Microglia are the first line of defense at the level of the central nervous system (CNS). Phenotypic change in microglia can be regulated by various factors, including the orexin system. Neuroinflammation is an inflammatory process mediated by cytokines, by the lack of interaction between neurotransmitters and their specific receptors, caused by systemic tissue damage or, more often, associated with direct damage to the CNS. Chronic activation of microglia could lead to long-term neurodegenerative diseases. This review aims to explore how tocopherol (vitamin E) and the orexin system may play a role in the prevention and treatment of microglia inflammation and, consequently, in neurodegenerative diseases thanks to its antioxidant properties. The results of animal and in vitro studies provide evidence to support the use of tocopherol for a reduction in microglia inflammation as well as a greater activation of the orexinergic system. Although there is much in vivo and in vitro evidence of vitamin E antioxidant and protective abilities, there are still conflicting results for its use as a treatment for neurodegenerative diseases that speculate that vitamin E, under certain conditions or genetic predispositions, can be pro-oxidant and harmful. Oxidative stress refers to a condition caused by the imbalance between oxidants and antioxidants in a biological system . Reactiv2O2; this study, in fact, has demonstrated that OXA is a neuroprotective molecule, partly because it reduces caspase-committed apoptosis and lipid peroxidation. There are also further studies regarding the orexin system and the reduction in oxidative stress, such as s study by Wang et al., which demonstrates how a neuroblastoma cell line reduces its oxidative stress induced by H2O2 following pretreatment with OXA [2O2 treatment is commonly used to induce oxidative stress [2O2-induced neurotoxicity; moreover, the treatment with H2O2 reduces the antioxidant activity of SOD. It has been seen that this was attenuated by the pretreatment with OXA, in line with a study by Bihamta et al. conducted on cardiomyocyte cells [2O2-induced increase in apoptosis and decrease in SH-SY5Y cell viability [The orexin system  consists of a population of neurons located at the hypothalamic level with the function of producing neuropeptides involved in the various regulatory processes, mainly of sleep and arousal ,27. The ectively . Both OXectively . A possiectively , narcoleectively ,33 and eectively ,34. Furtectively , which iectively . The funectively . The oreectively . There aectively ,40,41, wectively  that showith OXA . H2O2 tre stress . These rte cells . This isiability . In anotiability . TherefoMicroglial cells are monocyte–macrophage cells resident in the brain. In brain tissue, under physiological conditions, microglia cells are found in a quiescent state throughout the brain parenchyma, accounting for about 15% of the cell population , they ar TNF-α  IL-6 and TNF-α . Furthericroglia ,77. A sticroglia , i.e., Ond Il-10 . Therefond Il-10 ,79. Thesnd Il-10 .Vitamin E is a fat-soluble vitamin that plays the role of antioxidant against oxidative stress. As summarized in The neuroprotective roles of vitamin E have been well documented in both in vivo and in vitro studies ,94,95,96There is still little evidence for this concept, but several studies correlate vitamin E with the hypocretin system. For example, a work conducted by Sanita Masoudi et al. studies the effect of an AS03 adjuvanted influenza vaccine (Pandemrix) against H1N1 swine flu. ASO3, a squalene-based immunological adjuvant used in various vaccine products, also contains α-tocopherol . It has As summarized in This mini review provides further support for the potential protective effects of vitamin E on neuroinflammation, the orexin system and its correlation with neurodegenerative diseases. Therefore, vitamin E could help reduce the chances of developing neurodegenerative diseases thanks to its fundamental role on microglia and its correlation with the orexin system. Further studies need to be conducted to better understand the mechanisms involved in the neuroprotective role of vitamin E and the benefit of its integration in our diet.


This systematic review aims to describe 1) the epidemiology of the diseases indicated for treatment with growth hormone (GH) in Italy; 2) the adherence to the GH treatment in Italy and factors associated with non-adherence; 3) the economic impact of GH treatment in Italy; 4) the quality of life of patients treated with GH and their caregivers in Italy.Systematic literature searches were performed in PubMed, Embase and Web of Science from January 2010 to March 2021. Literature selection process, data extraction and quality assessment were performed by two independent reviewers. Study protocol has been registered in PROSPERO (CRD42021240455).We included 25 studies in the qualitative synthesis. The estimated prevalence of growth hormone deficiency (GHD) was 1/4,000–10,000 in the general population of children; the prevalence of Short Stature HOmeoboX Containing gene deficiency (SHOX-D) was 1/1,000–2,000 in the general population of children; the birth prevalence of Turner syndrome was 1/2,500; the birth prevalence of Prader-Willi syndrome (PWS) was 1/15,000. Treatment adherence was suboptimal, with a range of non-adherent patients of 10–30%. The main reasons for suboptimal adherence were forgetfulness, being away from home, pain/discomfort caused by the injection. Economic studies reported a total cost for a complete multi-year course of GH treatment of almost 100,000 euros. A study showed that drug wastage can amount up to 15% of consumption, and that in some Italian regions there could be a considerable over- or under-prescribing. In general, patients and caregivers considered the GH treatment acceptable. There was a general satisfaction among patients with regard to social and school life and GH treatment outcomes, while there was a certain level of intolerance to GH treatment among adolescents. Studies on PWS patients and their caregivers showed a lower quality of life compared to the general population, and that social stigma persists.Growth failure conditions with approved GH treatment in Italy constitute a significant burden of disease in clinical, social, and economic terms. GH treatment is generally considered acceptable by patients and caregivers. The total cost of the GH treatment is considerable; there are margins for improving efficiency, by increasing adherence, reducing drug wastage and promoting prescriptive appropriateness. Growth hormone (GH), also known as somatropin, is recommended as the main treatment option for growth failure in children with one of the following conditions: growth hormone deficiency (GHD), Turner syndrome (TS), growth retardation in infants born small for gestational age (SGA), Prader-Willi syndrome (PWS), growth retardation due to chronic kidney failure (CKF), and growth retardation associated with a defect in the Short Stature HOmeoboX Containing (SHOX) gene. This treatment has also been shown to be effective in improving body composition, muscle mass and strength, exercise capacity, glucose and lipid profile, bone metabolism, and quality of life in adults with GHD diagnosed in adulthood or childhood.Although there is a wide international literature on somatropin, focused on clinical effectiveness, safety and economic aspects, we were interested in verifying the available evidence specifically for Italy. Our aim was to identify studies conducted in Italy concerning GH therapy in non-adult subjects, through a systematic review of the literature. To our knowledge, there is not a published systematic review on this topic available.Specifically, we were interested in estimating the epidemiology of GH-treated growth failure conditions in Italy, verifying treatment adherence, assessing the economic impact of GH treatment and describing the quality of life of patients treated with GH and/or caregivers.This systematic review is based on a study protocol registered in PROSPERO (CRD42021240455). The reporting of the review follows the PRISMA reporting checklist .The research questions that this systematic review aims to address, are:to assess the prevalence and the incidence in Italy of pediatric conditions approved as indication for GH treatment: GHD, TS, CKF, PWS, SGA, SHOX gene deficiency;to assess adherence to GH treatment in Italy and identify factors that could facilitate or hamper the adherence;to assess the economic impact of GH treatment in Italy;to assess the quality of life of patients treated with GH and their caregivers in Italy.Systematic searches have been performed in PubMed, Embase and Web of Science, looking for papers published from January 2010 to March 2021, describing national or international studies. Only articles written in English or Italian were included. The complete search strategies are reported in Non-adult subjects  with an indication for the treatment with GH. All the following conditions were included: GHD, TS, CKF, PWS, SGA, SHOX gene deficiency.Growth hormone treatment.All the comparators  were considered.Research question 1 (epidemiology): epidemiological studies assessing the prevalence/incidence of the conditions with an indication for the treatment with GH in pediatrics;Research question 2 (adherence): studies of any design assessing adherence to GH treatment;Research question 3 (economic impact): economic studies assessing the economic impact of the treatment with GH, in terms of total costs, cost-effectiveness, cost-consequence, cost-utility, drug wastage costs, and other relevant economic measures.Research question 4 : studies of any design assessing the quality of life of patients treated with GH and their caregivers.For all the research questions, only studies performed in Italy, or international multicenter studies with at least one Italian center, were included.Research question 1 (epidemiology): prevalence and incidence of conditions treated with GH in Italy; in absence of Italian estimates, worldwide estimates were considered;Research question 2 (adherence): adherence to GH treatment;Research question 3 (economic impact): cost-effectiveness and other economic analyses of GH treatment;Research question 4 : quality of life of patients treated with GH and their caregivers.Research question 1 (epidemiology): prevalence and incidence;Research question 2 (adherence): medication possession ratio (MPR), proportion of days covered (PDC), proportion of doses administered vs prescribed, percentage of adherent patients;Research question 3 (economic impact): total costs, drug wastage costs, incremental cost-effectiveness ratio (ICER), quality-adjusted life year (QALY), etc.;Research question 4 : results reported in qualitative studies  aimed to assess the quality of life in patients treated with GH and their caregivers.The study selection process was performed by two independent reviewers. Any disagreement was solved through discussion and, when necessary, a third reviewer was contacted. Study selection was conducted in two phases. Initially, the reviewers assessed the records through the titles and abstracts screening, according to the predefined inclusion criteria. In the second phase, the reviewers evaluated the full-text of the potential eligible studies. The final studies included in the review were described in the main text and in the tables, while a list of excluded studies along with the reasons for exclusion have been published as . BibliogData extraction was performed by one reviewer and verified by another reviewer using a standardized form. The following information was extracted from the included studies: bibliographic data , study characteristics , participant characteristics , treatment characteristics , comparator characteristics , study outcomes .Specifically, for each research question we extracted the following information.Research question 1 (epidemiology)—secondary studies: number of included studies and their references, disease, epidemiological estimates ; primary studies: disease, study population (N), epidemiological estimates. None of the primary studies identified in this review are in common with those included in the secondary studies.Research question 2 (adherence)—definition of non-adherence / non-adherent patients (%), reasons for non-adherence, factors influencing (barriers / facilitators) the adherence.Research question 3 (economic impact)—type of economic analysis, type of costs / discount rate / economic perspective / reference year, results.Research question 4 —methods used for assessing QoL, subjects interviewed, results.The quality assessment was performed by one reviewer and verified by another reviewer, using appropriate checklists according to the study design of included studies. In particular, the following checklists were used: for prevalence studies, the checklist by Hoy et al. ; for coh2 statistic, considering a statistical significance level of p <0.05 for all analyses. The STATA 13/SE software was indicated for the statistical analyses. It was also specified in the protocol that, in all the cases where a meta-analysis was not considered feasible, the results would have been presented narratively.According to study protocol, a quantitative synthesis of the results through a meta-analysis would have been performed only in the case three or more (included) studies reported on the same outcome for any research question; moreover, to be combined, the studies would have to be similar in terms of PICO . Assuming a high level of heterogeneity between the included studies, it was planned to perform random effects meta-analyses, providing cumulative estimates along with 95% confidence intervals. The heterogeneity between the included studies would have been assessed using the IThe literature search performed in Medline (via PubMed), Embase and Web of Science identified 1,383 records overall. After removing duplicates, 924 records were screened through title/abstract and 50 of these were considered eligible for full-text evaluation. Twenty-five articles were excluded after full-text screening, while 25 articles –30 were The results are presented narratively, without attempting a quantitative synthesis  as the studies included in this review were heterogeneous in terms of study design, objectives and PICO .To address the research question 1 (epidemiology), we identified 9 studies , 28–30, Among the secondary studies, we included 3 narrative reviews , 25, 29,The 2 reviews by Cicognani  and NicoOther two included studies were those of Tornese , 30, relThe other study by Tornese et al.  was a coAnother secondary study included was the review paper by Spandonaro et al. , aimed tIn addition, our literature search retrieved 4 primary epidemiological studies , 17, 19 Chiavaroli et al.  describeThe paper by Genoni et al.  describeIn another epidemiological study included, Kodra et al.  presenteIn summary, in the included studies:the estimated prevalence of GHD in the pediatric population  is about 1/4,000–10,000, while the prevalence of GHD in Italy was indirectly estimated through the index of exposure to GH treatment in the Piedmont region , that is 9.44 subjects per 10,000 residents <18 years;the minimum hypothetical prevalence of SGA children is 2%, with a range of 3.1–5.5%; the hypothetical prevalence of SGA children with growth retardation at 2 years is 0.24%, while in a Japanese study it was 0.06%;the prevalence of SHOX-D in children with idiopathic short stature ranges between 1.1–15.0%, while prevalence in the general population of children is about 1/1,000-1/2,000 ;the birth prevalence of Turner syndrome is 1/2,500 ;the birth prevalence of PWS is about 1/15,000 , while the annual incidence in Italy is reported between 0.08 and 0.10 per 100,000 in registry studies;the birth prevalence of CKD in the general population is 0.001%, and the prevalence of growth failure among CKD children in Italy is 23.9%.The methodological quality of secondary included studies was not assessed because we assumed that it was low, based on study design (narrative review or commentary). The quality of primary studies was assessed through the Hoy checklist , a tool To address research question 2 (adherence), we identified 10 studies , 22, 23,The multicenter study by Bagnasco  was condTM auto-injector and the adherence to the treatment. Non-adherent patients, defined as those that missed more than two daily injections per month or six daily injections in a 3-month period, were 12.5% of the entire study population , of which 10.3% were naïve, and 18.3% experienced users. With regard to the Italian centers involved, non-adherent patients were 20.9% (15.4% naïve vs 34.4% experienced). Among the reasons for poor adherence, the following have been reported: forgetfulness (43.7%), device malfunction (18.2%), running out of cartridges/needles (12.9%), being away from home (12.6%).The international, multicenter study by Bozzola et al.  was condAnother study by Bozzola et al.  aimed toBuzi et al.  carried The paper by Cardinale et al.  describest year: 88.5%; 2nd year: 86.6%; 3rd year 85.7%).Centonze et al.  conducteThe paper by Giavoli  describeIn the pediatric GHD population, 82% of patients showed high adherence, while in the adult/transition population 94% declared an acceptable adherence. The reasons for poor adherence in the pediatric population were related to drug supply problems and motivational problems, while in the adult/transition population mainly drug supply problems.The paper by Loche et al.  describeMaggio et al.  conducteThe paper by Marcianò  reportedThe methodological quality of 5 included studies , 22, 23 The methodological quality of 3 included studies , 8, 18 wThe study by Bozzola et al. (2014)  was asseThe quality of the narrative review by Buzi et al.  was not To address the research question 3 (economic impact), we identified 5 studies , 26, 28.The paper by Drube et al.  describeIn the study by Foo et al. , patientThe paper by Lopez-Bastida et al.  aimed toPagani et al. conducted a study  includinThe study by Spandonaro et al.  was condThe quality of included studies was evaluated through the CHEC list, a 19-item checklist. The quality of Spandonaro et al.  was not To address the research question 4 , we identified 4 studies , 24, 27.The two studies by Lopez-Bastida  and RaguIn the international multicenter study of Bozzola et al. (2011)  which inThe paper by Lopez-Bastida , previouThe qualitative study by Marini et al. , conductThe multicenter study by Ragusa  et al., Two studies have been assessed through the CASP Qualitative checklist: Marini  and RaguWith regard to the epidemiology of diseases/conditions with an indication for GH treatment, we found mostly international estimates, with prevalence estimate for GHD in children ranging from 1/4,000 to 1/10,000 ; this esTurner syndrome occurs in about 1/2,500 girls  . An international included study  showed tAccording to a comparative study , the somIn a report from the Canadian Agency for Drugs and Technologies in Health (CADTH) , the ecoAn Italian study  showed tTM device was found to be acceptable by patients who were satisfied with its use; the only negative point concerns the pain experienced with the injection (38.5% of the answers). Another study https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  3 Jan 2022Journal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse to Editor: We ensure that our manuscript meets PLOS ONE's style requirements.2. Thank you for stating the following in the Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: Simona Granato, Giuseppe Novelli, Roberto Di Virgilio and Daria La Torre are employees of Pfizer.Barbara Polistena declares to have received in the last 5 years payments or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Allergan, Amgen, Astellas, Baxter, BMS, Boehringer-Ingelheim, Celgene, Eli Lilly, Janssen Cilag, Jazzpharma, Mylan, Nestlé HS, Novartis, Novo Nordisk, Pfizer, Roche, Sanofi, Servier, Shire, Takeda, Teva; in addition, she received consulting fees from UCB.Federico Spandonaro declares to have received in the last 5 years payments or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Allergan, Amgen, Astellas, Baxter, BMS, Boehringer-Ingelheim, Celgene, Eli Lilly, Janssen Cilag, Jazzpharma, Mylan, Nestlé HS, Novartis, Novo Nordisk, Pfizer, Roche, Sanofi, Servier, Shire, Takeda, Teva; in addition, he received consulting fees from Amgen.All other authors declare that they have no competing interests."We note that you received funding from a commercial source: "Allergan, Amgen, Astellas, Baxter, BMS, Boehringer-Ingelheim, Celgene, Eli Lilly, Janssen Cilag, Jazzpharma, Mylan, Nestlé HS, Novartis, Novo Nordisk, Pfizer, Roche, Sanofi, Servier, Shire, Takeda, Teva, and UCB."Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript is a systematic review of the epidemiology of disorders requiring growth hormone supplementation and specific other growth hormone treatment related issues. The authors focused on studies including Italian national data. The overview is well presented with detailed methodology and informative tables containing results. The discussion is mostly summarizing the results with a few major conclusions. These are particularly exposing the possibilities to decreasing costs of GH therapy, which is of public importance in light of very high treatment costs. In general, I have no major comments regarding the manuscript. Despite the fact that part of the authors are employed in pharmaceutical companies producing GH therapeutics, I did not notice any biased reporting.Response to Reviewer #1: We would like to thank the Reviewer #1 for his positive comments.Reviewer #2: Summary: This is a systematic review that addresses 4 major questions regarding GH use in children in Italy: the prevalence and incidence of pediatric conditions approved as indication for GH treatment: GHD, TS, CKF, PWS, SGA, SHOX gene deficiency, adherence to GH treatment, the economic impact of GH treatment, and the quality of life of patients treated with GH and their caregivers.Response to Reviewer #2: We would like to thank the Reviewer #2 for his comments, useful to improve the quality of our manuscript. Major comments:1. Abstract, Results section: In the paragraph discussing prevalence, state the population, i.e., general population, referred patients, etc.Response to Reviewer #2: We have specified that the prevalence of GHD and SHOX-D is in the general population of children; for Turner syndrome and PWS it is a birth prevalence, i.e. number of cases per live births.2. Page 7, Data extraction: Clarify that there was no overlap between primary and secondary studies.Response to Reviewer #2: We clarified this by adding the following sentence in the text: “None of the primary studies identified in this review are in common with those included in the secondary studies”.3. Page 13, 2nd paragraph: Add a discussion about the different definitions of SGA used in the studies.Response to Reviewer #2: We added a description of the different SGA definitions among the three studies: “This hypothetical prevalence is lower than the prevalence reported in the three population studies included in the review: 3.1% (Finland) [41] (SGA was defined as – 2 SDS of birth weight compared to a reference population), 5.5% (Sweden) [42], and 3.5% (Japan) [40] ”.4a. Page 14, 1st summary bullet: Does the general population mentioned in the prevalence of GHD include both children and adults? Response to Reviewer #2: Thank you for giving us the chance to clarify this point. Although in the article by Tornese et al.  this has not been clearly specified, we have assumed that the prevalence of GHD refers to the pediatric population, owing that the whole article is about SGA children. We amended the text accordingly. 4b. There is a striking difference between the prevalence noted in the international studies and that in Italy. (see below)Response to Reviewer #2: We agree that the difference between the international estimates and those deriving from the Piedmont Region  registry of patients treated with GH is notably; however, should be considered that the last one (registry estimate) is an indirect estimate of GHD prevalence. The Authors of this study  in the Discussion section state that their prevalence estimates are higher of those reported in previous studies, probably due to different patient’s selection criteria, the cut-off used for tests allowing the diagnosis of partial or complete GHD, and to the improvements of diagnostic techniques (higher sensitivity).We added in the discussion a text explaining this (see Response to comment n. 8).5. The majority of the epidemiological studies are international and most have only 1 study in Italy resulting in a wide prevalence range.Response to Reviewer #2: We pointed out in the Discussion that most of estimates come from international studies and the scarcity of Italian study. About SHOX-D, the review by Cicognani et al. states “The reason for this wide range of variability of the prevalence of SHOX-D in idiopathic short stature (ISS) is debated. Different factors may play an important role such as the various sizes of the samples, the different selection criteria, the ethnicity of the population, and the different methods used for the genetic analysis”. We added this sentence in the discussion: “This wide range could be due to different sample size of included studies, patient’s selection criteria, ethnicity, and different methods used for the genetic analysis”.6. Page 20, 1st paragraph: Include the various definitions of non-adherence in the text.Response to Reviewer #2: As suggested, we have described the non-adherence definitions for each study in the main text.7. Page 22, 2nd paragraph: Include the reasons for discontinuation in the paper by Marciano. Was this by choice or due to prescription/supply issues?Response to Reviewer #2: This study calculated the persistence to GH treatment based on administrative databases of drug prescriptions; therefore, the reasons for discontinuation can only be speculated. In the Discussion, Authors provided possible reasons for discontinuations. We added in the text the following description: “Most of those who discontinued were intermittent users (39.3%), that is, they restarted GH therapy after at least 60 days of interruption; in their paper, Marcianò et al. [23] have speculated that treatment discontinuation could be due to patients’ reduced compliance or lack of perceived benefit from GH therapy. Another possible reason for discontinuing GH therapy may be the occurrence of adverse reactions; in fact, more than 25% of patients who discontinued therapy had a new diagnosis of diabetes or cancer or has been hospitalized at least once after discontinuation. Another reason for discontinuation could be reaching final height (7.9% discontinued at age 15–17). The remainder of the subjects who discontinued therapy (27.7%) were thought to be probably related to the lack or loss of GH efficacy or to patients' lack of compliance”.8. Discussion: Explain the differences between international and Italian estimates of GHD and PWS prevalence.Response to Reviewer #2: About GHD, we added in the text the following sentence, according to the response to the comment n. 4b: “It should be noted that the GHD prevalence estimated by the Piedmont GH registry is higher than the prevalence reported in the literature; however, it should be considered as an indirect estimate which may differ from the real prevalence in the general population. The authors of the original study from which this estimate was reported [43], in the discussion state that their prevalence estimates are higher of those reported in previous studies, probably due to different patient selection criteria, the cut-off used for tests allowing the diagnosis of partial or complete GHD, and the improvements of diagnostic techniques (higher sensitivity)”.Regarding PWS, the international estimates are only apparently diverse from those from Italy. In fact, the PWS prevalence of 1/15,000  reported in the literature is a birth prevalence, while the reported annual incidence in Italy is referred to the entire Italian population. We added the following text in the Discussion in order to clarify this concept: “Regarding PWS, the international estimates are only apparently different from the Italian ones. In fact, the PWS prevalence of 1/15,000  reported in the literature is a birth prevalence, while the reported annual incidence in Italy refers to the entire Italian population (which was on average 60.2 million per year in the period 2012-2014). Estimating the birth prevalence from the annual incidence on the total population, it would be: 37 new PWS cases/year  out of 527,845 live births , that is 7 per 100,000, exactly the same birth prevalence reported in the literature”.9. Page 31: the quality of life assessment in patients with PWS does not seem to be specifically regarding GH treatment. Please clarify.Response to Reviewer #2: Even if the two papers on PWS have the main focus on the disease rather than on the GH treatment, we assumed that most of included patients were treated. We added the following text in the quality of life paragraph: “The two studies by Lopez-Bastida [21] and Ragusa [27] focus mainly on the disease (PWS) rather than on the GH treatment. We decided to include them in our analysis because we were interested in analyzing the quality of life in patients with PWS, which is one of the diseases for which GH treatment is approved in Italy, and we assumed that most patients were treated. In the Lopez-Bastida paper [21], information on medications used by PWS patients was obtained from questionnaires and their costs calculated. In the Ragusa study [27] there were references to GH treatment in interviews with various patients and caregivers”.Minor comments:1. Page 34, last paragraph: Expand “CAD.”Response to Reviewer #2: Thank you, we expanded it (Canadian Dollar).6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 10 Feb 2022Pediatric growth hormone treatment in Italy: a systematic review of epidemiology, quality of life, treatment adherence, and economic impactPONE-D-21-34417R1Dear Dr. Orso,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Mohamed A Yassin, MDAcademic EditorPLOS ONEAdditional Editor Comments : the manuscript can be accepted in its current formsReviewers' comments: 15 Feb 2022PONE-D-21-34417R1 Pediatric growth hormone treatment in Italy: a systematic review of epidemiology, quality of life, treatment adherence, and economic impact Dear Dr. Orso:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Tizanidine, an alpha-adrenergic substance with antinociceptive and antihypertensive effects, is extensively metabolized via cytochrome P450 (CYP) 1A2. Therefore, coadministration with potent CYP1A2 inhibitors, such as ciprofloxacin, is contraindicated. However, both drugs are broadly utilized in various countries. Their concomitant use bears an inherent high risk for clinically significant symptoms, especially in multimorbid patients experiencing polypharmacy. This study aims to investigate the impact of coadministration of tizanidine and ciprofloxacin using real-world pharmacovigilance data and to raise awareness of this potentially underestimated safety issue.We conducted a retrospective study including Individual Case Safety Reports (ICSR) registered until March 1, 2017, in the World Health Organization (WHO) global database. Demographic data, drug administration information, the course of the adverse drug reaction (ADR), its severity, and outcomes were analyzed for cases reporting ciprofloxacin comedication.n = 59, 64.8%) with a median age of 54 years (range 13–85 years). The countries contributing most reports were the USA  and Switzerland . ADRs reported most often affected the nervous system and the cardiac function, especially with large tizanidine doses or drugs with CNS and cardiovascular depressant effects. In two cases, a fatal outcome was reported.In 91 (2.0%) of the identified 4192 worldwide ICSR on tizanidine, coadministration of ciprofloxacin was reported. Most of the patients were female (Despite the existing formal contraindication, the concomitant use of tizanidine and ciprofloxacin can be observed in real-world clinical practice. Reactions mainly affected the central nervous and the cardiovascular system resulting in potentially severe adverse effects. The concomitant use of tizanidine and ciprofloxacin should absolutely be avoided. Tizanidine is an alpha-adrenergic substance reducing spasticity , 2. It aTizanidine has a low oral bioavailability with a high first-pass effect, considerable interindividual variability, and a narrow therapeutic range , 4. It iIt was shown that ciprofloxacin increases the peak plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC) of tizanidine 6–24-fold and 4–21-fold, respectively, resulting in clinical toxicity of tizanidine , 6. IncrBased on clinical observations, we hypothesized that despite of the formal contraindication, concomitant administration could occur, due to overlapping therapeutic indication. However, no international analysis of real-world data on concomitant use and on severity of this drug-drug interaction is available. Therefore, we conducted a retrospective analysis of the World Health Organization (WHO) global database for Individual Case Safety Reports (ICSR) using a case-non-case approach to analyze this potentially underestimated drug safety issue.TM. Since 1968, VigiBaseTM, the WHO global database of ICSR, collects, processes, and homogenizes worldwide ICSR [TM includes mainly post-authorization unsolicited or spontaneous reports [TM include details on the demographic characteristics of the patient , reporting country, reporter qualification , medications , and the reported suspected drug reactions. The suspected ADRs are coded using the Medical Dictionary for Regulatory Activities , and all medications are coded using WHODrug. The MedDRA® terminology follows a hierarchical structure comprising terms in five ascending orders with higher orders including more generic terms. Drug-drug interactions are included in the MedDRA® terminology as low level (LLT) and preferred terms (PT) [TM.In this retrospective descriptive study, we searched for ICSR on suspected ADR in the WHO global database VigiBaseide ICSR , 11. Therms (PT) . In caseTM until March 1, 2017. On March 1, 2017, it contained more than 14.6 million ICSR from 126 participating countries. We received the coded data elements of all international tizanidine-associated ICSR from the WHO global database VigiBaseTM in the form of a Microsoft Excel file. ICSR of interest contained tizanidine (ATC: M03BX02) as a suspected or interacting drug. Combination therapy ICSR were extracted. Number of events was defined as number of unique ICSR, as identified by the report ID. All reported terms and corresponding MedDRA terms were included in the analysis as multiple ADRs can be reported in a single ICSR.We included all ICSR of tizanidine reported to VigiBaseIn a first step, we screened the data for ciprofloxacin as comedication. That data was then analyzed for demographic data (age at ADR onset and gender), completeness, reported ADRs, clinical outcome, concomitant drugs, seriousness, and seriousness criteria. Two subgroups were categorized by the daily dose of tizanidine with less than 12 mg and 12 mg or more per day, respectively. Other reported comedications were analyzed for CYP interactions and classified based on their effects as central nervous system or cardiovascular depressants according to the labelling in the respective summary of product characteristics (SmPC).For descriptive analyses, we used Microsoft Office Excel (2010), IBM SPSS Statistics (version 23), and STATA  for Windows software. Depending on the distribution of the values, we used the arithmetic mean with standard deviations or the median with ranges.Worldwide, 4192 ICSR on tizanidine were identified in the WHO database in the study period. After selection of cases reporting concomitant tizanidine and ciprofloxacin administration, 91 (2.2%) international ICSR reporting in total 444 ADRs remained for analysis  and Switzerland . The demographic characteristics of the investigated population are summarized in Table n = 22, 24.2%).In 59 cases (64.8%), the patients were female, and 28 patients (30.8%) were male (in 4 ICSR (4.4%), gender was not reported). Age was reported for 65 patients resulting in a median age of 54 years (range 13–85 years). The countries contributing most reports were the USA . Of those, most ICSR  were qualified as serious. The most frequent reason for severity was “caused/prolonged hospitalization” . Two ICSR reported a fatal outcome.A severity assessment was performed for most cases , “drug interaction” , “asthenia” , and “urinary tract infection” . All coded MedDRA terms with a frequency of ≥ 4 are displayed in Table Among the 444 reported ADRs, the most frequently observed reactions were “hypotension” n = 5, 5.6%, n = 18, 19.8%) followed by oxycodone  and fampridine and gabapentin . Among the most frequently reported comedications, no other CYP1A2 inhibitor besides ciprofloxacin was identified. In 25 ICSR (27.5%), patients received only tizanidine and ciprofloxacin without further comedication. In 66 ICSR (72.5%), further drugs were coded. In 66 ICSR (72.5%), tizanidine was reported as suspected and in 25 ICSR (27.5%) as interacting drug. Ciprofloxacin was reported 48 times (52.7%) as suspected, 21 times (23.1%) as interacting, and 22 times (24.2%) as concomitant drug. The most frequently used nine coadministered drugs  were furthermore classified as antihypertensive and/or CNS depressive acting agents. Except for fampridine, all these nine drugs could cause antihypertensive ADRs, and except for paracetamol, all of them could cause CNS depressive ADRs.The comedication was found to be very heterogenic. Among the 91 ICSR, 265 different drugs were used as comedications. The median number of administered drugs was five Table . The mosThe daily dose of tizanidine in milligram was reported in 46 ICSR (50.5%). The median administered dose was 8 mg (range 1–48 mg). Regarding the dose-dependent character of the interaction, two subgroups with available dosage information in milligram were analyzed: 26 patients received a tizanidine dose of less than 12 mg/day and 20 patients received 12 mg or more of tizanidine daily. Patient characteristics were similar in both groups . Proportions of serious ADRs were similar in both groups (55.0% vs 53.8%). In addition, the number of comedications was comparable in the subgroups (5 vs 6 concomitant drugs).The daily dose of ciprofloxacin was reported in 37 ICSR (40.7%). The median daily dose patients were receiving was 1000 mg (500–1500 mg).Of the identified 91 ICSR, 68 (74.7%) reported both tizanidine and ciprofloxacin as either suspected or interacting drugs. In only 23 ICSR (20.9%), the interaction between tizanidine and ciprofloxacin was coded as an ADR. When comparing the 68 ICSR to all 91 ICSR, the proportion of reporters’ qualification was found to be similar.For two patients receiving the combination ciprofloxacin and tizanidine, a fatal outcome was reported. The first was an elderly male patient of 71 years who received tizanidine in a dosage regimen of 24 mg together with ciprofloxacin for treatment of a urinary infection. He developed muscle weakness and a decreased muscle tone, hypotension, and organ failure that eventually led to his death. The only concomitant coded drug was clonazepam—exhibiting CNS depressive effects. The second patient was a 49-year-old male with a medical history of gastrointestinal carcinoma, HIV disease, chronic viral hepatitis, and alcohol and tobacco use. He had received 2 mg tizanidine daily together with 500 mg ciprofloxacin twice daily for the treatment of a bacterial pneumonia. The number of concomitant drugs was extensive and contained morphine, zolpidem, oxazepam, clindamycin, prednisone, thiamine, levothyroxine, gabapentin, sodium picosulfate, acetylcysteine, esomeprazole, and a vitamin B supplement. Notably, morphine, zolpidem, oxazepam, and tizanidine were reported as interacting, whereas ciprofloxacin was coded as concomitant drug. The ADR that led to his death was respiratory distress.The concomitant use of tizanidine with CYP1A2 inhibitors, such as ciprofloxacin, is formally contraindicated . NeverthAnother pharmacokinetic aspect is not related to drug dosage but to patients’ physiologic properties. Tizanidine undergoes an extensive hepatic metabolism. According to the product information, the therapy of patients with decreased liver function therefore is contraindicated. The results of a retrospective survey and case report  and a noDrugs with similar ADR profiles administered together may cause synergistically amplified reactions and higher numbers of concomitant drugs may increase the risk to develop toxicity. Common ADRs of tizanidine include antihypertensive and CNS depressive effects , 9, 20. Most reports analyzed in the current study originated from the USA and Switzerland. Different from the other reporting countries, only the US and Swiss reports included reports from consumers and non-healthcare professionals. Compared with other countries, the USA has the pharmacovigilance system in which patients are the most involved already since 1969 . This miNot only because of the pharmacodynamic aspects of the possible ADRs but also due to the relatively narrow therapeutic range of tizanidine , 20, theAs previously shown, the concomitant use of ciprofloxacin with tizanidine is frequently observed in the outpatient setting . ConsideTM, the WHO’s pharmacovigilance database, collects spontaneous reports on suspected ADRs of authorized drugs from countries all around the world with the aim to increase patient’s safety. Data are collected as ICSR representing mainly post-authorization unsolicited reports [Our study’s most important strength is the comprehensive dataset we used for the analysis. VigiBase reports . TherefoTM contain neither narrative nor clinical or laboratory data. Therefore, this data do not allow for a formal assessment of causal drug-ADR relationship, but reports are based on the suspicion of the reporter. Since ICSR can only be extracted from the database, a verification of coding or modifications, such as recoding, is not possible. Completeness of documentation is highly dependent on individual reporter. Due to this, the ICSR are heterogeneous regarding completeness. Due to potentially missing timing and duration of use of comedication, simultaneous administration could be misclassified. Given the small number of the coded term “drug interaction,” it can be hypothesized that the contraindicated combination could have remained unrecognized as such. On the one hand, due to missing data on exact timing of administration, it cannot be ruled out that the drug use did not result in a major CYP interaction based on the timing. This might lead to an overestimation of the interaction problem of tizanidine and ciprofloxacin. On the other hand, an unrecognized interaction might lead to reporting only of the suspected drug, without reporting of comedication, leading to an underestimation of the issue. Overall, incidence rates cannot be provided solely based on pharmacovigilance data. Further observational studies are needed to clarify the prevalence of this contraindicated drug combination and potential country-specific prescribing. This might lead to a bias, which cannot be accounted for due to missing data. Another, commonly described, limitation in pharmacovigilance is the underreporting of ADRs in a spontaneous reporting system, which amounts to up to 94% [However, the main limitation of this study is the data characteristics of the retrieved ICSR. The ICSR comprised in the register database VigiBasep to 94% . FrequenConditions usually treated with tizanidine can be associated with accompanying symptoms resulting in ciprofloxacin treatment. Results from this analysis of real-world pharmacovigilance data show that despite the existing formal contraindication, both drugs are concomitantly prescribed in clinical practice.Most reported adverse effects, such as hypotension, somnolence, fatigue, and asthenia, are related to the central nervous and the cardiovascular system. Concomitant use of cardio- or CNS-depressant drugs might aggravate these effects. The combination of ciprofloxacin with tizanidine should be absolutely avoided.


SMARCA4 gene are supposed to play the major role in SCCOHT oncogenesis and their identification is crucial for the diagnosis. Adequate genetic counselling of the patients and their families seems to be of great importance. Optimal management and treatment approaches are not known yet but may extremely influence the prognosis of young female patients that suffer from this very resistant disease. Nowadays, a translational research seems to be the key for the further diagnostic and treatment strategies of SCCOHT. The purpose of the case report is to provide practical information and useful recommendations on the diagnosis, management, and treatment of SMARCA4-deficient carcinoma of the uterine cervix resembling SCCOHT.Small cell carcinoma of hypercalcemic type (SCCOHT) is a rare gynaecological neoplasm, originating mostly in the ovaries. Cervical origin of this very aggressive malignancy with unknown histogenesis is an extremely rare condition, without published management recommendations. Alterations in  Small cell carcinoma of the ovary (SCCO) is a very lethal malignancy. It contains two separate subtypes: the pulmonary type (SCCOPT), and hypercalcemic type (SCCOHT) .SCCOHT is one of the most frequent malignant undifferentiated ovarian tumors in women younger than 40 years. It is a rare and highly lethal tumor that typically affects young women. Many women are diagnosed at early stage. However, the aggressive behaviour of SCCOHT leads to a significantly bad prognosis, while most women die within 1 year after the diagnosis is established. The prognosis is bad even for very early stage SCCOHT.SCCOHT was first described in 1982 in a series of 11 patients with concurrent SCCO and hypercalcemia .Familial SCCOHT cases may be more often bilateral ,5. HyperA NCDB study  showed a high frequency of elevated CA125 (84% of cases) . Serum CSCCOHT is well described in the miscellaneous ovarian tumors chapter in the recent WHO Classification of Female Genital Tumours (2020). Grossly, the tumor is usually unilateral, large, solid, and tan to white to grey. Foci of haemorrhage, cystic degeneration, and necrosis are common. Microscopically, the tumor is composed of monomorphic cells, arranged mostly in solid and, less commonly in follicular, trabecular, or nested growth pattern. The follicle-like spaces may contain eosinophilic or basophilic secretions. In most cases, the predominant tumor population consists of cells with round, ovoid, or occasionally spindled hyperchromatic nuclei with coarsely clumped chromatin and small nucleoli and with scant cytoplasm . Mitotic activity is typically brisk and proliferation index Ki67 is regularly high. In about half of the tumors, larger cells with eccentric vesicular nuclei with prominent nucleoli and with abundant eosinophilic cytoplasm  are present. If significantly predominant, a “large cell variant” of the tumor may be considered. In up to 15% cases, a minor mucinous component either in the form of benign glands or cysts, or rarely as malignant signet-ring cells is present. The stroma of the tumor is usually minimal, it may be, however, myxoid or edematous in occasional cases . ImmunohSMARCA4 gene, was reported in more than 90–95% of cases and is currently considered an important diagnostic finding. However, not all tumors are negative as occasional neoplasms show loss of both SMARCB1 and SMARCA2 with retained SMARCA4 expression. Dual loss of SMARCA4 and SMARCA2 is not uncommon in these tumors, the latter occurring due to epigenetic inactivation rather than mutation [Recently, loss of SMARCA4 (a.k.a. BRG-1) expression, due to germline or somatic mutation of mutation .Differential diagnosis depends on the proper localization of the tumor and includes, in principle, any poorly differentiated malignant tumor. Regarding malignancies of the uterine cervix, particularly poorly differentiated adenocarcinoma, adenosquamous or squamous cell carcinoma, malignant melanoma, small cell neuroendocrine carcinoma, SMARCA4-deficient undifferentiated uterine sarcoma, dedifferentiated endometrial carcinoma, non-Hodgkin malignant lymphomas, high-grade leiomyosarcoma and rhabdomyosarcoma, Ewing sarcoma, and germ cell tumors  must be considered. As regards more common ovarian localization, particularly the juvenile variant of granulosa cell tumor needs to be distinguished , as wellBRCA1/2 germline or somatic mutations play the major role in development of high-grade serous adenocarcinoma, the most common malignancy of the ovary [BRCA1/2 mutations. Similarly, classic oncogene mutations  have not been identified in SCCOHT yet. On the other hand, SCCOHT is distinctively characterized by SMARCA4 mutations (germline or somatic), or mutations in other subunits of the SWI/SNF complex [SMARCA4 gene, that is involved in the SWI/SNF complex [As regards ovarian carcinoma, e.g., he ovary . However complex ,17. The  complex –18.SCCOHT has typically a low mutation load with low PD-L1 (Programmed Death Ligand-1) expression, which makes this cancer less suitable for immune checkpoint blockade treatment. However, the response of SCCOHT to modern immunotherapy has to be studied, with some positive response already reported .Next generation sequencing (NGS) technologies as Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) can distinguish driver and passenger mutations or non-pathogenic normal gene polymorphisms in ovarian carcinomas .SMARCA4 mutations have been studied to reveal the presence of different types of gene alterations [SMARCA4 specific mutations consisted of frameshift (36.4%), stop/nonsense (32.2%), splice-site (20.3%), missense (5.9%) and in-frame deletions (5.1%), with no concrete mutational hotspot defined.More than a hundred of pathogenic erations . These SIn all mentioned studies, immunohistochemical SMARCA4 (a.k.a. BRG1) protein expression was lost in 82–95% of SMARCA4 altered tumors, confirming inactivating mutations of SMARCA4 gene with defective protein production. For comparison, only 0.4% of other primary ovarian tumors have negative SMARCA4 protein expression.Other SCCOHT tumors with intact SMARCA4 protein expression may carry mutations in other SWI/SNF complex subunits, e.g., inactivating SMARCB1 , or ARID1A mutations . HoweverTo maintain the chromatin remodeling activity of the SWI/SNF complex in human, three core subunits  and two helicases/ATPases (BRM/SMARCA2 and BRG1/SMARCA4) are essential –25. To cARID1A, PBRM1, SMARCB1, SMARCA4, and ARID2 genes) are frequently mutated in human cancers. SWI/SNF complex was found to be mutated in circa 20% of human malignancies according to a meta-analysis [The mammalian SWI/SNF complex functions as a tumor suppressor, and the subunits of the mammalian complex  is applied in most SCCOHT patients, with no clearly proven benefit . Howeverin vitro studies showed SCCOHT cell lines quite resistant to platinum chemotherapeutic drugs. On the contrary, epothilone demonstrated a strong anti-proliferation effect in vitro and in xenografts in vivo [On the other hand, some  in vivo ,35.Multi-modality intensive treatment approaches combining surgery, high-dose multiagent chemotherapy , and postoperative radiotherapy may be an adequate treatment option for most SCCOHT patients ,37. SurvSeveral agents such as bortezomib, pazopanib, or PARP inhibitors were described as possible treatment options for SCCOHT . Immunotin vitro and in vivo [in vitro, therefore CDK4/6 inhibitors should be also studied in this indication [SCCOHT displays notable genomic stability and does not seem to acquire additional mutations after exposure to chemotherapy . Combine in vivo –47. At tdication .The available literature about radiotherapy in SCCOHT consists of smaller case series and single case reports. Some series suggest that postoperative radiotherapy may play an important role in the primary treatment of SCCOHT ,33. Addide facto resembling the uterus  of the pelvis revealed a tumor measuring 80 × 90 × 80 mm, growing from the cervical region, e uterus , fillingDuring explorative laparotomy, the removal of lymphadenopathy with histologically confirmed metastases of hypercalcemic small cell carcinoma was successful. However, radical hysterectomy without residual disease was impossible, unless the patient underwent anterior pelvic exenteration. The patient was immediately indicated for chemotherapy via our multidisciplinary team. In the absence of any treatment recommendations, first-line cisplatin plus etoposide combined chemotherapy was selected, with regular pelvic MRI before each subsequent cycle to monitor the treatment response. After 2 cycles of chemotherapy, progression of the disease in the uterus was found, with infiltration of bilateral parametria, the bladder wall and anterior rectal wall infiltration. Distant dissemination on whole-body CT was not clearly demonstrated, except for non-specific small lesions of both lungs up to 5 mm.Due to gynaecological bleeding and micturition problems, the patient underwent extended-field pelvic radiotherapy with retroperitoneal lymph-nodes up to 45 Gy in 25 fractions, with a subsequent boost to the tumor, uterus and vagina up to a total dose of 59.4 Gy in 33 fractions. The pattern of tumor growth outside the uterus was not suitable for intracavitary brachytherapy. The patient managed radiotherapy without complications with clinical relief from both bleeding and urinary symptoms. The partial regression of both the tumor and lymphadenopathy was confirmed by MRI .Three months after radiotherapy, 7 months from the initial diagnosis, the patient was admitted to the hospital for general deterioration, breathlessness, fever, loss of appetite, and pain. Immediate CT of the head ruled out brain metastases. Although partial regression of the pelvic tumor was confirmed on the whole-body CT scan, there was massive systemic progression of the disease with mediastinal and neck lymphadenopathy, multiple metastatic lesions of the lungs and pleura with bilateral pleural effusions, multiple liver metastases (largest 52 × 36 mm), omental involvement with soft tissue masses (largest 72 × 56 mm), and ascites . The patIn the present case, the diagnosis of hypercalcemic small cell carcinoma .Microscopically, the tumor was composed entirely of large cells with vesicular, less commonly hyperchromatic nuclei, frequently with prominent nucleoli, and with abundant eosinophilic cytoplasm, sometimes imparting a rhabdoid appearance . Small cImmunohistochemically, the tumor cells showed diffuse or nearly diffuse expression of broad-spectrum cytokeratins (CK), CK18, PTEN, β-catenin (membranous), vimentin, and GATA3. Expression of MLH1, PMS2, MSH2, and MSH6 was retained in tumor cell nuclei, indicating the absence of microsatellite instability (MSI). There was focal expression of p16. Isolated tumor cells showed expression of PAX8, CD10, calretinin, and synaptophysin. Detection of CK5/6, CK7, CK19, CK20, EMA, p63, p40, ER, PR, CEA, chromogranin, CD56, WT1, SALL4, Oct3/4, CD30, glypican-3, inhibin, SOX10, melan A, HMB-45, and LCA gave negative results. The expression of PD-L1 was negative in the tumor. The most diagnostically beneficial finding was the complete loss of SMARCA4 (a.k.a. BRG-1) expression . ExpressHPV DNA detection and genotypization was performed by qualitative real-time PCR with the AmoyDx Human Papillomavirus Genotyping Detection Kit . In a DNA extract of the tumor tissue, no HPV DNA was detected.Both germline and somatic WES of the patient and the tumor was performed. Library for whole exome capture and sequencing was prepared from DNA extracted from patient´s blood and tumor using TruSeq Exome Kit according to the manufacturer´s instructions. Prepared library was loaded onto NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) and sequenced on the NextSeq 500 instrument . Sequencing coverage for exomes was >20× at > 90% of captured regions.The mutation burden of the tumor was low (4 mutations/Mb). Examination of both the germline and somatic exome revealed the following selected variants :ARID1A (wild-type), SMARCA2 (wild-type), SMARCB1 (common variables in non-coding sequencies), and others.There were no other variants of the genes encoding SWI/SNF complex found, including PALB2, BRCA2, SMARCA4), genetic testing was recommended also to the patient’s family.As several germline likely pathogenic or pathogenic gene variants were detected by WES . Mother’s familial history was also negative for malignancy.A heterozygous c.3976G>T/p.E1326* variant in the PALB2 gene. Father’s father (a heavy smoker) died of lung cancer at the age of 51; the father’s mother died of an unknown malignancy at the age of 64 years; and the father’s brother died of pancreatic cancer at the age of 38 years.The patient’s father  showed a heterozygous c.509_510delGA/p.R170fs variant in the PALB2 variant inherited from the father.The patient’s brother  showed a familial heterozygous Here we report the very first published case of SMARCA4-deficient carcinoma of the uterine cervix resembling SCCOHT and our challenging diagnostic process and dismal treatment experience. The histological picture of the disease, as well as the severity of its behavior and insufficient response to treatment does not seem to differ from the experience we have with the ovarian type of the disease. It was an aggressive tumor that affected a very young woman, with an advanced stage at diagnosis, with a rapid progression and a typical severe course of the disease, with early dissemination to the liver, peritoneum, and lungs, which resulted in death within 8 months from the first symptoms.The main uncertainity of this case study is whether the uterine cervix was the place of origin of such a rare histology. Uterine cervix is rarely the site of metastatic spread of other malignancies, but this cannot be excluded. On the other hand, vaginal metastases constitute the majority of vaginal malignancies, which mainly originate from the cervix, endometrium, or ovary, among other locations. Since the status of ovary/ovaries has never been examined histologically in our patient, we cannot exclude primary ovarian SCCOHT with cervical manifestation. There was no tumor mass present in the patient on ultrasound, CT, or MR imaging. Theoretically, the ovarian lesion can be subtle and may not be picked up by imaging. However, this possibility seems highly unlikely.The patient was a virgin at diagnosis, excluding HPV associated tumorigenesis. Moreover, the detection of HPV DNA was negative in tumor tissue. Clinical manifestation was otherwise not atypical, instead of the primary tumor growing more around the uterus, than infiltrating it. It is crucial to distinguish SCCOHT of the uterine cervix from the much more common squamous cell carcinomas or adenocarcinomas, that is possible only after careful histopathological examination. The most diagnostic is the loss of SMARCA4 (rarely SMARCB2 or ARID1A) expression immunohistochemically.However, the microscopic differential diagnosis is very broad and includes any poorly differentiated/high-grade malignant tumor of both the uterine cervix and the uterine corpus. Absence of HPV DNA excludes the diagnosis of both HPV-associated squamous cell carcinoma and HPV-associated adenocarcinoma. Moreover, markers of squamous differentiation, i.e., CK5/6, p63, and p40, were completely negative and the absence of CK7 and CEA expression would be unusual in cervical HPV-associated adenocarcinoma. Morphology of the tumor excludes the diagnosis of any of HPV-independent cervical adenocarcinomas, i.e., of gastric type, of clear cell type, and of mesonephric type. Large cell neuroendocrine carcinoma shows significant expression of CD56, chromogranin, and synaptophysin. In the present tumor, the expression of CD56 and chromogranin was completely negative, and synaptophysin was detected only in isolated tumor cells, making the diagnosis of large cell neuroendocrine carcinoma unlikely. Germ cell tumors should be also considered. However, the expression of SALL4 as well as of other markers  was completely negative, arguing against this diagnosis. Absence of expression of LCA and of “melanoma” markers  excludes the diagnosis of hematologic malignancy and malignant melanoma, respectively. As regards the uterine corpus, particularly dedifferentiated/undifferentiated endometrial carcinoma and recently described SMARCA4-deficient uterine sarcoma enter the differential diagnosis –53. DediSMARCA4 c.3976G>T/p.E1326* variant in a heterozygous form. This variant was inherited from the mother. It was present in the tumor, also in a heterozygous form. Other germline polymorphisms detected in the SMARCA4 gene were also heterozygous in the tumor. For this reason, the loss of heterozygosity in the tumor, e.g., due to deletion of the second unmutated SMARCA4 alelle, cannot be confirmed only considering WES data. However, a complete loss of SMARCA4 protein expression was confirmed immunohistochemically. Therefore, another processes that led to the loss of function of the second unmutated allele of the SMARCA4 gene cannot be excluded .The overall mutation load of the tumor was typically low, as expected in SCCOHT. The most probable pathogenic variant associated with SCCOHT that was found in the patient was only the germline SMARCA4 and BRCA2 genes from her mother and at the same time a heterozygous mutation in the PALB2 gene from her father. This very unlikely mutational load with an autosomal dominant inheritance might probably cause, by its nature, such a rare malignancy at such a young age. SCCOHT is described as a monogenic disease, the most probable cause of an extremely rare histological type of the tumor in the presented case, was the inherited likely pathogenic SMARCA4 variant, with the loss of SMARCA4 protein function and expression. However, overall DNA repair instability, caused by BRCA2 and PALB2 variants could play a role in much more difficult “game of genes.”The patient inherited a heterozygous mutation in the SMARCA4 imply familial SCCOHT with a requirement of genetic counselling, that should be recommended for all family members. However, there is no clinical management consensus on SMARCA4 mutation carriers. Patients carrying germline mutations are usually younger  than noncarriers. In patients younger than 18 years old, the absence of germline mutation is unlikely. Considering the rapidly progressive tumor growth and high mortality of SCCOHT, germline mutation carriers should be counselled about prophylactic bilateral oophorectomy. However, the early onset of SCCOHT  indicates that prophylactic surgery should be performed at a younger age than in hereditary BRCA 1/2 variant carriers. Therefore, prophylactic bilateral oophorectomy may not be acceptable for young SMARCA4 variant carriers. An improvement on in vitro fertilization may facilitate prophylactic surgery in these carriers. Cervical/uterine SCCOHT is such a rare condition that prophylactic hysterectomy in young SMARCA4 variant carriers, in our opinion, would not be justified. Our recommendation is to consult SMARCA4 variant carriers about the risks considerately and omit hysterectomy in patients contemplating in vitro fertilization. For in vitro fertilization, pre-implantation genetic diagnosis should be recommended to prevent genetic transmission of the deleterious mutations.Germline variants in Effective treatment options for SCCOHT are lacking. The rarity of SCCOHT limits the design of randomized clinical trials. Available evidence suggests the benefit of multi-modality treatment with radical surgery, high-dose multiagent chemotherapy with eventual stem cell transplantation. Postoperative radiotherapy may be advocated, considering retrospective case series and otherwise high rates of tumor recurrence and progression. However, clear evidence for the standard use of adjuvant irradiation is lacking and when used, we advise for the careful implementation of IMRT techniques sparing as much as healthy tissue as possible. Palliative directed radiotherapy may provide some treatment response with no proven curative potential. In our patient, a dose of 59.4 Gy in 33 fractions lead to a partial, incomplete response. Therefore, we strictly do not recommend definitive radiotherapy of SMARCA4-deficient carcinoma of the uterine cervix resembling SCCOHT with curative potential, as otherwise standardized in the treatment of advanced squamous cell carcinoma or adenocarcinoma.Overall response of SCCOHT to standard chemotherapy regimens is poor, with rapid progression of the disease during or shortly after chemotherapy. In our patient, there was an early progression of the disease during platinum-based chemotherapy. Combined cisplatin/etoposide chemotherapy is a standard of care in lung small cell carcinoma. However, due to our experience, we do not recommend combined cisplatin plus etoposide as an effective treatment of cervical SCCOHT.