Radiotherapy (RT) is an effective treatment for dogs presented with neurologic signs caused by pituitary tumors. However, its impact on the outcome of concurrent pituitary‐dependent hypercortisolism (PDH) is controversial.Determine whether dogs with PDH have longer survival after pituitary RT compared with dogs with nonhormonally active pituitary masses and to evaluate whether clinical, imaging, and RT variables affect survival.Ninety‐four dogs divided into 2 groups: PDH and non‐PDH, based on the presence of hypercortisolism. Forty‐seven dogs were allocated to the PDH group and 47 to the non‐PDH group.Retrospective cohort study in which clinical records of dogs undergoing RT for pituitary macroadenomas between 2008 and 2018 at 5 referral centers were retrospectively evaluated.P = .4). A definitive RT protocol was statistically associated with longer survival compared with a palliative protocol . The only factor statistically associated with survival from multivariate Cox proportional hazard analysis was total radiation dose (Gy) delivered (P < .01).Survival was not statistically different between PDH and non‐PDH groups (median survival time [MST], 590 days; 95% confidence interval [CI], 0‐830 days and 738 days; 95% CI, 373‐1103 days, respectively; No statistical difference in survival was identified between the PDH and non‐PDH groups, and longer survival was associated with higher Gy delivered. ACTHadrenocorticotropic hormoneACTHstadrenocorticotropic hormone stimulation testCTVclinical target volumeCTcomputed tomographyDIdiabetes insipidusDMdiabetes mellituseACTHendogenous ACTHGyGraysGTVgross tumor target volumeHAChypercortisolismKCSkeratoconjunctivitis siccaLDDSTlow‐dose dexamethasone suppression testMSTmedian survival timeMRImagnetic resonance imagingPDHpituitary‐dependent hypercortisolismPBRpituitary‐to‐brain ratioPTVplanning target volumePUPDpolyuria and polydipsiaRTradiotherapyUCCRurine cortisol‐to‐creatinine ratio1Pituitary tumors are considered uncommon in dogs.For functional pituitary tumors, pituitary‐dependent hypercortisolism (PDH) is the most common endocrine disease, resulting from hypersecretion of adrenocorticotropic hormone (ACTH).Neurological abnormalities in dogs with pituitary tumors have been associated with tumor dimension, but neurological signs have been described even when tumors are <10 mm in height.Regardless of whether pituitary tumors are functional or not, ~50% of dogs develop neurological signs, reflecting altered thalamic, hypothalamic, and optic chiasm function. Clinical signs range from altered mentation and behavior to more complex neurologic abnormalities leading to blindness, ataxia, head tilt, cervical hyperesthesia, cranial nerve deficits, seizures, or a combination of these.Adenoma resection is the treatment of choice for PDH in humansPituitary radiotherapy (RT) is an alternative strategy to manage neurological signs caused by large pituitary tumors in dogs.Definitive and palliative RT protocols previously have been associated with different outcomes in dogs with pituitary macroadenoma,Our primary aim was to determine whether the presence of PDH affected survival in dogs undergoing pituitary RT. We hypothesized that dogs with pituitary tumors and PDH would have improved survival because the clinical signs of HAC would lead to earlier detection. A second aim was to evaluate clinical, RT, and imaging factors that might affect the outcome.22.1In this multicenter, retrospective, cohort, observational study, we recruited dogs presented for investigation of neurologic or endocrine signs, or both, that underwent RT for pituitary macroadenoma at 5 referral centers between January 2008 and January 2018. Pituitary macroadenoma was defined as a tumor localized in the pituitary region with suprasellar extension on brain imaging. No specific numerical pituitary height, volume, or pituitary‐to‐brain ratio (PBR) cut‐off was used to define a pituitary tumor as macroadenoma.Data were obtained from available medical records, by contacting owners using a written questionnaire or both. Clinical presentation and endocrine test results were reviewed by a single veterinarian at each participating institution. Clinical data collected included signalment, clinical presentation, presence of co‐morbidities, medications administered at the time of diagnosis, and any endocrine test performed. Date and cause of death or euthanasia were recorded, or date of last follow‐up if date of death was not available.Dogs were divided into 2 groups, a PDH group and a non‐PDH group. Dogs were included in the PDH group if a diagnosis of PDH was made on the basis of appropriate clinical signs and compatible endocrine test results and diagnostic imaging findings. A positive urine cortisol‐to‐creatinine ratio (UCCR) alone was not considered sufficient for the diagnosis of HAC. Dogs were included in the non‐PDH group on the basis of absent signs of HAC or endocrine test results for HAC not compatible with excess cortisol production or both, together with a pituitary mass identified during diagnostic imaging. Endocrine testing to exclude HAC was not a requirement if clinical signs of hypercortisolemia were absent. Dogs were excluded if insufficient information was available to adequately classify dogs into the above groups, or if data regarding RT was incomplete.The study received ethical approval from the Animal Health Trust Ethical Committee in February 2019.2.23), presence of pituitary cysts, hemorrhage, lateralization of the lesion, presence of peritumoral edema, flattening of the basisphenoid, and presence of hydrocephalus. In each dog, the height and width of the pituitary were measured on the image that contained the largest cross‐section of the gland.All diagnostic imaging studies were reviewed by a single author (SM), using Horos v4.0.0 RC5. Where available, both pre‐treatment and post‐treatment studies were compared. All studies were evaluated using window parameters identified as ideal for pituitary assessment in post‐contrast computed tomography (CT) or magnetic resonance imaging (MRI) studies.Pituitary volume was calculated using the Horos v4.0.0 RC5 closed polygon tool: the region of interest (ROI) was marked on all transverse slices from the most rostral to the most caudal part of the pituitary mass and the ROI/ROI volume/compute volume tool calculated the volume of the lesion by summing the volumes of the ROIs of each slice.2.3Radiotherapy was prescribed to decrease tumor size, ameliorate neurologic signs associated with the tumor mass effect or both in all cases. The following RT variables were recorded: type of linear accelerator, planning software, RT protocol, frequency, presence of treatment interruptions, Gray (Gy) dose per fraction, total Gy dose, field number and angulation, Gy per field, gross tumor volume (GTV), clinical target volume (CTV), planning target volume (PTV), and occurrence of any acute or delayed adverse effects after RT. An RT protocol was defined as definitive when the treatment was delivered either 5 days/week (Monday to Friday) or on a Monday‐Wednesday‐Friday schedule, depending on clinician preference. An RT protocol was defined as palliative when the treatment was delivered once per week, typically for 4 or 5 doses in total. None of the included cases underwent stereotactic radiotherapy (SRT). The RT variables included in our study followed previously published guidelines,2.4χ2 tests were used to assess proportions in each group. The Kaplan‐Meier method with log rank tests was used to estimate the effect of individual clinical variables, imaging characteristics, and RT variables with survival defined as the difference between the first day of RT treatment and the date of death or last follow‐up. Dogs lost to follow‐up or that were alive at the time of statistical analysis were censored and time between first day of RT and last contact with a veterinarian was used for analysis. Logistic regression was used (Mantel‐Cox log‐rank test) to assess the association of endocrine status, clinical findings, RT, and imaging variables with survival. Variables with P < .2 in univariate Cox analysis were used for the multivariate analysis using a backward elimination approach.Statistical analysis was performed using Microsoft Excel 16.46, SPSS Version 28.0.1.1, and GraphPad Prism Version 9.4.1. for Mac. Normality was assessed using histograms and Shapiro‐Wilk tests. Fisher's exact or 33.1Ninety‐four dogs met the inclusion criteria, with 47 assigned to the PDH group and 47 to the non‐PDH group.Endocrine testing had been undertaken in 60/94 (63.8%) dogs (47/47 [100%] in the PDH and 13/47 [27.6%] in the non‐PDH group).In the PDH group, 36/47 dogs (76.6%) were diagnosed based on an adrenocorticotropic hormone (ACTH) stimulation test (ACTHst) only, 7/47 (14.9%) had a combination of ACTHst and low‐dose dexamethasone suppression test (LDDST), 3/47 (6.4%) had ACTHst and endogenous ACTH (eACTH) assay and 1/47 (2.1%) had ACTHst, LDDST, and eACTH.Within the non‐PDH group, 7/47 (14.9%) dogs had ACTHst assessment only, 3/47 (6.4%) had ACTHst and LDDST, 1/47 (2.1%) had LDDST only, 1/47 (2.1%) had eACTH only, 1/47 (2.1%) UCCR only, and 34/47 (72.4%) of non‐PDH dogs had no endocrine tests performed.P = .68), and median body weight being 16.75 kg in the PDH group and 19.2 kg in the non‐PDH group (P = .55). The proportion of females and males was 42.6% and 57.4% in both groups (P = 1.00).No significant differences in age or body weight at diagnosis were found between groups, with the mean age in both groups being 8.5 years (Crossbreed dogs were most commonly identified within the study population (n = 14), followed by Staffordshire Bull Terrier (n = 11), Greyhound (n = 8), Jack Russell Terrier (n = 8), Labrador Retriever (n = 8), Cocker Spaniel (n = 6), Boxer (n = 6), Cavalier King Charles Spaniel (n = 3), Bichon Frise (n = 3), Border Terrier (n = 3), French Bulldog (n = 3), West Highland White Terrier (n = 2), English Bull Terrier (n = 2), Lurcher (n = 2), and Golden Retriever (n = 2). Other breeds were represented by 1 case only. As previously reported, a high prevalence of terrier breeds  was observed.χ2 = 1.23; P = .25) nor in the proportion of dogs with other presenting signs.Presenting clinical signs are summarized in Table Lethargy was the second most common presenting sign after neurologic abnormalities and was reported in 25/94 dogs (26.6%), 13 in the PDH (27.6%), and 12 (25.5%) in the non‐PDH group.Polyuria and polydipsia (PUPD) were described in 28/94 (20.2%) dogs, of which 19 were in the PDH group (40.4%) and 9 in the non‐PDH group (19.1%).The most frequent medications administered before RT included trilostane in 37/94 (39.3%) dogs, prednisolone in 26/94 (27.7%), levothyroxine in 9/94 (9.6%), analgesics in 9/94 (9.6%), and desmopressin in 6/94 (6.4%).All 37 dogs that received trilostane before RT were in the PDH group. Of those, the trilostane dose remained unchanged post‐RT in 8, was decreased in 9, and was discontinued in 12 dogs. Two dogs had their trilostane dose increased at subsequent follow‐up, and for 6 dogs it was not possible to determine the post‐RT dose prescribed. The reason for trilostane dose discontinuation was available for 5 dogs, and was attributed to hypoadrenocorticism (n = 2), lethargy and anorexia (n = 2), and relapse of neurologic signs (n = 1). In this last dog, it was explicitly recorded that the decision for trilostane discontinuation was the potential anti‐inflammatory effects of endogenous corticosteroids in decreasing peritumoral edema. The rationale behind dose changes in other cases could not be determined from the medical records.One dog in the PDH group had not been treated with trilostane before RT, but was prescribed trilostane after RT. Two dogs classified as non‐PDH received trilostane a few months after RT treatment. One of these dogs did not have any reported signs of HAC before RT and no endocrine tests were performed before RT. The remaining dog had PUPD at the time of pituitary macroadenoma diagnosis but both ACTHst and LDDST were not consistent with HAC. The rationale for starting trilostane in these 2 dogs was not available.Fifteen of 94 dogs (15.9%) had concurrent endocrinopathies, 9/15 in the PDH and 6/15 in the non‐PDH group. Of these, 2 dogs had concurrent diabetes mellitus (DM): 1 in the PDH and 1 in the non‐PDH group. Six dogs had a previous diagnosis of diabetes insipidus (DI), 2 of which were in the PDH and 4 in the non‐PDH group. Fifteen dogs had a prior diagnosis of hypothyroidism, 6 in the PDH, and 9 in the non‐PDH group. One dog in the non‐PDH group was diagnosed with hypersomatotropism, concurrent DM, and hypothyroidism.No difference was found between the 2 groups in the proportions of dogs with concurrent endocrinopathies or in those receiving medications. The endocrine testing leading to diagnosis of the concurrent endocrinopathies was not available for review.3.2P = .78). No significant difference was observed in any of the imaging variables between the PDH and non‐PDH groups (Table Pre‐radiotherapy images were available for review in 76/94 (88.3%) dogs, with diagnostic imaging reports available for the remaining 18. In 49 of these, a macroadenoma had been diagnosed by CT, 35 by MRI and 10 dogs had both CT and MRI imaging before RT. Whenever dogs underwent both imaging modalities, CT was used for RT planning. The type of pre‐RT imaging did not have a statistically significant effect on overall survival , consequences , or lateralization are summarized in Table 3.3χ2 = 5.7; P = .27), or in the proportions of PDH or non‐PDH dogs between machines , or in pituitary volume treated by each machine (P = .61). Additional details on RT protocol, schedule, and doses are summarized in the Table Eighty dogs (85.1%) underwent a definitive treatment protocol, of which 41 were in the PDH and 39 in the non‐PDH group. Of these, 14 had a Monday‐Wednesday‐Friday protocol , whereas 66 had a Monday‐to‐Friday protocol . Fourteen dogs (14.9%) underwent palliative RT, of which 6 were in the PDH and 8 in the non‐PDH group. No significant difference was found in any of the RT protocols or variables between the 2 groups (Table P = .16). The number of fractions prescribed was 5 in all dogs undergoing the palliative protocol, and median was 10  for the definitive protocol. The number of fractions delivered ranged from 1 to 5 in the palliative group, with 2 dogs in the non‐PDH group having an incomplete treatment. In the definitive protocol, the number of fractions administered ranged from 3 to 20, with 4 dogs having incomplete treatment (2 in the PDH group and 2 in the non‐PDH group). Median total radiation dose delivered for dogs undergoing palliative RT was 37 Gy  and 45 Gy  in dogs undergoing definitive RT. Median total RT dose was 43.5 Gy  in the PDH group and 44.85 Gy  in the non‐PDH group.Median time before initiation of RT treatment after pituitary mass diagnosis was 12 days  and no significant difference was found between PDH and non‐PDH groups (P = 1.00).Twenty‐five dogs (26.6%) experienced adverse RT effects. Twenty dogs, all undergoing definitive RT, had acute adverse RT effects, occurring hours or days after RT treatment. Five dogs experienced delayed adverse RT effects, occurring weeks to months after RT, of which 2 dogs received definitive and 3 dogs had received palliative RT. Adverse RT effects and their frequency are listed in Table Radiotherapy protocol interruptions occurred in 6/94 (6.3%) dogs, because of vomiting (n = 1), abscess development (n = 1), lethargy (n = 1), and the linear accelerator being out of order (n = 3). The RT protocol was not completed in 7/94 dogs (7.4%) because of deterioration of neurological signs (n = 5), euthanasia (n = 1), or severe vomiting (n = 1).Varian 2100C (19 in the PDH group and 17 in the non‐PDH group), 30 using Varian 600C (12 in the PDH group and 18 in the non‐PDH group), 12 with Elekta Precise (5 in the PDH group and 7 in the non‐PDH group), 10 using Siemens Oncor Impression Plus (8 in the PDH group and 2 in the non‐PDH group), and only 1 dog was treated using Dynaray (in the PDH group).The model and brand of linear accelerator used was available for 89/94 dogs (94.7%), of which 36 were treated using Prowess v 4.71, v 5.1 or v 5.51 in 10 (7 in the PDH group and 3 in the non‐PDH group), and Oncentra (Elekta) in 43 dogs (19 in the PDH group and 14 in the non‐PDH group).The software used for RT planning was available for 90/94 dogs (95.7%): Addenbrooke's Radiotherapy Planning System (ARPS) was used in 37 (20 in the PDH group and 17 in the non‐PDH group), 3.43). The same dog experienced relapse of neurologic signs 22 months after RT, and repeated CT at this time showed increased PBR and pituitary volume . It was not possible for the other cases to determine retrospectively whether repeated imaging was performed during routine re‐assessments or because of progression of clinical signs.Follow‐up images after RT were available for only 7/94 (7.4%) dogs, 4 in the PDH group and 3 in the non‐PDH group. The interval between RT and repeated brain imaging ranged from 2 to 22 months. Tumor volume reduction and decreased PBR were documented in all dogs in which post‐RT imaging was available. One dog had 4 CT studies post‐RT, which showed progressive decrease in PBR and tumor volume over a period of 13 months  for all dogs was 514 days . No significant difference was found in survival between dogs in the PDH and non‐PDH groups .No significant difference was found in survival between PDH and non‐PDH dogs that received definitive or palliative RT . Deterioration or relapse of neurologic signs was the most common cause (44/60 [73.3%] dogs). Other causes unrelated to pituitary neoplasia are listed in Table P = .05) difference in MST was found between the definitive protocol group  and the palliative protocol group .A significant (3.6P < .2) are presented in Table P < .01).The univariate Cox analysis variables that met the inclusion criteria for the multivariate analysis  in this cohort. The retrospective nature of our study made it difficult to assess neurological examination findings or severity of neurological presentation. Furthermore, presence of HAC‐associated systemic signs, complications, and costs associated with trilostane treatment might influence owners' perception of quality of life and might have shortened survival in the PDH group.Our data adds to the conflicting veterinary literature on the association between hormonal status and survival of dogs with pituitary macroadenomas, using both fractionated RT and stereotactic RT protocols. In agreement with our findings, a previous studyIn the previous study,The PBR has been described as the variable having the highest discriminatory power in distinguishing enlarged from nonenlarged pituitary glands.In our study, tumor dimensions  were not associated with survival. This finding contradicts another study of dogs with pituitary masses treated with RT, in which those with smaller tumors  lived longer than those with larger tumors.In our study, the finding that definitive RT protocols were associated with prolonged survival is not surprising. Definitive RT protocols allow a higher number of fractions at shorter intervals, maximizing cell repair, redistribution, reoxygenation, and repopulation, and allowing a larger therapeutic window between the minimal tumor control dose and the maximum tolerable dose to the healthy surrounding tissues. On the other hand, definitive protocols are intensive and might be more likely to be recommended in stable patients, thus resulting in a selection bias.P = .004).P = .01).To our knowledge, previous studies have not evaluated how differences in total Gy administered impacts survival in pituitary RT. However, comparing the veterinary literature available, higher total delivered RT dose seems associated with a longer MST Table . A previIn the included cohort, a decrease in PDH signs, or a consistent decrease in trilostane dosage in those dogs receiving this drug, could not be determined because of the retrospective nature of the study. Endocrine testing was not performed at regular intervals after RT, and limited data were available to assess the endocrine response to RT. Although some dogs experienced a decrease in trilostane dosage, or discontinuation, the rationale for this adjustment was not always available, and these changes could have been secondary to resolution of HAC signs, hypoadrenocorticism, concurrent diseases, owner finances, or relapse of neurologic abnormalities.In view of these considerations, a prospective study design might help resolve existing conflict in the veterinary literature about the role of RT in improving clinical signs associated with HAC. In people, more aggressive RT protocols are required to overcome hormonal production compared with doses used for mass reduction.A surprisingly low proportion of dogs in the PDH group had PUPD . Dogs in this group most likely were diagnosed with HAC and started on medical treatment before being referred for RT, and their HAC signs, including PUPD, might have been controlled at the time of presentation for RT. It is also possible that PDH dogs might have displayed other signs  that were considered more severe than PUPD and that, despite the presence of PUPD, this concern was not explicitly included in the clinical notes.Our study had several limitations, mainly because of its retrospective nature across multiple hospitals. Primarily this design meant there was a lack of standardization of diagnostic testing and RT protocols, including endocrine testing and post‐RT monitoring. Despite our efforts, a misclassification of dogs between the 2 groups is possible because of secondhand evaluation of the content and detail of the clinical notes as well as the intrinsic limitations of endocrine testing. As in similar studies on pituitary RT, no pituitary histology was available. Different histological characteristics may affect responsiveness to treatment, but adenohypophyseal hyperplasia and adenoma are the most common pituitary diagnoses in dogs, with other sellar neoplasms being rare.Considering these limitations and current gaps in the veterinary literature, prospective, multicenter studies are needed, with standardized diagnostic testing, RT protocols and planning, and appropriate follow‐up, to assess the clinicopathologic effects of RT on pituitary secretory properties.In conclusion, we did not identify clinical characteristics of pituitary tumors associated with survival. However, RT protocol did affect outcome in our cohort, independently of patient hormonal status, and a higher total radiation dose (Gy) was associated with longer survival.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Approved by Animal Health Trust Ethical Committee, project number 39‐2018.Authors declare human ethics approval was not needed for this study.Table S1. Radiotherapy planning, doses and delivery parameters.Click here for additional data file.


Cell membrane encapsulation is a growing concept in nanomedicine, for it achieves the purpose of camouﬂage nanoparticles, realizing the convenience for drug delivery, bio‐imaging, and detoxification. Cell membranes are constructed by bilayer lipid phospholipid layers, which have unique properties in cellular uptake mechanism, targeting ability, immunomodulation, and regeneration. Current medical applications of cell membranes include cancers, inflammations, regenerations, and so on. In this article, a general bibliometric overview is conducted of cell membrane‐coated nanoparticles covering 11 years of evolution in order to provide researchers in the field with a comprehensive view of the relevant achievements and trends. The authors analyze the data from Web of Science Core Collection database, and extract the annual publications and citations, most productive countries/regions, most influential scholars, the collaborations of journals and institutions. The authors also divided cell membranes into several subgroups to further understand the application of different cell membranes in medical scenarios. This study summarizes the current research overview in cell membrane‐coated nanoparticles and intuitively provides a direction for future research. This article presents a literature analysis of cell membrane‐coated nanoparticles covering 11 years of evolution based on the data from the Web of Science Core Collection, including the annual publications and citations, most productive countries/regions, most influential scholars, the collaborations of journals and institutions. It gives a current research overview in this field and intuitively provides a vision for researchers. Cell membrane biomimetic nanomedicine was first brought up by Zhang et al. in 2011 by coating nanoparticles with an erythrocyte membrane. The nanoparticle's circulation time increased to 40 h in vivo, greatly extending the half‐life period, and the red blood cell membrane (RBCM)‐encapsulated nanoparticles can be used multiple times without irritating an immune response. Since then, cancer cell membranes (CM) were applied to design anticancer vaccination and drug delivery in 2014; platelet membranes (PM) were applied to escape macrophage phagocytosis in 2015; leukocyte membranes as one of the immune cell membranes (IM) were purified to avoid immune system clearance in 2013; T‐cell membranes were exploited to target tumor, using their distinct targeting characteristics in 2017, and other cell membranes‐coated research flourished like bamboo shoots after a spring rain.Stability and targeted drug delivery are urgent problems for nanoparticles in medical applications. The traditional solution is to add polymer materials on the surface of nanoparticles but hard to avoid immune system recognition.1 Cell It offers a global perspective, which can more comprehensively evaluate the phylogeny of a certain field, and provides a basis for the prediction of future research hotspots.Bibliometric analysis helps scholars to gain an overall view of a complex field quickly including acknowledgment of the main publishing institutions, the most influential scholars, and the most representative research. It was able to assist with developing approaches focused on prognosticating the current and future directions of certain areas. It also structured in a more newfangled display mode, compared with systemic reviews which mainly aim at the content of research produced in a certain field.10 It SchemeMany original articles have been published from 2011 to 2021 in the field of cell membrane‐encapsulated nanoparticles research. In this article, we provided an overview and scientific analysis of research about cell membrane‐encapsulated nanoparticles via data collected from the Web of Science Core Collection (WoSCC). The analyzed indexes include annual publications and citations, most productive countries, most influential and high‐cited authors, and collaborated journals, as well as institutions. These were demonstrated and visualized by several bibliometric software. Moreover, the profiles of different membranes were analyzed independently, including RBCM, PM, CM, IM, and other cell membranes, to understand the different applications in this field by comparing the publications of these five types of cell membranes Scheme1. This22.1Figuren = 179) compared with that in 2011 to 2019 (n = 170), indicating the potential of cell membrane‐coated nanoparticles was gradually accepted by researchers in biomedicine. The mean total citation rate has increased steadily over the years. As shown from the global productivity in Figure TableFive hundred and eighty‐three articles associated with cell membrane‐coated nanoparticles were screened out from the database of WoSCC. These documents have been cited 2644 times until December 31st, 2021, with a mean of 11.3 times per publication. The flowchart of the inclusion criteria is demonstrated in 2.2FigureFrom 2011 to 2021, a total of 31 countries were included in the analysis of cell membrane‐coated nanoparticles. First, we conducted the collaborative connections among countries in 2.3TableFiguren = 5492), Che‐Ming J Hu (n = 4207), Weiwei Gao (n = 4193), Brian T Luk (n = 3944), Diana Dehaini (n = 2193), Pavimol Angsantikul (n = 1924), Ashley V Kroll (n = 1647), and Wei Liu (n = 1602). Local citations measure the times an author (or a document) included in this collection has been cited by the documents also included in the collection. Liangfang Zhang was also the most locally cited author (n = 1046), followed by Ronnie H Fang (n = 894), Weiwei Gao (n = 737), Che‐Ming J Hu (n = 719), Brian T Luk (n = 700), Diana Dehaini (n = 378), Pavimol Angsantikul (n = 328), Wei Liu (n = 302), Ashley V Kroll (n = 297), and Lang Rao , and the leading scholars were Wei Zhang, Wei Liu, Qianfang Meng, and Lang Rao. Their main research directions were the wound‐targeting function of platelet to achieve fixed‐point illumination of the wound and hybrid cell membrane (HM)‐coated nanoparticles for disease diagnosis and treatment. The visualization of the collaborations among institutions is presented in Figure The University of California San Diego had the greatest contribution, publishing 93 articles concerning cell membrane‐coated nanoparticles Figure . Its leareatment.13 The2.4Q = 0.7088, weighted mean silhouette S = 0.8601). As presented in FigureK values  being the most productive journal in this field. It was followed by ACS Nano (n = 30), Advanced Functional Materials (n = 26), Journal of Nanobiotechnology (n = 26), and Biomaterials (n = 25). The above journals were of high quality in the field of biomedical science and material science. It revealed that cell membrane‐coated nanoparticles attracted academic attention and exerted a great scientific impact on scholars and their studies. From the developing trend in this field, ACS Nano first rose in 2014, publishing a work entitled “Erythrocyte membrane is an alternative coating to polyethylene glycol for prolonging the circulation lifetime of gold nanocages for photothermal therapy” (https://doi.org/10.1021/nn503779d). Next, it came with Advanced Functional Materials in 2015, ACS Applied Materials & Interfaces and Biomaterials in 2016, and Journal of Nanobiotechnology in 2020 ,” “Macrophage cell membrane (n = 107),” “Platelet membrane (n = 43),” “T cell (n = 47),” and “Breast cancer cells (n = 61).” To further understand the specific medical application scenarios of each cell membrane, we enumerated the research related to RBCM in Figure To decipher the frontier hotspots of cell membrane‐coated nanoparticles field more adequately, we performed hotspot foam‐tree analysis via a website‐based analyzing tool—Carrotrs Figure6A. The 2.7n = 166, Figuren = 169); IM had slow growth from 2016 to 2019, followed by moderate growth after 2019 ; PM was more niche than other types . Although it started early, the research has grown rather slowly. Others included HM, stem cell membranes, and cardiomyocytes . It was worth mentioning that publications in others were growing fast since 2019. It indicated that the applications of cell membrane‐coated nanoparticles have expanded to broader medical treatment. In the aspect of global citations, RBCM had the most cited work in 2011 ; PM reached its citation peak in 2015 (n = 1194), while IM had it in 2020 (n = 1041); the popularity of CM had not diminished since 2014, and there have been three peaks in citations in 2014 (n = 751), 2016 (n = 1584), and 2020 (n = 1250); while others had a trend of becoming popular on citations since 2015.To properly facilitate researchers to choose the suitable cell membrane for their research direction, we continued to explore how medical applications of different cell membrane‐coated nanoparticles varied in the number of publications, citations, and topic trends. After categorizing the cell membranes into five types, namely RBCM, PM, CM, IM, and others, respectively, we found that RBCM was with the greatest number of publications from 2011 to 2018  from others to see the developing trend. Figure To further analyze the influence of different cell membranes in the field, we added up the citations of each type of membrane each year to observe the overall citation changes. As presented in Figure 15 FurConcerning the analysis of other cell membrane‐coated nanoparticles, China, the USA, and France were the top three most productive countries  presentation and the low specificity of the extracted tumor antigens, coating CM perfectly addressing the dilemma through targeting APCs, promoting homologous recognition between materials and tumor cells, and precise targeting of tumor cells. Gou et al. reported that engineered peptide‐expressed biomimetic CM‐encapsulated STING agonists can significantly enhance type I interferon (IFN) secretion in dendritic cells and inhibit PD‐1 unresponsive tumor growth without causing obvious side effects. Furthermore, Wang et al. camouflaged Ce6 with CM to perform a combination of photodynamic therapy and hyperthermia for tumor remote metastasis, which resulted in a synergistic effect in tumor inhibition by triggering immunogenic cell death.Despite various cell sources for cell membranes, the extraction and purification process, as well as the coating methods are quite similar Scheme . Moreove18 Pla pH‐sensitive nanomaterials coated by the macrophage membranes can be stably released in the blood circulation (pH 6.5), but the rapid release occurs after membrane fusing in the intratumoral acid microenvironment (pH 5), which helps to precisely kill tumor cells. Studies on NK cell membrane‐encapsulated nanoparticles have exhibited that this biomimetic strategy can utilize receptor molecules on the surface of NK cell membranes. This camouflage not only prolongs the circulation time in vivo but also excites significant tumor‐homing potential, which is expected to play a role in tumor bioimaging and immune monitoring. As the specific immune recognition receptors are expressed on the surface, T cell membrane coating is promising. Zhang et al. designed nanoengineered CD4+ T cell membrane‐coated nanoparticles to integrate with HIV‐1 strains and to target gp120 high‐expressing cells to reduce HIV sanctuaries. Other membranes include stem cell membranes, out membrane vesicles (OMVs), endothelial cell membranes, islet beta cell membranes, and hybrid membranes. Stem cell membranes tend to have low immunogenicity and possess the ability to repair and regenerate, which decreases the toxicity of nanoparticles in the lung and rescues damaged myocardium by reducing apoptosis. On the opposite, OMVs have strong immunogenicity and are suitable for the preparation of antibacterial vaccines. It activates dendritic cells in the lymph nodes in vivo and promotes the maturation of DCs, triggering durable antibody responses, inducing the production of IFN‐γ and IL‐17, and stimulating T cells to respond. Islet beta cell membranes can utilize the interaction between β cells to promote the growth and reproduction of residual β cells, to provide therapeutic ideas for effectively solving insufficient insulin production. Due to the affinity and adhesion between endothelial cells and innate immune cells, wrapping the endothelial cell membranes around anti‐infective drugs can effectively deliver drugs to the site of inflammation.As for IM, APCs represented by macrophages, natural killer cells (NKs), and cytotoxic lymphocytes (CTLs) all belong to the category of immune cells. The mechanism of macrophage membranes to fight tumors is based on the recognition between integrin α4 and vascular cell adhesion molecule (VCAM) expressed on the surface of CM.26 pH‐ HM not only significantly improves the specific recognition of nanoparticles to certain cell lines and accelerates the intra‐cellularization but also extends the blood retention time of nanoparticles. The circulation lifetime of HM‐encapsulated nanoparticles was 1.39 times and 3.88 times longer than CM‐encapsulated nanoparticles and pure nanoparticles after 24‐h injection. Hybrid biomimetic coating of chemotherapy regimen doxorubicin and photosensitizer CuS exhibits great synergistic photothermal/chemotherapy with ≈100% inhibition of melanoma growth. Combinations of CM and OMV achieve a better clinical therapeutic effect by enhancing the innate immune response and residing in lymph nodes, reducing the risk of metastasis. Cell membrane surface engineering can serve different purposes. One of its most prominent applications is the introduction of targeting moieties  Escherichia coli modified with an integrin αvβ3 peptide targeting ligand and indocyanine green (designated IP‐OMVs) acted as combo punch to treat cutaneous melanoma. With the assistance of near‐infrared irritation, it can not only arouse photothermal–photodynamic responses against primary melanoma but also activates TRAIL‐related apoptosis in disseminated tumor cells, leading to a complete eradication of the tumor. At present, cell membrane‐encapsulated nanoparticles are also gradually transforming into clinical applications, among which erythrocyte membranes are the most widely used strategies for regimen encapsulation (Table l‐asparaginase (GRASPA) have launched phase II/III trials in acute lymphoblastic leukemia and acute myeloid leukemia (NCT01518517 and NCT01810705). Ery‐Dex (dexamethasone sodium phosphate encapsulated in autologous erythrocytes) was also applied in nervous system disorders and genetic syndrome (NCT01255358 and NCT02380924). Apart from the treatment of erythrocyte membranes, debridement + NanoPRP coverage treatment based on PM is currently recruiting patients with diabetic foot ulcers (ChiCTR2100046769). Preclinical trials, including delivery of immunotherapy agents and delivery of chemotherapy agents, are ongoing . Clinical trials of cell membrane‐coated therapeutics are emerging, indicating their relatively promising medical translational prospects.Through our analysis, we found that HM and EM have the therapeutic effect of fusion and multi‐function, which is one of the main development directions of cell membrane‐encapsulated nanomaterials in the future Figure .35]35[3s Figure . By provAccording to different diseases or research purposes, choosing different membranes to fuse or to modify membrane surface proteins through gene editing can exert a variety of cell membrane functions, which is an important development direction in this field.4For better diagnosis approaches, disease prevention, and treatment, cell membrane‐encapsulated nanoparticles have broad prospects in clinical translation. Bio‐inspired nanotherapeutic strategies provide an innovative platform for this purpose. Drug delivery, detoxification, and immunomodulation are the three main research directions for current medical applications of cell membrane‐encapsulated nanoparticles. We identified historical and future research trends by analyzing the number of publications and citations, the productive countries, influential and high‐cited authors and institutions, mainstream journals, representative works, co‐occurrence keywords, and frontier hotspots in different cell membranes during the past 11 years of expeditious development in this field. As noted in the result section, institutions from the China and USA play leading roles with other countries and institutions steadily participating. From the trends concerning global cooperation, it becomes more and more tight and prosperous as time goes by. Moreover, Liangfang Zhang and his team contributed the most in this field, with the University of California San Diego being the institution with the highest output. Recent researches pay more attention to the applications of HM and EM‐encapsulated versatile nanoparticles in complicated medical scenarios by taking advantage of the specific underlying mechanism of molecules on the membrane. Different from meta‐analysis and general review, bibliometric analysis is the basis of our study, which offers a more comprehensive and unique aspect of the growth of cell membrane‐coated nanoparticles. Given that this field is gradually becoming marketable, we are looking forward to better and more cutting‐edge research.5n = 322), meeting articles (n = 71), meeting abstracts (n = 16), book chapters (n = 6), editorial material (n = 4), revisions (n = 2), online publications (n = 2), retraction (n = 2), data papers (n = 1), reprint (n = 1), retract content (n = 1), and non‐English (n = 16) were removed. Finally, the title, abstract, or full text of these publications were read to screen out articles closely related to the topic studied (cell membrane‐coated nanoparticles), and the criteria for the collected data included article type, publication year, institution, country, authorship, abstract, keyword, citation, and references. After careful selection, a total of 583 publications were retrieved and analyzed. The publication list of the 583 articles is provided in Table Data were collected from WoSCC with the algorithm of TS = (nanoparticles*) AND ( ( ( (TS = (coat*)) OR TS = (camouflage*)) OR TS = (biomimetic)) AND TS = (cell membrane)) on July 26th, 2022. The literature research schedule ranged from January 1st, 2011 to December 31st, 2021. A total of 3478 publications were retrieved, while 390 articles, including reviews (2 workbench 3.10.2, and VOSviewer 1.6.18 were applied in this bibliometric analysis. Citespace was used to perform cluster analysis containing references, keywords, and timeline views. A modularity Q > 0.3 and a mean silhouette > 0.5 were considered qualified for the clustering results. Citespace was used to illustrate the top six productive countries, and R was used to show the top ten influential authors. The collaborative connections between countries, institutions, authors, and journals were also produced by R. R bibliometrix package was used to conduct three‐field plots and the bibliometric geographic map. Moreover, the keyword density maps of cell membrane‐coated nanoparticles field worldwide were generated by Vosviewer. Graphs of publications and citations presented in this study were conducted by GraphPad Prism 8.3.0.CiteSpace.6.1.R3 and 5.8.R1, R bibliometrix package V 3.2.1, Carrotne views.40 A mThe authors declare no conflict of interest.Y.Z. and S.J. contributed equally to this work. Conceptualization: Y.Z., S.J., X.F., Q.M.; Methodology: S.J., Y.Z., G.C., Y.C.; Investigation: Q.X., S.J., D.L., Y.L.; Visualization: S.J., Y.Z.; Supervision: H.L.; Writing–original draft: Y.Z., S.J.; Writing–review & editing: Y.Z., J.Y., X.F., H.L.Supporting InformationClick here for additional data file.


Our case report demonstrates extremely uncommon data associated with MIS‐A, such as cholestatic jaundice, anemia, and quickly progressing pneumonia. IVIG and pulse steroid medications are the best treatments for improving clinical outcomes.We report a case of multiple organ dysfunctions due to MIS‐A in an adult with a history of suspected COVID‐19. Our case demonstrates extremely uncommon data associated with MIS‐A, such as cholestatic jaundice, anemia, and quickly progressing pneumonia. IVIG and pulse steroid medications are the best treatments for improving clinical outcomes. Its true pathophysiology is still a subject of debate.The aim of the current study was to better comprehend and interpret MIS‐A in low‐ and middle‐income countries, where unique challenges exist between limited diagnostic and treatment options and the requirement to rule out a broader differential diagnosis that might resemble the features of MIS, such as severe/critical COVID‐19, classic Kawasaki syndrome, sepsis, and septic shock. Moreover, this report is a part of the global effort to establish a clear picture of this syndrome to enable further development of evidence‐based clinical guidelines to detect and treat affected patients early and properly.2A 22‐year‐old man was admitted to Almaza Fever Hospital, Cairo, Egypt, suffering from fever and mild upper respiratory symptoms 8 weeks prior with neither alarming basic laboratory investigations nor chest computed tomography (CT), which is routinely done on causality for fever cases. The patient has been vaccinated with 2 doses of the Sputnik‐V COVID‐19 vaccine, given 12 and 16 weeks prior, respectively. On March 10, he presented to the causality department with a fever of 38.5°C, a dry cough, and a sore throat for 1 day and was otherwise hemodynamically stable.a.m. on March 13th, the patient became drowsy, oliguric, and had unstable vital signs . Chest examination showed bilaterally reduced air entry with scattered coarse crepitation. He was transferred to the intensive care unit (ICU), where supplementary oxygen was given via a mask with a reservoir bag, alternating with a CPAP mask. A central venous line was inserted (CVP: 8 cm H2O). Norepinephrine (3.3 mg/kg/min) was used as a vasopressor, and IV fluids were guided by CVP measurement.The patient was admitted to the ward, where basic laboratory investigations were performed. SARS‐CoV‐2 PCR swabs were requested as well as a chest CT . The summary of clinical presentations, laboratory measurements, and interventions over timeline of illness was presented in Table The electrocardiogram (ECG) showed only sinus tachycardia. An echocardiogram was performed, which revealed mild mitral regurgitation. Abdomino‐pelvic U/S showed hepatomegaly and grade I nephropathy.Emergency laboratory investigations showed hepatic and renal dysfunction, coagulopathy, leucocytosis, thrombocytopenia, increased inflammatory markers, a high D‐dimer level (1979/500 ng/mL), elevated total creatine kinase (CK) (2058/170), and an increased IL‐6 level (453 pg/mL), while CKMB was 72/16. The brain CT was irrelevant. Chest CT was evaluated and quantified using Yang et al.,Mycoplasma pneumoniae, Bordetella pertussis, Influenza A, A H1 and B and Legionella pneumophila was performed, and all of them came back negative. Pan‐cultures  were ordered on day 3 and revealed no growth on day 6. However, screening for COVID‐19 antibodies (IgM/IgG) was positive for IgG. Additionally, we performed auto‐immune screening , screening for viral hepatitis , testing of quantiferon—TB Gold and all were negative. Procalcitonin was nonspecific (0.08 ng/mL). The differential diagnosis was performed, including:Severe COVID: studies and case reports on MIS‐A reveal that the disorder has a heterogeneous clinical presentation that frequently resembles severe COVID‐19. When PCR results are negative in patients with suspected acute COVID‐19, SARS‐CoV‐2 antibody testing (IgM antibodies) is pertinent for the diagnosis of acute COVID‐19‐related illness,Multisystem Inflammatory Syndrome in Adults (MIS‐A): According to the most recent CDC definition,Kawasaki disease: the diagnosis is established by a fever lasting five or more days. At least four of the following criteria should exist: polymorphous rash excluding bullous or vesicular eruptions, conjunctival injection, oropharyngeal mucous membrane changes, extremity changes, and lymphadenopathy. Symptoms common to MIS‐C but not typical of classic Kawasaki disease include abdominal pain, which is often more severe than in classic Kawasaki disease; thrombocytopenia, anemia, and lymphopenia; and elevated levels of ferritin, troponin, and D‐dimer,Septic shock: symptoms include fever, tachypnea, tachycardia, hypotension, and signs of tissue hypoperfusion. Its diagnosis is based on the recovery of the pathogen by culture.Scarlet fever: rash, fever, and lymphadenopathy are present. A positive rapid streptococcal test or the culture result is diagnostic, andStaphylococcus aureus, while the diagnosis is confirmed by the isolation of group A Streptococcus in the case of streptococcal toxic shock syndrome.Toxic shock syndrome: case definitions include hypotension and multisystem involvement in the presence of a history of retained foreign bodies  and one or more sites of soft tissue necrosis in the case of Testing for a variety of common upper respiratory pathogens such as Coronavirus 229E and HKUI; Rhinovirus/Enterovirus, Boca virus; The criteria of our case were not consistently met with classic Kawasaki but were matched with MIS‐A. The reported case is a 22‐year‐old male who was admitted to the hospital with a fever that lasted for the first 3 days of hospitalization. The patient showed characteristics of MIS‐A, including conjunctivitis, skin rash, cardiovascular affections , hepatic and renal disorders ,Anakinra (100 mg/0.67 mL) was given in a dose of 300 mg once daily intravenously for 4 days, followed by 100 mg once daily till clinically improved,On March 15, the patient received methylprednisolone 1 g/d and IV immunoglobulin 2 g/kg over 24 h of medication. The D‐dimer level was found to be 1979 ng/mL. There were no abnormalities in the blood film. The result of Coomb's test was negative. At 18000 plates, he received 12 packs or one platelet pack. The patient toxicological screening was unrestricted.2 levels were above 94% in room air. Follow‐up on the chest CT. On March 20, the patient showed an improved CT‐SS (17/40). On March 21, the norepinephrine was replaced with midodrine 5 mg TID to support blood pressure. While taking desmopressin, the urine output began to decrease. An MRI of the brain was performed on March 24 in order to rule out central causes of polyuria, with unremarkable results. By the end of a couple of weeks, the patient was shifted to the ward, under oral desmopressin, prednisolone, and prophylactic apixaba. A follow‐up chest CT showed a CT‐SS of 5/40.The patient started to improve clinically on March 16. The infusion rate of norepinephrine decreased while the blood pressure was elevated. As a result, both his oxygen consumption and saturation improved. On March 19, because of the patient's ongoing polyuria and normal urine‐specific gravity testing, desmopressin 120 mg biweekly was started. The oxygen supplementation was stopped when SOFigure 3An increase in fever‐related inflammatory biomarkers ,Laboratory confirmation of COVID‐19 infection as a new infection (including RT‐PCR test positive and/or SARS CoV‐2‐positive antibody) approximately 12 weeks prior to onset,Any infectious agents other than COVID‐19 should be excluded, andTo exclude the outcome of organ dysfunction and related tissue hypoxia, severe respiratory illness should not be present.MIS‐A is a severe hyper‐inflammatory syndrome that manifests with extra‐pulmonary multiorgans failure a few weeks just after the development of acute SARS‐CoV‐2 infections. The important constructive syndrome characteristics that were used for the syndrome case definition were the followingRash, plus or minus Conjunctivitis includes the nonpurulent type plus or minus mucocutaneous inflammation,Decreasing blood pressure plus or minus shock,Cardiac disorders that appear include valvulitis, myocarditis, or pericarditis. It also includes echocardiography abnormalities or increasing proBNP and troponin indicated in laboratory examinations.Coagulation defects are confirmed by clinical or laboratory test results and/or liver injury. The results included high D‐dimer, increased prothrombin time, and increased active partial thromboplastin time.Abdominal pain, vomiting, and/or diarrhea as gastrointestinal symptoms recently occurred.Besides the above criteria, at least two of the following essential characteristics should be included:Behzadi et al. investigated 36 instances of MIS‐A and concluded that the median patient age was 33 years old, 63% of the patients had no significant prior medical history, and 47% had SARS‐CoV‐2 infection, confirmed via PCR, antibody screening, or clinically. Fever was recorded in 86% of patients, while gastrointestinal symptoms were less frequently reported. A sore throat was observed in five patients (14%), and unilateral cervical pain or swelling was reported in four other cases (16%). In particular, tachycardia (61%) and hypotension/cardiogenic shock with verified reduced ejection fraction (64%) were reported as signs of cardiovascular failure.There is no agreement on the mechanism causing MIS‐A following the COVID‐19 infection. Several acute organ damages and systemic vasculitis are caused by MIS‐A, which is considered to be an extraordinary immune response. The significant improvement as a response to IVIG and aspirin confirms the existence of vasculitis.Several incidences of pulmonary affection in MIS‐A had been documented .In the present case, the Sputnik‐V COVID‐19 vaccine was administered twice, 2 months apart, and MIS‐A was discovered 3 months after the second dose. According to Ehikhametalor et al., MIS‐A emerged following a COVID‐19 infection and 3 weeks after the second dose of the vaccination. Further research is required to understand whether immunization may impact the COVID‐19 clinical picture.After reviewing the literature, a large variety of management strategies and therapeutic approaches are suggested.MIS‐A is a significant fatal illness that can affect multiple organs. Our case demonstrates extremely uncommon data associated with MIS‐A, such as cholestatic jaundice, anemia, and quickly progressing pneumonia.Anti‐IL‐1 receptor antagonists, pulse corticosteroids, and IVIG have all been used to treat MIS‐A; these extremely effective treatments need to be further evaluated.The current reported case is the third MIS‐A report from the Middle East, after the reported cases from Israel and the United Arab Emirates,4MIS‐A is a SARS‐CoV‐2 infection characterized by a delayed immunologic response and hyper‐inflammation. It should be suspected and identified by the clinical and public health communities using clinical evidence and empirical treatment to decrease morbidity and mortality outcomes. Moreover, the treatment should begin as soon as possible, with an accurate diagnosis and close monitoring. To avoid exacerbating the clinical condition, MIS‐A should be treated using the most effective methods. Anti‐IL‐1 receptor abtagonists, IVIG and pulse steroid medications are the best treatments for improving clinical outcomes. Although our presented case findings and management may not be broadly applicable to adults who may have MIS‐A, they do provide perspective on the difficulties in selecting a therapeutic option. Further research is needed to provide a comprehensive picture of this illness and to develop evidence‐based clinical guidelines. Long‐term follow‐up studies are necessary to investigate the potential consequences of MIS‐A.Mohamed Abdel‐Salam Elgohary: Conceptualization; data curation; investigation; visualization; writing – original draft; writing – review and editing. Mohamed Emam Mohamed: Data curation; investigation; writing – original draft. Mahmoud Moustafa Hamada: Data curation; investigation; writing – original draft. Jaklin S. Fahim: Data curation; investigation; writing – original draft. Thanaa A. El‐Masry: Investigation; visualization; writing – original draft. Ahmed Mohamed Ibrahim: Data curation; investigation; writing – original draft. Ahmad Gad Taha: Data curation; investigation; writing – original draft. Mohamed G. Seadawy: Conceptualization; data curation; investigation; visualization; writing – original draft; writing – review and editing. Athanasios Alexiou: Investigation; writing – original draft. Marios Papadakis: Investigation; writing – original draft. Gaber El‐Saber Batiha: Investigation; writing – original draft.The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.The authors declare no competing interests.After informed written consent from the participant, the study has been approved by the Research Ethics Committee of the Faculty of Pharmacy, Tanta University (REC‐TP code: TP/RE/04/22p‐0034), and the ethical committee office of the Medical Military Academy in agreement with the Helsinki Declaration Roles.The patient was delighted with the highly skilled medical service. He was surprised by the rapid response of the medical staff, and finally, the kind social support provided by the medical team.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Tumor size is an important prognostic factor in lung cancer in dogs, and the canine lung carcinoma stage classification (CLCSC) recently has been proposed to subdivide tumor sizes. It is unclear if the same classification scheme can be used for small‐breed dogs.To investigate whether the tumor size classification of CLCS is prognostic for survival and progression outcomes in small‐breed dogs with surgically resected pulmonary adenocarcinomas (PACs).Fifty‐two client‐owned small‐breed dogs with PAC.Single‐center retrospective cohort study conducted between 2005 and 2021. Medical records of dogs weighing <15 kg with surgically resected lung masses histologically diagnosed as PAC were examined.The numbers of dogs with tumor size ≤3 cm, >3 cm to ≤5 cm, >5 cm to ≤7 cm, or >7 cm were 15, 18, 14, and 5, respectively. The median progression‐free interval (PFI) and overall survival time (OST) were 754 and 716 days, respectively. In univariable analysis, clinical signs, lymph node metastasis, margin, and histologic grade were associated with PFI, and age, clinical signs, margin, and lymph node metastasis were associated with OST. Tumor size classification of CLCS was associated with PFI in all categories, and tumor size >7 cm was associated with OST. In multivariable analysis, tumor size >5 cm to ≤7 cm and margin were associated with PFI, and age was associated with OST.The tumor size classification of CLCS would be an important prognostic factor in small‐breed dogs with surgically resected PACs. AEsadverse eventsCBDCAcarboplatinCIconfidence intervalCLCSCcanine lung carcinoma stage classificationCTcomputed tomographyHRhazard ratioIQRinterquartile rangeJSACCJapan Small Animal Cancer CenterMDCTmultidetector‐row computed tomographyOSToverall survival timePACprimary pulmonary adenocarcinomaPDprogressive diseasePFIprogression‐free intervalPPCprimary pulmonary carcinomaPPNprimary pulmonary neoplasiaTOCtoceranib phosphateVCOG‐CTCAEVeterinary Comparative Oncology Group Common Terminology Criteria for Adverse Events1Primary lung cancer is a common malignancy in peoplePrognostic factors for lung cancer in people include histologic classification,Although a staging system for lung tumors in dogs was proposed by the World Health Organization in 1980, this staging system did not include an assessment of tumor size. However, tumor volumeHowever, unlike humans, dog breeds have large differences in body size. In the 2 reports that evaluated CLCSC, the median weight of the dogs was >20 kg.Therefore, we aimed to investigate whether the tumor size classification of CLCS is prognostic for survival and progression outcomes in small‐breed dogs with surgically resected PACs. We also explored other potential risk factors related to progression outcomes and survival times.22.1A retrospective cohort study was performed at the Japan Small Animal Cancer Center (JSACC) between April 1, 2005, and March 31, 2021, in dogs weighing <15 kg that had undergone surgical resection of pulmonary masses diagnosed as PACs by histologic examination. Dogs with a prior history of adenocarcinoma and concomitant malignancies other than PACs on the day of initial examination were excluded.2.2Information from medical records was obtained on signalment, clinical signs at initial examination, clinical pathology results, including hematology and urinalysis, radiography, abdominal ultrasonography, computed tomography (CT), cytology, histology, postoperative chemotherapy, date that tumor recurrence occurred, and presence of distant metastases. Staging, including history, physical examination, blood test results, urinalysis, thoracic and abdominal radiography, and abdominal ultrasound examination, was performed in all cases. The diagnostic methods and date and cause of death  also were collected. When follow‐up data were limited, medical records were obtained from the primary veterinarian, or updates were obtained by telephone from the owner. The presence of clinical signs was defined as the initial presentation with any potential clinical sign associated with a lung tumor, including coughing, tachypnea, dyspnea, lethargy, exercise intolerance, anorexia, or hemoptysis, whereas no clinical signs at diagnosis were defined as incidental identification of a lung tumor on imaging with no clinical signs associated the lung tumor.2.3For the CT scanner at the JSACC, we used a 4 multidetector‐row (MD) CT scanner  from 2005 to January 2015 and an 80MDCT scanner  from February 2015 to 2021. Contrast‐enhanced CT of the chest was performed under general anesthesia. The CT scanning and reconstruction parameters used at the JSACC for the 4MDCT scanner were 120 kVp, 120 mA, rotation time = 0.75 seconds, slice thickness = 1‐2 mm, beam pitch = 1.374, reconstruction interval = 1‐2 mm, and a standard algorithm, whereas for the 80MDCT scanner these were 120 kVp, 350 mA, rotation time = 0.5 seconds, slice thickness = 0.5 mm, beam pitch = 1.388, reconstruction interval = 0.5 mm, and a standard algorithm. Iopamidol  was used as the contrast agent for CT. This contrast agent was administered at a dosage of 2‐2.5 mL/kg (600‐750 mg/kg) for >10 seconds via the cephalic vein, and images were taken 120 seconds after administration.2.4Progression‐free interval (PFI) was defined as the time from surgery to progressive disease (PD). Progressive disease was defined as recurrence, dissemination, metastasis, or malignant pleural effusion noted postoperatively and was confirmed when the diagnosis was based on imaging, cytologic findings or histologic findings. Dogs were censored from PFI analysis if no detectable PD was noted at the last follow‐up or death. Overall survival time (OST) was defined as the time from surgery to death from any cause. Dogs that were lost to follow‐up or remained alive were censored from OST analysis at the date of the last contact.P < .05 was obtained in univariable analyses. These results were presented as hazard ratio (HR) and 95% confidence intervals (CI). The potential risk factors between the dogs receiving or not receiving adjuvant CBDCA were compared using Fisher's exact test. P values <.05 were considered significant. Statistical analysis was performed using EZR software .The Kaplan‐Meier method was used to estimate and depict PFI and OST. Interval or survival between groups was compared using the log‐rank method. Age, weight, clinical signs at the initial examination, lymph node status , completeness of tumor resection (margin), histologic grade (grade 1‐3), histologic patterns (papillary vs other), tumor size classification of CLSC , and adjuvant chemotherapy were analyzed as variables to identify potential risk factors associated with PFI and OST. These data were reported as median and interquartile range (IQR). Lymph node metastasis was evaluated by histopathologic examination, and as in previous reports, for dogs in which lymph node resection or biopsy was not performed, lymph node status was recorded as N0.33.1Fifty‐two client‐owned small‐breed dogs met the criteria for inclusion. There were 23 castrated males, 26 spayed females, and 3 intact males. The median age was 12 years . Seventeen dog breeds were included: Toy Poodle (n = 10), Miniature Dachshund (n = 10), Chihuahua (n = 7), Maltese (n = 4), Shih Tzu (n = 4), and 17 dogs of other breeds, each with fewer than 3 dogs per breed (n = 17). The median body weight was 5.6 kg  and the numbers of dogs ≤5 kg, >5 kg to ≤10 kg, and >10 kg to ≤15 kg were 19, 29, and 4, respectively. Clinical signs at initial presentation were noted in 33 dogs (63.5%): coughing (n = 32), anorexia (n = 9), exercise intolerance (n = 9), lethargy (n = 3), hemoptysis (n = 2), tachypnea (n = 1), and dyspnea (n = 1).3.2Contrast‐enhanced thoracic CT was performed in all cases. Three patients had CTs performed at other institutions. The CT data were partially or totally lost for 2 dogs and could not be reviewed, and thus the description from the original diagnostic imaging report was used. Those reports described the anatomic location and the longest diameter of the mass but did not describe the details of the lymph nodes. The median longest diameter of the lung masses was 41.0 mm . The lung mass was solitary in 49 dogs, and satellite nodules were found in the same lobes as the lung mass in 3 dogs. The numbers of dogs with tumor size ≤3 cm, >3 cm to ≤5 cm, >5 cm to ≤7 cm, or >7 cm were 15 (28.8%), 18 (34.6%), 14 (26.9%), and 5 (9.6%), respectively. Lung masses occurred in the cranial lobe of the right lung in 8 dogs (15.4%), the middle lobe of the right lung in 5 dogs (9.6%), the caudal lobe of the right lung in 13 dogs (25.0%), the accessory lobe in 3 dogs (5.8%), the cranial lobe of the left lung in 15 dogs (28.8%), and the caudal lobe of the left lung in 8 dogs (15.4%). Fourteen of 50 dogs (28.0%) were diagnosed with suspected lymph node metastasis. Other CT findings included a small amount of effusion in 4 dogs, 1 of which had lung lobe torsion. Pleural fluid examinations were performed in 2 of 4 dogs; both dogs were found to have transudative pleural effusion and no tumor cells. Distant metastases were not found in any of the cases.3.3Forty‐six dogs underwent resection of 1 lung lobe. Because of the anatomical location of the tumor and the pulmonary vessels and trachea, 3 dogs underwent resection of the accessory lobe and right caudal lung lobe, and 3 dogs underwent left lung resections. Lymph nodes were removed in 37 dogs (71.2%); the regional tracheobronchial lymph nodes were removed in 35 dogs. In the other 2 dogs, cranial mediastinal and sternal lymph nodes were removed in 1 dog, and cranial mediastinal lymph nodes were removed in the other dog. One dog had a nodule in the caudal mediastinum that was removed. Lymph nodes were removed in 12 of 14 dogs with suspected metastases on CT. Intraoperative complications occurred in 6 (11.5%) dogs, postoperative complications occurred in 19 (36.5%) dogs, and perioperative death was observed in 4 dogs . Six dogs showed no improvement in coughing after surgery, and the suspected causes of coughing (including causes affecting >1 dog) were tracheal or bronchial‐related disease (n = 3), myxomatous degenerative mitral valve disease (n = 3), carcinomatous pleuritis (n = 1), pneumonia (n = 1), and pneumothorax (n = 1). The remaining 3 dogs, in which improved coughing could not be confirmed, died during the perioperative period, and therefore it was not possible to assess whether coughing had improved.3.4The primary PAC was completely resected in 45 dogs (86.5%). The histologic grades of PAC were grade 1, 2, and 3 in 35 (67.3%), 15 (28.9%), and 2 (4%) dogs, respectively. Vascular invasion was noted in 8 (15.4%) dogs. There were 14, 17, 13, and 8 dogs for the T1, T2, T3, and T4 stages of CLCSC, respectively. In 5 dogs, T stage did not correlate with the tumor size classification of CLSC because of the presence of either intrathoracic organ invasion or a lung nodule. Lymph node metastases were observed in 9 of 37 dogs (24.3%), of which 6 dogs had suspected lymph node metastases on CT. Thirteen, 28, and 11 dogs were classified as CLCSC stages 1, 2, and 3, respectively. No dogs were classified as stage 4.3.52 for 30‐60 minutes. The CBDCA administration had been proposed to the owner at the discretion of each attending or referring veterinarian. A median initial dose of 225 mg/m2  was administered, and dogs received a median of 4 doses . Grade 4 neutropenia and thrombocytopenia occurred in 10 (58.8%) and 8 (42.1%) dogs, respectively. Neutropenia with fever occurred in 6 dogs (31.6%), with all dogs recovering. Grade 3 or 4 gastrointestinal toxicity occurred in 2 dogs (10.5%). Differences in baseline prognostic factors were present between dogs that did and did not receive chemotherapy  was used in all dogs. The CBDCA was administered to 3 of the 19 dogs by a referring veterinarian. At the JSACC, CBDCA was administered IV 4‐6 times at an interval of every 3 weeks and at a dosage of 200‐250 mg/mOne dog did not receive adjuvant CBDCA but was given thalidomide postoperatively. The affected dog had multiple lung metastases on Day 193 and died on Day 220.3.6Regarding outcomes at the time of analysis, 41 dogs had died (78.8%), 6 were still alive (11.5%), and 5 were lost to follow‐up (9.6%).Progressive disease of PAC was reported postoperatively in 24 dogs (46.2%). The sites of PD after surgery were as follows (including sites affecting multiple dogs): lung (n = 13), malignant pleural effusion (n = 6), pleura (n = 3), humerus (n = 2), ribs (n = 2), dorsal mediastinum (n = 1), subcutaneous (n = 1) and right eye (n = 1). Progressive disease was diagnosed by diagnostic imaging alone in 8 dogs, and in the remaining 16 dogs, PD was diagnosed by diagnostic imaging and cytology in 14 dogs and diagnostic imaging and histopathology in 2 dogs. The median time from surgery to PD was 208 days . Twenty‐two of the dogs with PD had died by the date of the last confirmation. The causes of death in these dogs were PAC (n = 18), non‐neoplastic diseases (n = 2), and other neoplastic diseases , and in 1 case, the cause of death was unknown. Non‐neoplastic diseases were congestive heart failure (n = 1) and sepsis (n = 1) associated with pyelonephritis.Except for dogs that died in the perioperative period, 16 of 25 dogs without PD had died. The causes of death in these dogs were suspected to be non‐neoplastic disease (n = 9) and other neoplastic diseases , and the cause of death in 6 dogs was unknown. Non‐neoplastic diseases included congestive heart failure (n = 2), seizures (n = 2), immune‐mediated hemolytic anemia (n = 1), obstructive hydrocephalus (n = 1), pneumonia (n = 1), chronic kidney disease (n = 1), and protein‐losing enteropathy (n = 1).Median PFI and OST were 754  and 716 days , respectively. Median PFIs for dogs with tumor sizes ≤3 cm, >3 cm to ≤5 cm, >5 cm to ≤7 cm, or >7 cm were not reached, 754, 365.5, and 95 days, respectively  and margin , and the only potential risk factor associated with OST was age .Results of univariable and multivariable analyses investigating the association of PFI and OST with potential risk factors are shown in Tables 3.7Of the 24 dogs with PD, 12 dogs received rescue treatment: toceranib phosphate , surgery (n = 2), and CBDCA (n = 1). Surgeries on 2 dogs were performed by referring veterinarians; 1 dog underwent right eye removal, and the other underwent lobectomy of the right caudal lung lobe, both of which were diagnosed as PAC metastases on histopathologic examination. One dog that had received CBDCA as the primary rescue treatment had TOC as a further rescue treatment after recurrence of malignant pleural effusion.4In our study, Median PFI and OST for surgically resected PACs in small‐breed dogs were 754 and 716 days, respectively. In multivariable analysis, tumor size >5 cm to ≤7 cm and margin were associated with PFI, and age was associated with OST. Risk factors associated with progression outcomes and survival time in small‐breed dogs with surgically resected PACs have not been reported previously.In small‐breed dogs with surgically resected PACs, tumor size classification of CLCS was associated with PFI. No significant difference was found between tumor sizes >3 cm to ≤5 cm and >5 cm to ≤7 cm, and between >5 cm to ≤7 cm and >7 cm. However, these results potentially could be attributed to Type II statistical error resulting from inadequate sample size. In addition, because the inclusion criteria in our study were limited to dogs in which surgical resection was performed, non‐surgical treatment might have been selected for dogs with larger tumor sizes. Overall survival time was not associated with tumor size classification of CLCS. In our study, the association between tumor size classification of CLCS and OST may have been influenced by the fact that 16 of 48 dogs (33.3%), excluding deaths in the perioperative period, died without PD of PACs and half of the PD cases received rescue treatment.The median OST for our study was 716 days, indicating relatively long survival. The median survival time for surgically resected PPCs in dogs has been reported to be 92‐399 daysSurgical margins were associated with PFI in both univariable and multivariable analyses in our study. Previous studies have reported that surgical margins of PPCs or primary pulmonary neoplasia (PPN) are associated with OSTThe lymph node metastatic rate determined by histology in our study was 24.3%, which was comparable to previously reported lymph node metastatic rates ranging from 22.4% to 32.7% for PPCs.In small‐breed dogs with surgically resected PACs, we found PD in 46.2% of the dogs. The incidence of PD in dogs with PPC has been reported previously to be 43.3%‐50.7%,Our study had some limitations. First, the study was conducted at a single institution with a relatively small sample size. Second, because of the retrospective study design, some diagnostic methods were incomplete; The CT equipment was modified during the study, and some data was lost; thus, there was incomplete reassessment of CT images. The distance between the surgical margins and the tumor on histologic tissue sections was not evaluated, and therefore the prognostic impact could not be assessed. Third, the PD evaluations were problematic; 9 dogs were diagnosed with PD using only thoracic radiography to identify lung masses. In these dogs, no cytologic or histologic assessments of the lung masses were performed. We also did not perform necropsies on these dogs. It is possible that the lung masses diagnosed as PD were diseases other than PAC. Routine monitoring was recommended, but the follow‐up schedule was not standardized. Fourth, because of the long study period, treatment methods were not consistent; surgical techniques for lobectomy were changed  and rescue therapy options after PD were added .In conclusion, the tumor size classification of CLCS can be an important prognostic factor in small‐breed dogs with surgically resected PACs. Given that postoperative PD was found in 46.2% of the dogs in our study, a randomized trial with a larger sample size is warranted to determine the benefit of adjuvant chemotherapy for dogs with potential risk factors.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


Cardiovascular diseases (CVDs) constitute one of the significant causes of death worldwide. Different pathological states are linked to CVDs, which despite interventions and treatments, still have poor prognoses. The genetic component, as a beneficial tool in the risk stratification of CVD development, plays a role in the pathogenesis of this group of diseases. The emergence of genome‐wide association studies (GWAS) have led to the identification of non‐coding parts associated with cardiovascular traits and disorders. Variants located in functional non‐coding regions, including promoters/enhancers, introns, miRNAs and 5′/3′ UTRs, account for 90% of all identified single‐nucleotide polymorphisms associated with CVDs. Here, for the first time, we conducted a comprehensive review on the reported non‐coding variants for different CVDs, including hypercholesterolemia, cardiomyopathies, congenital heart diseases, thoracic aortic aneurysms/dissections and coronary artery diseases. Additionally, we present the most commonly reported genes involved in each CVD. In total, 1469 non‐coding variants constitute most reports on familial hypercholesterolemia, hypertrophic cardiomyopathy and dilated cardiomyopathy. The application and identification of non‐coding variants are beneficial for the genetic diagnosis and better therapeutic management of CVDs. Two main challenges of mapping are the amount of recombination and allelic diversity. Only allelic diversity within the recombinant inbred lineWhat also continues to present a challenge is functional annotation.1.1Deoxyribonucleic acid (DNA) is composed of both genic and intergenic regions. Exonic regions encode amino acids and are generally well conserved.2http://varnomen.hgvs.org/). In addition, we conducted a comprehensive search on published articles on non‐coding variants and some variants were extracted through this method. After collecting all the variants, total number and common variant of each separate gene were reported in our study  . Defects in the LDL‐receptor adapter protein (LDLRAP) gene lead to the recessive inheritance (AR) form of familial hypercholesterolemia. Patients with familial hypercholesterolemia have higher levels of low‐density lipoprotein (LDL). If such patients do not receive pharmacological treatments, they develop different disorders, including coronary heart diseases, premature and progressive atherosclerotic cardiovascular diseases, xanthomas, myocardial infarction and premature coronary artery diseases.LDLR, APOB and rarely PCSK9; however, variations in other genes such as STAP1 and APOE have been reported.LIPA), patatin‐like phospholipase domain containing 5 (PNPLA5), hepatic lipase (LIPC), cytochrome P450 (CYP27A1), steroid 27‐hydroxylase, cerebrotendinous xanthomatosis (subfamily XXVIIA), polypeptide 1, endothelial lipase (LIPG) and cytochrome P450 family 7 subfamily A member 1 (CYP7A1) have been identified through pedigree analysis.LDLR, APOB and PCSK9. Talmud et al. proposed a model called ‘12 SNP’, which comprised 12 common SNVs in 11 genes: NYNRIN, APOB, ST3GAL4, SORT1, ABCG8 HFE, SLC‐22, SLC‐22, LDLR, APOE, MYLIP and PCSK9. These variations were discovered through GWAS conducted by the Lipids Genetics Consortium .Familial hypercholesterolemia (OMIM ID‐143890) is an inherited disease with an autosomal dominant (AD) inheritance mode that is mainly the consequence of defects in three main genes  was associated with LDL‐C, and the second variant, located between GOT2 and APOOP5 , had a robust association with HDL‐C. The third variation was rs138326449, which was previously replicated as a splice variant in APOC3 and was associated with triglycerides and HDL‐C.LDLR, APOB and PCSK9, a novel LDLR deep intronic variant  co‐segregated with LDL‐C and the familial hypercholesterolemia phenotype.APOB that functioned as a regulatory element and contributed to serum apolipoprotein B levels. In the past 5 years, the main focus of genome analysis has been on three regions: cis‐ and trans‐regulatory elements, enhancers or promoters, and regulatory transcribed non‐coding regions.The implementation of WES in recent years has proffered better clinical management and diagnosis of less common genetic disorders. Despite such promising improvements, however, WES is capable of investigating and examining only 30% of the genome, which underscores the role of the other parts of the genome such as regulatory regions.LDLR . Moreover, LDLR had an overlap with another gene, AS1, and 34 variants that reside in this region . Three variants were reported for LDLRAP1, which is important in the rare forms of familial hypercholesterolemia  are responsible for almost 70% of the identified HCM mutations. Other genes involved in HCM pathogenesis, with a frequency of 1%–5%, are TNNI3, TNNT2, MYL2, TPM1, ACTC1 and MYL3.CSRP3) and α‐actinin 2 (ACTN2), which encode proteins vital for sarcomere function and structure, lead to HCM.VCL, PRKAG2 and TTN. These variants, with a threefold frequency, disrupted TFs and splicing sites. Analysis with different bioinformatics tools such as Genome‐Wide Annotation of Variants (GWAVA), Combined Annotation‐Dependent Depletion (CADD) and Genomiser revealed that two intronic variants  had high frequency rates in the recruited probands by comparison with a healthy population. Eight intronic variants were identified in TTN. Among them, c.31484‐286G>T (rs142156368) and c.32077+31C>G (rs72650063) were highly enriched in the cohort. The findings suggested that these intronic variants might contribute to HCM.MYBPC3, with an overall frequency of 24/1644 (2.2%), led to a frameshift and a stop codon. Additionally, c.1224‐52G>A was surprisingly frequent in that study (1.1%) in comparison with p.Arg502Trp, which was the most common variant of HCM with a prevalence rate of between 1.4% and 2%. The reason for this high frequency is not clear; nonetheless, this common variant could be the result of proband Asian ethnicity. Also in that study, a novel variant, c.1898‐23A>C, was identified in two mutation‐negative families segregated by phenotype. The reverse transcription‐polymerase chain reaction (RT‐PCR) amplification of the RNA indicated the presence of additional 473 base pairs (bp) containing the intron 19 sequence. This mid‐splice variant caused premature stop codons and mediated mRNA decay, resulting in haploinsufficiency.MYBPC3 intronic variants  act in HCM pathogenesis. Moreover, c.393‐5C>A, located in SCN5A, leads to exon skipping and one small in‐frame deletion in the S1‐D1 transmembrane of the α subunit of the cardiac sodium channel. Interactions between S1–S3 and S4 segments are a vital factor in cardiac function, and this deletion leads to the loss of function of sodium channels.Hypertrophic cardiomyopathy, characterized by asymmetric hypertrophy in the ventricular wall, is the most prevalent Mendelian cardiomyopathy (≈1:500).MYBPC3 constituted 132/470 (28.08%) of the reported variants. Three other genes  had more variants than the other involved genes  constitute the majority of candidate causal and likely candidate causal alterations in DCM.TTN accounts for 12%–15% of sporadic DCM cases and 25% of familial DCM cases.TNNI3K). Segregation analysis was conducted for the other family members, and the result indicated that this variant co‐segregated with the phenotype. Functional analysis and in silico tools categorized this variant as a deleterious one. The TNNI3K gene directly regulates cardiac contraction through the phosphorylation of 2 troponin I serine residues.TNNI3K had direct interactions with three DCM‐linked sarcomeric proteins ; therefore, the loss of function of TNNI3K resulted in myocardial structural disorders and arrhythmias.TNNI3K has a key role in both sarcomere organization and heart development; hence, decreased TNNI3K mRNA levels might lead to DCM and cardiac conduction disease.TTN, and it was associated with a cardiomyopathy logarithm of the odds (LOD score) of 3.6.FLNC truncating mutations in 20 DCM probands. Among these mutations, six mutations  were located in the non‐coding part of the genome. Previous investigations indicated that FLNC splice site variants may lead to haploinsufficiency and mediate candidate causality via this mechanism.FLNC encodes filamin‐C, which is responsible for sarcomere attachment to the plasmatic membrane.DMD (221/476 [46.43%]), and TTN (162/476 [34.03%]) comprising the majority of the reported variants, respectively  had a robust association with advanced heart failure. In contrast to rs1739843, which was associated with both ischemic and nonischemic heart failure, rs6787362 was associated only with ischemic heart failure. The results of one prospective meta‐analysis pooling the data of four previously published cohorts of the Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium indicated that 2 of 14 high‐signal SNPs were located in intronic regions: rs11118620 in LOC100129376 and rs11880198 in GNA15.Heart failure is a public health problem affecting 1%–2% of the adult population in developed countries.4.3JUP), desmocollin‐2 (DSC2), desmoplakin (DSP), plakophilin‐2 (PKP2) and desmoglein‐2 (DSG2).LMNA A/C, CTNNA, MEM43, TTN, PLN and DES have been associated with ARVC.DES mutations and identified 4 non‐coding variants: c.579‐38C>T, c.735+20C>T, c.736‐35C>A, and c.736‐11A>G. Mechanisms affecting subcellular organelles, desmin cytoskeleton and myofibril degradation can explain why these mutations result in desminopathies or myopathies.DSG2, c.2146‐1G>C and c.337‐2A>T) and 1 candidate causal variant  were reported in PKP2. That study was the largest ARVC investigation by NGS to seek causative variants linked to ARVC.DSG2 in a genetically unexplained patient. Further investigations revealed that DSG2 protein levels in the carriers of ‐317G>A were reduced compared with those in the healthy controls. Luciferase reporter gene assays also indicated that transcriptional activity was diminished in the carriers of the DSG2_A minor allele because of the impact on TGF‐β1 and an activator of the protein kinase C pathway. The presence of the DSG2_A cAMP response element‐binding protein results in the activation of TF (CREB/ATF) binding and changes in the interactions of c‐JUN.PKP2 gene (Table Another type of cardiomyopathy is arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/ARVC), characterized by sudden death, syncope, heart failure, ventricular arrhythmias and palpitations. ARVC affects between 1/1000 and 1/5000 in the general population, and it usually affects young people, especially athletes. The first mutation reported for ARVC was a defect in plakoglobin. Recent advances in genetic testing have revealed that mutations in desmosomal genes constitute more than 50% of the affected patients.ne Table .TNNT2, FLNC and MYPN are associated with RCM.+) levels, and it participates in the regulation of muscle contraction. Cardiac troponin contains three subunits , which prevent the interaction between actin and myosin in the absence of Ca2+. Defects in the troponin complex affect Ca2+ affinity, and the interactions between these proteins lead to cardiomyopathy development.ALMS1 (26/54 [48.15%])  Table .5GPC5 gene. Glypicans are a group of proteoglycans with six members binding to the outer surface of the plasma membrane through a glycosyl‐phosphatidylinositol anchor. Glypicans are involved in many intracellular pathways, including the Wnt pathway, Hedgehog developmental pathways and morphogenetic pathways; they are, therefore, candidate genes for major processes in heart development.TBX5, which is a TF expressed in the heart, was sequenced in patients with congenital heart diseases and 10 variants were identified. Among these variants, the prevalence of 1 variant, c.*1101C>T (rs6489956), was considerably different between the case group and the healthy controls. TBX5 mRNA expression was evaluated using quantitative RT‐PCR conducted on cardiac tissue samples. The results demonstrated that TBX5 rs6489956 genotypes were correlated with transcription and translation levels.GATA4 has conserved motifs that may play a role in post‐transcriptional regulation. The localization of mRNAs and proper configuration both rely on 3′‐UTR, and any defects in this region may lead to a pathological state.ELN. Supravalvular aortic stenosis is found in 60%–84% of individuals due to ELNhaploinsufficiency. This variant affects the 3′ acceptor splice‐site region in intron 17 and leads to premature splicing.CREBBP 51/264 [19.32%] and KMT2D 38/264 [14.4%]) were more frequent. For 33 genes, only one non‐coding variant was reported , BMPR2 (63/552 [11.41%]) and VHL, LOC107303340 (53/552 [9.6%]) constituted a considerable portion of the reported variants  had a significant association with sudden cardiac death (odds ratio = 4.05), and four patients had intronic variants in the SCN5A gene.The definition of sudden cardiac death by the World Health Organization is death within 1 h after symptom manifestation or 24 h after categorization as an asymptomatic patient.MYL4, NPPA and KCNQ1 are responsible for this cardiac condition.PITX2 gene were 60% more susceptible to this abnormal heart rhythm.GLA|RPL36A‐HNRNPH2 ,10The current literature features a few non‐coding candidate likely candidate causal variants associated with Mendelian disorders.PCSK9, which has a role in the inhibition of LDL‐C circulation, can be a treatment target.Functional non‐coding regions such as promoters/enhancers, introns, miRNAs, 5′/3′ UTRs and lncRNAs constitute a significant proportion of the genome harbouring candidate causal variations.In conclusion, novel genetic approaches and technologies, data sets, and the results of GWAS studies can be drawn upon to unravel the complex genetic architecture of cardiovascular diseases. The ultimate goal in the identification of non‐coding variants is to provide both a better understanding of the pathophysiological mechanisms involved in cardiovascular diseases and effective treatments.Katayoun Heshmatzad: Investigation ; methodology ; software ; writing – original draft . Niloofar Naderi: Investigation ; methodology ; software ; writing – original draft . Majid Maleki: Conceptualization ; data curation ; resources ; validation ; visualization . Shiva Abbasi: Investigation ; methodology ; resources ; software . Serwa Ghasemi: Investigation ; methodology ; software ; visualization . Nooshin Ashrafi: Methodology ; resources ; software . Amir Farjam Fazelifar: Investigation ; resources ; validation ; visualization . Mohammad Mahdavi: Investigation ; methodology ; resources ; validation ; writing – review and editing . Samira Kalayinia: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .The authors declare that they have no competing interests.Tables S1–S10Click here for additional data file.