and skin rash. The authors concluded that, although sorafenib has activity in ATC, this is at a low frequency and is similar to the previous experience with fosbretabulin [Sorafenib was also administered in a phase 2 study  in patieetabulin .p < 0.001) with 4 complete responses (CR) in lenvatinib-group. A sub-analysis confirmed that PFS was also longer in lenvatinib-group when patients receiving a previous TKI therapy were considered. Although OS was not different in the two groups, when a sub-analysis stratified the patients by age, an OS advantage in patients older than 65 years old was demonstrated. Lenvatinib, as well as sorafenib, is an oral TKI with a high anti-angiogenic activity. Lenvatinib is able to inhibit VEGF1–3, FGF1–4, PDGF, KIT, and RET receptors . The effA post-marketing multicenter, randomized, double-blind phase 2 study was conducted to evaluate if a lower starting dose could demonstrate the same clinical benefits with a lower AEs incidence . The study is still active but is no longer recruiting and 152 participants have been enrolled. They were randomly assigned to treatment with 1 of 2 blinded dosages of lenvatinib in a 1:1 ratio to receive lenvatinib 18 mg or 24 mg orally once daily. The estimated study completion date was September 2020 and, even if the full results have not yet been presented, in a press release from EISAI company topline the results were shown. According to these results, the 18 mg dose did not show non-inferiority in efficacy in respect with the 24 mg dose as measured by ORR at week 24, and the incidences of grade 3 or higher treatment-emergent AEs through week 24 were similar between dose arms.Before and after the commercialization of the drug, several studies were published about the real-life use of the drug showing the same safety results with a lower PFS ,85. An eLenvatinib was also demonstrated to be effective in a retrospective, single center analysis of 18 Korean patients with confirmed ATC . Six patTo date, lenvatinib is approved for the treatment of RAI refractory DTC, hepatocellular carcinoma, and, in combination with everolimus, for renal cell carcinoma, but not for ATC.p < 0.001), disease control rate (p < 0.001), and biochemical response (p < 0.001). The use of this drug was also approved for children with advanced hereditary MTC thanks to the results obtained in a phase 2 clinical trial [Vandetanib is a once-daily oral multitargeted drug with a recommended daily dose of 300 mg/die, additionally adjusted in case of unmanageable toxicity. Its main molecular targets are represented by VEGFR-2, VEGFR3, EGFR, KIT, and RET as shown in al trial .The treatment was generally well tolerated, and its side effect profile was similar to that of other TKIs. With the daily use of vandetanib in real life, some retrospective studies were published in order to find predictive factors for a more durable drug response ,92: a yoRET mutation which showed a significant increase of OS  [Cabozantinib is an oral multiple TKI approved by the FDA in 2012 and EMA in 2014 for the treatment of progressive and metastatic MTC. It is able to inhibit three main TK receptors: MET, VEGFR2, and RET as shown in  months) . MoreoveThe most frequent AEs occurring at any grade in the EXAM study are reported in -RET status and after randomization they have been treated until disease progression or intolerable toxicity. The study is still ongoing, and no data have been presented so far.Another clinical trial with cabozantinib in MTC patients has been conducted and nowadays is still active but no longer recruiting. The randomized, double blind, multicentric EXAMINER clinical study (NCT01896479) has been designed to evaluate two different doses of cabozantinib (60 mg vs. 140 mg) in progressive and metastatic MTC in order to test if the lower dose has the same results in terms of PFS and overall response rate compared to higher dose but with fewer AEs. According to the study design, previously other TKI treatment was permitted, and patients have been stratified based on M918TEML4-NTRK3 in a manner similar to the action of MAPK pathway inhibitors [NTRK fusions are rare but can be found in several solid tumors, including PTC, PDTC, and ATC. When present in TC, this fusion is usually mutually exclusive with other oncogenic driver mutations. Larotrectinib is a highly selective inhibitor of tropomyosin receptor kinase (TRK) A, B, and C , and is hibitors .Entrectinib is another selective inhibitor of TRKA, TRKB, and TRKC, but it is also able to inhibit ALK and ROS1 TK . A recenRET inhibitor  has been conducted in 65 centers in 12 countries to evaluate the efficacy of LOXO-292 in solid tumors with RET alterations (RET mutations and RET fusions) among with MTC and TC derived from follicular cells  [RET mutations, previously treated with vandetanib or cabozantinib or both, and 88 patients with RET mutations not previously treated with other TKI. Selpercatinib showed a marked and durable anti-tumor activity. In fact, the percentage of patients experiencing a response was 69%  in the first cohort and 73%  in the second one and interestingly, the first cohort was independent by the number of previous multitargeted treatments administered. The 1-year progression-free survival was 82%  in the first group and 92%  in the second one. Responses were observed across all qualifying RET mutations, including in patients who had tumors harboring the drug resistant RET mutation V804.Selpercatinib, known also as LOXO-292, is a highly selective ATP-competitive small molecule nhibitor , with a sistance  and showand ATC) . The priRET fusion–positive DTC, the percentage who had an objective response was 79%, and 1-year PFS was 64%. Of two patients with ATC, one had a durable response for 18 months, with the response ongoing at the end of the study.Although the majority of patients enrolled had MTC, 19 DTC patients  and 2 ATC patients were also included . In 19 pRET-mutant TC, both MTC and DTC/ATC, who require systemic therapy [The most related AEs were of grade 1–2. Most frequent any grade AEs are reported in  therapy . The appRET-mutant MTC  [RET mutation status, safety profile, and pharmacokinetics.An interventional phase 3 trial with selpercatinib has recently started and is recruiting all over the world. This is a multicenter, randomized, open-label study comparing selpercatinib to physicians’ choice of cabozantinib or vandetanib in patients with progressive, advanced, kinase inhibitor naïve, 4211337) . PatientRET-alterations: an expanded access trial (NCT03906331) for patients with TC with RET activation who have been previously treated with vandetanib or cabozantinib or other TKI and cannot enter in the phase 3 trial and a phase 2 trial dedicated exclusively to Chinese patients (NCT04280081).Currently, a few other phase 1–2 studies are ongoing, aiming to confirm the efficacy and tolerability of selpercatinib in different types of solid tumors with RET inhibitor targeting oncogenic RET alterations , in particular it was 74% in treatment-naïve patients and 60% in those previously treated with cabozantinib or vandetanib. The 18-months PFS and duration of response were 71% and 90% in patients previously treated with other TKI while in treatment-naive patients they were 85% and 86%, respectively. Responses occurred regardless of RET genotypes.Pralsetinib, also known as BLU-667, is another highly selective erations . Previoug VEGFR2 . In the re shown . At 13 FRET fusions which showed an ORR of 75% with a median duration of response of 14.5 months.Preliminary data showed that pralsetinib was also highly active in patients with TC with RET-fusion positive NSCLC and, very recently, for RET fusion-positive TC and RET mutation-positive MTC. Currently, it is under regulatory review in Europe for RET fusion-positive NSCLC.Regarding pralsetinib’s safety, most treatment-related AEs were grade 1–2. The most common of any grade AEs are summarized in BRAF kinase inhibitor , pyrexia (37%), and nausea (35%). In 2018, in ESMO meeting, Keam et al. proposed an updated report of the above-mentioned trial, including 28 patients [V600EBRAF mutated RAI-refractory PTC who had evidence of disease progression within 13 months prior to randomization [Dabrafenib is a selective inhibitor of E kinase . FDA app in 2018 . In thispatients . Accordimization . This phn = 25). Twenty-one patients had received only one prior VEGFR-targeted therapy , and four patients had received two of such therapies. Of the 25 patients, 40% had a PR, 52% SD, and 8% had non-evaluable disease. The median PFS and OS were 12.7 months and 34.7 months, respectively, demonstrating that cabozantinib had a clinically significant, durable objective response activity in patients with RAI-refractory DTC who experienced disease progression while taking prior VEGFR-targeted therapy.Cabozantinib, as mentioned above, is an oral TKI approved for the treatment of advanced MTC but its efficacy was also tested in phase 1  and phasA phase 3, multicenter, randomized, double-blind, placebo-controlled study of cabozantinib in subjects with RAI-refractory DTC after prior VEGFR-TKI therapy is currently ongoing . The study provides for the enrollment of 300 patients. Estimated study completion date is December 2022.p < 0.0001). ORR was 55.56% and 2.27%, respectively, while median OS was 29.9 months in the placebo arm, and not reached in apatinib arm (p = 0.0356). The most frequent treatment-emergent ≥ grade 3 AEs in two arms were hypertension (34.8% vs. 0%), hand–foot syndrome (17.4% vs. 0%), and proteinuria (17.4% vs. 2.2%).Apatinib, an oral inhibitor of VEGFR2, showed clinical activity in preliminary studies involving patients with RAI-refractory DTC. These first results led to a randomized, double-blind, multicenter phase 3 trial that aimed to evaluate the efficacy and safety of apatinib in 92 patients with RAI-refractory DTC (NCT03048877). The results of the study were recently presented at ESMO meeting 2020 by Lin et al.  and theyRET. Its antitumor effects were demonstrated in various types of carcinoma in a phase 1 clinical trial [p < 0.0001). The ORR was 59.21% in anlotinib arm and no response was observed in placebo arm (p < 0.0001). In addition, significant disease control rate benefit was observed for anlotinib treatment . The incidence of treatment-related AEs of two groups was 100% and 86.49% (p = 0.003). Serious treatment-related AEs occurred in 15.79% of patients receiving anlotinib. The most common AEs in anlotinib arm were hypertension (84.21%) and hypertriglyceridemia (68.42%).In the last few years, the attention was focused on another TKR inhibitor, anlotinib (AL3818), a novel oral drug targeting VEGFR2 and 3, FGF 1–4, PDGFR α and β, c-Kit, and al trial , and, real trial , both inal trial . A totalMEK and BRAF inhibitors showed some positive results.In the past, several drugs have been evaluated to redifferentiate metastatic TC through restoration of the NIS activity . It is still active but with no more recruiting and with no preliminary available results.Selumetinib is a e in DTC . In the e in DTC , 20 patiA phase 3 study (NCT01843062) evaluating the CR rate for selumetinib in the setting of adjuvant treatment with RAI has been recently reported . The resBRAF kinase inhibitor and it was tested in a pilot study aimed at evaluating the proportion of patients in whom vemurafenib increased RAI incorporation [BRAF mutated cases were enrolled but only four patients were 124iodine responders on vemurafenib and treated with RAI. A tumor regression was observed 6 months after the treatment. Analysis of research of tumor biopsies demonstrated that vemurafenib inhibition of the MAPK pathway was associated with increased thyroid gene expression and RAI uptake.As mentioned above, vemurafenib is an oncogenic poration . Twelve BRAF inhibitor tested to determine whether it can stimulate RAI uptake in V600EBRAF mutated unresectable or metastatic RAI-refractory PTC [131I whole body scan. Patients whose scan showed new sites of RAI uptake remained on dabrafenib for a longer time, and then were treated with 150 mCi (5.5 GBq) of RAI. Sixty percent of patients demonstrated sites of RAI uptake and all showed a clinical benefit, either PR or SD, on standard radiographic restaging at 3 months. Serum thyroglobulin decreased in 4/6 treated patients.Dabrafenib is another selective tory PTC . Ten patAlthough other studies confirmed that targeted therapy may downregulate MAPK signaling and sensitize tumors to RAI , so far RET kinases and has promising preclinical activity in models of RET-driven MTC [RET mutated tumors. The drug was administered at a daily dose of 45 mg. The estimated study completion is June 2021.Ponatinib (AP24534) is an oral multitargeted TKI that potently inhibits several targets, among which are both the native and mutant forms of BCR-ABL chimeric protein. Quite recently, it has been demonstrated that the drug is also a potent inhibitor of iven MTC ,127. A pp = 0.0289). The most common AEs in anlotinib arm were hand–foot syndrome, hypertension, hypertriglyceridemia, and diarrhea.Anlotinib (AL3818) is a novel multi-targeted TKI, inhibiting tumor angiogenesis and proliferative signaling. After the results of a phase 2 single-arm trial (NCT01874873) demonstrating that anlotinib has a durable antitumor activity with a manageable adverse event profile in locally advanced or metastatic MTC, a subsequent randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in China to compare the efficacy and safety of anlotinib versus placebo in MTC patients . The primary endpoint was the PFS. Previous TKI therapies were not allowed. At the 2019 ASCO congress, some results were presented : 91 patiRET-driven cancer models [RET-810 mutation. Hopefully, in vivo clinical trials will confirm these in vitro studies.TPX-0046 is a 3rd generation, highly selective TKI tested, so far, in drug-resistant and naïve r models . This drTo date, no immunotherapy has been approved for advanced TC, but several preclinical and clinical trials together with case reports have been reported. A few studies are ongoing, using immune checkpoint inhibitors alone or in combined strategy.In the phase Ib KEYNOTE trial, the use of pembrolizumab (anti PD-1) in monotherapy was studied in TC including PTC and FTC showing an ORR of 9% and reduction of tumor size of 35–50% . The useBRAF inhibitor), binimetinib (MEK inhibitor), and nivolumab is under evaluation in V600EBRAF mutated TC in a randomized open label clinical trial still recruiting (NCT04061980). Combined approaches with radio and chemotherapy were also evaluated in ATC: the association of pembrolizumab and chemoradiation [Considering the slow onset of immunotherapy efficacy and the evidence that immune checkpoint inhibition alone is not curative in most patients, several combined approaches with chemo and target therapy are under investigation. For example, the association of encorafenib (adiation  (doxorubadiation .BRAF mutation (vemurafenib plus cobimetinib), RAS, or NIF1/2 (only combimetinib) or the absence (bevacizumab plus paclitaxel) (NCT03181100).Based on the evidence that VEGF inhibition can reverse the immune suppression in the tumoral microenvironment, multiple clinical trials with VEGF and/or VEGF inhibitor and immune checkpoint inhibitors have been designed. In an ongoing phase 2 clinical trial, the use of pemproblizumab+lenvatinib was investigated in unresectable ATC (NCT04171622) and also in a randomized study in a small group of advanced ATC and PDTC  with an RET inhibitors (LOXO292 and BLU-667) with immune checkpoint inhibitors.Few studies are ongoing for MTC. A phase 2 trial of pembrolizumab is ongoing in recurrent or metastatic MTC (NCT03072160) as well as the use of a cancer vaccine GI-6207 targeting the CEA in a randomized phase 2 clinical trial (NCT01856920). A future prospect for MTC patients might be the combination of specific So far, immunotherapy is not yet applicable in clinical practice for patients with advanced TC but, as demonstrated by the numerous active trials, there is great interest and hope.Despite good results, both from clinical trials and clinical practice, some problems are still present in the use of these therapies. For example, problems are the balance between drug efficacy and management of AEs, the drug resistance, and the absence of recognized predictors of response. While for AEs, thanks to the daily use of these drugs in clinical practice, some preventive strategies have been implemented to avoid toxicity and to prolong the use and efficacy of the treatment , the queRET mutation in tumor sample seems to be associated with better overall survival [RET mutation [The availability of predictive markers would help to select patients with higher probability to benefit from the treatment, but the research into these genetic and/or biological markers is still ongoing. The pre-treatment analysis of the mutational pattern of tumor samples might have a role in the selection of patients, but, in almost all clinical trials, the drug response was demonstrated to not be strictly related to the mutational status of tumors with some exceptions. For examples, for cabozantinib the presence of M918T somatic survival , while omutation . Furthermutation , while tmutation , for hypmutation , and promutation . In addimutation . The sammutation .Drug resistance is one of the most critical and unavoidable problems for the clinician. Many mechanisms limit drug efficacy but especially the tumor heterogenicity, which is related not only to a genetic intratumor heterogenicity but also to epigenetic alterations induced by the molecules secreted by the tumor microenvironment. Much evidence shows that a genetic heterogenicity is present in TC and related to different subclones within the same tumor . This heThyroid cancer has different histologies and molecular identities. The initial treatment is always represented by total thyroidectomy plus/minus lymphadenectomy followed by, whenever appropriate, RAI treatment. This initial treatment can cure the vast majority of TC but the subgroups of multimetastatic, poorly differentiated, and anaplastic require other therapies varying from local treatments to systemic therapies. Local treatments are always preferred when the disease is oligometastatic or, if multimetastatic, with only one specific lesion that is growing. When the disease is advanced, multimetastatic, and progressive, the systemic therapy should be initiated and nowadays there are already four different types of TKIs approved; two for MTC and two for RAI refractory DTC and PDTC. The TKIs therapy changed the outcome of advanced metastatic TC patients but, although a prolonged PFS was well documented, an improvement of the OS has yet to be established. Clinicians should bear in mind that TKIs are not curative and a continuative treatment is needed to maintain a response to the disease. For this reason, it is very important that other studies will be performed and other therapeutic strategies will be explored. Moreover, the first generation of TKIs, being multikinase inhibitors, has a lot of side effects that may reduce the quality of life of these patients, frequently leading to a discontinuation of the drug with an important impact on neoplastic disease recurrence. For this reason, new therapeutic approaches are needed and, according to preliminary studies, a new second generation of selective TKIs seems to be a good option able to balance efficacy and AEs in patients with advanced, metastatic, and progressive TC.


Many details concerning the modulation of macrophage functionality by melatonin are obviously also applicable to microglial cells. Of particular interest is the polarization toward M2 subtypes instead of M1, i.e., in favor of being anti-inflammatory at the expense of proinflammatory activities, which is well-documented in macrophages but also applies to microglia.Melatonin interacts in multiple ways with microglia, both directly and, via routes of crosstalk with astrocytes and neurons, indirectly. These effects of melatonin are of relevance in terms of antioxidative protection, not only concerning free-radical detoxification, but also in prevention of processes that cause, promote, or propagate oxidative stress and neurodegeneration, such as overexcitation, toxicological insults, viral and bacterial infections, and sterile inflammation of different grades. The immunological interplay in the CNS, with microglia playing a central role, is of high complexity and includes signaling toward endothelial cells and other leukocytes by cytokines, chemokines, nitric oxide, and eikosanoids. Melatonin interferes with these processes in multiple signaling routes and steps. In addition to canonical signal transduction by MT The multiple and partially divergent activities of microglia are of crucial importance to the initiation, course, and termination of inflammation in the central nervous system. Crosstalk between microglia and other cells, especially but not exclusively astrocytes and neurons, are also involved in these processes. Melatonin has been shown to act in protective and mostly anti-inflammatory ways under various conditions of high- and low-grade brain inflammation, including age-related impairments in the course of immunosenescence, neurodegenerative disorders, and bacterial and viral infections as well as in processes caused by ischemia/reperfusion or brain trauma ,2,3,4. T+, CD68+ expressing microglia with amoeboid morphology was shown to be distinguished from infiltrated macrophages by the presence of TMEM119 (transmembrane protein 119), which may serve as a reliable microglial marker absent in macrophages [+ cells were reported to not consistently express M1 and M2 markers [Such a mode of polarization in functional differentiation also exists in microglia, although it does not seem to be fully identical to the one observed in macrophages. The differences in functionality can be traced by determining surface and expression markers, which, however, also reveals the necessity of discriminating M2 subtypes  that arerophages . However markers ,13, with markers ,15,16. GWhen comparing the roles of microglia over the decades, it becomes evident that earlier studies regarded these cells as comparably uniform, mainly differing in their states of quiescence or activation. The roles of actively counteracting inflammatory responses remained largely unconsidered for quite some time. This view was also evident in the earlier interpretation of markers that were only used for categorizing cells as quiescent or activated regardless of whether they were of the proinflammatory M1 type or one of the anti-inflammatory M2 subtypes. Some of the markers were, in fact, unsuitable for discriminating the opposite roles in inflammation, such as Iba-1 and CD68, which are expressed in both M1 and M2 microglia . With reThe mechanisms that control inflammation are of utmost importance to numerous pathologies ,3,4. TheMeanwhile, a substantial body of evidence has accumulated concerning the actions of melatonin in microglia. Basically, most of these findings are in good agreement with the earlier data although the aspect of polarization has not yet been generally considered. The suppression of iNOS by melatonin has now been repeatedly confirmed in microglia ,25,26,27N-acetylcysteine, which may indicate a role of ROS scavenging [Suppression of the formation and activity of the NLRP3 inflammasome caused by melatonin was observed after LPS treatment in natural microglia in vivo  and in Bavenging . The preavenging ,36,37,38avenging . Downstravenging , and theavenging ; the preavenging ,41; and avenging ,36,42. Aavenging ,46,47,48avenging .A rather substantial body of evidence exists for the suppression of proinflammatory cytokines by melatonin in microglia. While most studies have only considered IL-1β and TNFα, additional data have been published for IL-6 and IL-18 ,53,54,55Although a substantial body of data exists on the upregulation of anti-inflammatory M2-typic cytokines by melatonin without the identification of microglia as their source , respectAlong with the secretion of inflammation-promoting cytokines, various chemokines are also released to attract other leukocytes that infiltrate the site of injury. As an anti-inflammatory agent, melatonin should attenuate this process. Existing data are partially compatible with this assumption. However, the microglia-specific information is, again, rather limited. The downregulation of CCL5 and CCL9 were reported in conjunction with other anti-inflammatory actions , but theAn article on retinal microglia and/or macrophages concerning a murine model of wet macular degeneration reported that melatonin upregulates CCL3 and CCL5 along with producing an increase of iNOS expression and TNFα release . These f1 receptor [Despite the deviating effect in retinal microglia that may reflect an unexpected change in polarization towards M1, the majority of the observations are in favor of melatonin’s anti-inflammatory actions in the regulation of microglial functions. With regard to this overwhelming evidence, an earlier cytokine-based conclusion that melatonin may not be an important modulator of macrophage and microglia function  appears receptor . This woreceptor ,74,75. CA detailed listing of pathologies in which anti-inflammatory effects have been observed would be space-consuming and would not reveal novel insights since the protective actions that are now specifically attributable to microglia are largely identical to those known from earlier studies without the specification of cellular cytokine sources. Again, all of the previously known conditions, such as toxicity by LPS and other inflammation-inducing agents, bacterial infections, brain trauma, ischemia/reperfusion, hemorrhage, aging, and neurodegenerative disorders are covered The actions of melatonin on microglia cannot be seen independently of other cells in the CNS. The interplay of neurons, astrocytes, and microglia has been repeatedly addressed in articles on neuroprotection and neurodegeneration as well ,78,79,80Under various neuroinflammatory conditions, astrocytes and microglia are concomitantly activated ,97,98,99With regard to melatonin research, the understanding of these crosstalks and the orchestrating role of melatonin are still in their infancy. Along with the multiple documented reductions of proinflammatory cytokines discussed in +/CD45+) and the infiltration of macrophages/microglia (CD11b+/CD45+) into the damaged site by about a half. This effect may be caused by several factors, such as the attenuation of cytokine release, including that of CXCL8 (=IL-8). CXCL8 can be released by macrophages and microglia, but it can also be released by various other cell types. Concerning the attraction of macrophages or microglia, the CD11b+/CD45+ criterion does not yet allow conclusions on polarization [An influence of melatonin on the infiltration of other cells was reported in rat transient focal cerebral ischemia . It redurization , but theSeveral other studies shed light on neuronal/microglial interactions. This became even more evident in primary murine glial cultures and BV2 cells, in which NLRP3 activation and the release of proinflammatory cytokines were shown to depend on the suppression of nAChR (α7 nicotinic receptor), i.e., on the strength of a neuronal signal . MelatonA recent investigation related neuronal/microglial protection by melatonin to properties of exosomes . In ratsA rather divergent inflammatory interaction has been described for the relationship between macrophages/microglia and pinealocytes, in which melatonin synthesis itself is affected ,110. In N1-acetyl-5-methoxykynuramine) [A further aspect that may gain considerable future importance concerns the role of another still insufficiently considered type of glia, the NG2 cells . These cells, which had previously only been regarded as oligodendrocyte precursors, constitute the fourth large population of glia in addition to microglia, astrocytes, and oligodendrocytes . The NG2uramine) ,18,19,91uramine) , but theuramine) .Sirt1 siRNA [The notion that several protective actions of melatonin are mediated by the upregulation of sirtuins, most particularly, but not exclusively, the upregulation of SIRT1, has opened a new field of melatonin research. The criteria for the involvement of SIRT1 are convincing when effects by melatonin are suppressed by SIRT1 inhibitors such as sirtinol or, more specifically, by EX527, or by downregulation using t1 siRNA ,74,113. t1 siRNA ,74,113. t1 siRNA ,74,113. +-cells  was suppressed by melatonin, an effect abolished by SIRT1 inhibition [+ cells of the corpus callosum. Another study on LPS-stimulated BV2 cells reported SIRT1-dependent inhibition of the TLR4/MyD88/NF-κB pathway by melatonin, along with a suppression of HMGB1 release [42-treated HMC3 cells, a human microglial cell line, melatonin was recently shown to not only prevent NF-κB nuclear translocation, but to also cause prolonged upregulations of SIRT1 and BDNF (brain-derived neurotrophic factor) [Meanwhile, this relationship between melatonin and SIRT1 has become evident in the control of microglia, too. In murine microglia, NLRP3 activation in Iba1hibition . Similarhibition . In a hyhibition . In a ra release . This la release ,116. In  factor) . Notably factor) .The list of publications referring to the melatonin–SIRT1 nexus in microglia is still relatively short. However, this should be regarded as a starting point for an emerging trend that will gain attention and importance in the near future.Sirt1 mRNA, were downregulated by melatonin, thereby preventing a blockade of SIRT1 expression. Although the expression of these miRNAs is known from macrophages, their investigation in microglia is still in its beginning. In the case of miR-155, its proinflammatory role in the microglia has been confirmed [MicroRNAs have become another hot topic in cell biology. In the context of melatonin and SIRT1 as its downstream factor, several microRNAs have been identified that play a role in inflammation control ,117. Amoonfirmed ,120,121.onfirmed .The importance of melatonin-modulated microRNAs to microglia may be assumed with regard to the above-mentioned protective changes in exosome composition by melatonin treatment . The desIn the melatonin literature, the topic of polarization preferably concerns macrophages . Althoug+ (arginase-1+) cells. As Arg1 is a reliable marker of M2 microglia [+/CD206+ cells and decreased that of iNOS+/CD16+ cells [In the visual cortex of mice, inflammation by microelectrode implantation was attenuated by melatonin, which also increased the number of Arg1icroglia , a shifticroglia . In a st6+ cells  using tw6+ cells . In the 6+ cells . While i6+ cells . Therefo6+ cells  appears 6+ cells . CD86 is6+ cells , but, at6+ cells . Despite6+ cells . In both6+ cells ). In BV26+ cells .Collectively, these studies are in favor of an anti-inflammatory action of melatonin via microglia M2 polarization . HoweverMost of the information summarized in this article indicates that the effects by melatonin on microglia largely resemble those known for macrophages. They are consistent with numerous findings obtained in studies on the CNS, in which microglia was not specifically addressed although the suppression of TLR4 and NLRP3 activation and downstream factors as well changes in pro- and anti-inflammatory cytokines were the most easily interpretable as effects on microglia.Nevertheless, a number of uncertainties and gaps have remained at the present state of knowledge. On the one hand, it seems obvious that melatonin favors the M2 polarization of microglia at the expense of M1, in a way that is similar to the one known for macrophages (cf. ref. ). HoweveA particular problem exists in the case of iNOS. In fact, iNOS is a marker of M1 but not M2 microglia . HoweverAnother gap exists with regard to the subtypes of M2 microglia. As these subforms are involved in a sequence of functions in the course of healing and reorganization after injury, a profound and complete understanding of M2 properties under the influence of melatonin would require differential considerations of the changes in the subtypes. In principle, subtypes can be identified by certain ensembles of markers, such as CD206, CD209, TGFβ, and CCL22 in M2a; CD16, CD32, CD64, and IL-10 in M2b; and CD163 and IL-10 in M2c [Moreover, attention should be increasingly directed toward the routes of the secondary signaling downstream of melatonin receptors, in particular, by SIRT1 and SIRT1-associated pathways, and to the wide field of noncoding RNAs, microRNAs in particular, but also lncRNAs, circRNAs, and, with regard to melatonin’s chronobiological role, circadian-associated snoRNAs, asRNAs (antisense RNAs), and the various types of enhancer RNAs . Despite