Understanding the genetic basis of convergence at broad phylogenetic scales remains a key challenge in biology. Kingfishers (Aves: Alcedinidae) are a cosmopolitan avian radiation with diverse colors, diets, and feeding behaviors—including the archetypal plunge-dive into water. Given the sensory and locomotor challenges associated with air-water transitions, kingfishers offer a powerful opportunity to explore the effects of convergent behaviors on the evolution of genomes and phenotypes, as well as direct comparisons between continental and island lineages. Here, we use whole-genome sequencing of 30 diverse kingfisher species to identify the genomic signatures associated with convergent feeding behaviors. We show that species with smaller ranges  have experienced stronger demographic fluctuations than those on continents, and that these differences have influenced the dynamics of molecular evolution. Comparative genomic analyses reveal positive selection and genomic convergence in brain and dietary genes in plunge-divers. These findings enhance our understanding of the connections between genotype and phenotype in a diverse avian radiation. An analysis of 30 diverse kingfisher species identifies genomic signatures associated with their convergent feeding behaviors, including plunge-diving. Despite considerable research on morphological changes associated with behavioral convergence4, less is known about the genomic basis of behavioral convergence5. Part of what makes this such a challenge is the complex mapping between genotype and phenotype6. Because of this many-to-one mapping, there are many ways that genomic convergence can play out in evolutionary time. For example, we might see convergence in the same amino acid sites 7, the same genes but different amino acids 8, or the same pathways but entirely different genes9. Furthermore, the expected number of convergent substitutions across a clade is expected to be low8 owing to species-specific differences in the genotype-phenotype map10.Novel behaviors can promote phenotypic diversity by allowing organisms to use resources in new ways, thereby causing selection towards different adaptive peaks11. For example, recent work in aquatic mammals found genomic convergence in response to selective pressures associated with diving and thermoregulation13. Similarly, loss of function in genes important for taste perception in penguins was suggested to be a consequence of swallowing fish whole14. Although many birds eat fish, diving into water at high speeds  is rare, evolving in only a few avian families: Pelicanidae (pelicans), Laridae (terns), Phaethontidae (tropicbirds), Sulidae (gannets and boobies), and Alcedinidae (kingfishers). Kingfishers in particular comprise a species-rich clade of birds (116 recognized species16) with diverse diets–ranging from aquatic to terrestrial prey and including both invertebrates  and vertebrates 17–and multiple evolutionary origins of plunge-diving behavior18. Evidence for morphological convergence in beak18 and brain shape19 concomitant with these repeated shifts to plunge-diving makes kingfishers an ideal group for studying the genomic basis of convergent phenotypes.Transitions from air to water pose major sensory and locomotor challenges that affect the evolution of both genes and morphological structures involved in these functions19 and elevated speciation rates20. Species on islands experience different evolutionary pressures than those on continents21. Divergent selection among islands or between the mainland and islands might therefore drive phenotypic changes  and genomic changes. Alternatively, genetic drift might reduce adaptive potential on islands. For example, genetic diversity of island species is positively correlated with island size22. Correspondingly, genetic drift should have an outsized effect on island populations and thus decrease overall genomic convergence. Yet, comparisons of effective population size between continental and island taxa through time, as well as patterns of genomic convergence, have not been thoroughly tested across a species-rich clade. Nearly neutral theory23 predicts that small populations should experience higher rates of nonsynonymous substitutions than large populations24. This has been demonstrated empirically in island birds25, as well as more recently across a broader range of taxa26. Because of this, selection is expected to be less efficient in small island populations28.In addition to diverse foraging behaviors, many kingfishers are geographically isolated on oceanic islands recently associated with rapid rates of brain shape evolution29 might drive increased rates of positive selection on islands. We anticipate our results will have implications for our understanding of convergent evolution across scales and provide a blueprint for further functional genomics work on the sensory capabilities of birds.Here, we use whole-genome data from 30 kingfisher species to assess whether convergent evolution of plunge-diving behavior is associated with convergence at the genomic level. First, we compared temporal patterns of population demographics to test the prediction that island species would have more fluctuations and lower effective population sizes than continental species. Second, we compared genomic convergence associated with a phenotype  that has evolved in both continental and island systems to test the prediction that genetic drift associated with small island populations should lessen the strength of genome-wide convergence. Third, we compared levels of positive selection between continental and island plunge-diving species to weigh relative support for drift and selection operating on islands. Given that plunge-diving behavior has emerged independently in both continental and island-dwelling kingfishers, we hypothesized that positive selection would act more strongly on genes related to plunge-diving behavior in continental kingfishers as compared to island species. An alternative hypothesis is that smaller body sizes or environmental heterogeneity among islandsTodiramphus chloris collaris) genome30, we fit neutral models of sequence evolution in PAML (model M0) to estimate molecular branch lengths for each annotated gene30. We then used synonymous mutation rates for each species as input in a pairwise sequentially Markovian coalescent (PSMC) analysis of demographic history through time. Using a recent time-calibrated phylogeny of kingfishers20, we identified four origins of plunge-diving behavior and five origins of island-dwelling , yet there was significant divergence in curve shape between continents and islands  might be driven by convergent genomic changes, as has been recently documented in boas34. However, reduced population sizes on islands might also result in lower convergence in genomes of island taxa. This is because stronger genetic drift in small populations reduces the effectiveness of selection, decreasing the probability of independently evolving the same phenotypic or genetic mutations35. Based on this idea, we predicted that island kingfishers would show little evidence for genome-wide convergence.Using a large-scale genomic dataset of 30 kingfisher species, including a newly annotated Collared Kingfisher  for plunge-diving behavior was 0.08  and the number of divergent substitutions . Bayesian phylogenetic analyses showed that plunge-diving lineages have significantly more convergent amino acid changes than would be expected under a neutral model of sequence evolution in which convergent substitutions scale with divergent substitutions similarly for both plunge-diving and non-plunge-diving lineages Fig. , Table 138 and protein convergence tests39 , cognition , chromosome segregation , retina homeostasis , and microtubule-based movement   have been linked to lower visual acuity in mice46. Other adaptively convergent genes include CENPJ, a gene that has been linked to decreased brain size in primates47, and MAPT, a gene that codes for tau proteins that stabilize microtubules in the brain48. Proper functioning of MAPT has been suggested to mitigate concussion-related brain injuries during foraging in woodpeckers49. This is interesting given that kingfishers show convergent evolution in brain shape that is further tied to their diversification19, along with evidence of convergence at the genomic level  showing support for convergent rate shifts in plunge-diving kingfishers  and in what parts of the genomes are assessed . Overall, studies showing high numbers of rate shifts in genic and regulatory regions tend to be those involving either extreme morphological changes 63 or behaviors expected to have strong downstream effects on morphology 65. Changes in behavior likely involve several trait systems , which might explain the emerging trend of morphological convergence being linked to stronger signatures of molecular convergence than behavioral convergence. Consistent with past work, we find overlap but also differences in sets of genes identified by different methods . Together, these results elucidate genomic bases of behavioral convergence and set the stage for further work on functional genomics and sensory system evolution.https://github.com/ibest/HTStream). We then used FASTP v. 0.20.166 to assess read quality. The majority of reads had low adapter content (<1%) and were of high quality (Q > 30).We were loaned tissues from frozen samples at the Field Museum of Natural History . Bases with low coverage (<5X) or low mapping quality (Q < 30) were N-masked in the final consensus sequences. We used the “−I” option in BCFTOOLS mpileup to exclude INDELS because we wanted genomic coordinates to be the same as the reference genome72. This allowed us to use the reference genome annotations (GFF format) obtained from GeMoMa and extract coding sequences for each individual. All scripts for data analyses are available at Zenodo73.We aligned cleaned reads to the draft collared kingfisher genome74 in which we N-masked all variable sites and re-mapped reads of three species differing in their phylogenetic divergence to the reference collared kingfisher  from a recent study on penguin genomes that used a similar assembly approach to ours72. To plot effective population sizes (Ne) as a function of time, we used published R code76 to input data into R with a generation time of 4.8 years (IUCN 2021) and mutation rates taken from the average substitution rate estimated with the PAML M0 model (see below). These rates were comparable to the value used for the common kingfisher  in a larger study on the evolution of mutation rates77. To statistically test whether the shapes of PSMC curves differ between islands and continents, we used a multivariate clustering approach. Briefly, we first determined the time range with Ne data for all 31 species. Next, we log-transformed the Ne values. Finally, after converting these data into an array, we used the procD.pgls function32 in the R package geomorph78 to run a distance-based phylogenetic generalized least squares analysis, with either insularity or feeding behavior as a predictor and a recently published kingfisher timetree20.To understand population demographic fluctuations over time, we used PSMC v. 0.6.59. Briefly, we determined the total number of convergent amino acids  and divergent amino acids  for each pair of lineages. In calculating convergent and divergent sites, we opted to treat lineages as the whole path from an ancestor to tip rather than single branches79. We then used phylogenetic linear mixed models implemented in MCMCglmm80 to test whether the number of convergent changes was explained by feeding behavior, island dwelling, divergence time, number of divergent amino acid substitutions, or the interaction between divergent substitutions and divergence time. Given potential inflation of effect sizes due to non-independent samples , we treated each lineage in a pairwise comparison as a random effect in the model, after shuffling lineage pairs to ensure even occurrence of each lineage in a comparison81. We also accounted for phylogenetic non-independence by treating the MRCA of each pair as a random effect, with an expected covariance structure determined by the phylogenetic tree calculated with the ginverse function in MCMCglmm. We ran the model for 106 generations and discarded the first 25% as burn-in. Phylogenetic signal was calculated as the posterior mean of the node random effect divided by the sum of all random effects scaled by (1/π2/3)81.We tested the hypothesis that plunge-diving behavior results in genome-wide convergence following Sackton et al.39. This model tests for signatures of convergence in all residues of a protein. We set up the cutoff of the posterior probability that a given residue is evolving convergently in plunge-diving lineages to 0.8. Genes that showed support for convergence were retained and used in downstream enrichment analyses (see below).To further test which genes were convergently evolved in plunge-diving kingfishers, we used the program PCOCdN) nucleotide substitutions to that of synonymous substitutions (dS) for aligned homologous protein sequences37. Support for ω > 1 is interpreted as evidence for positive selection, whereas ω < 1 suggests purifying selection  and ω = 1 indicates neutral evolution. For each aligned coding sequence, we used PAML37 to fit a null model (model M0) of sequence evolution assuming a single ω across the genome and phylogeny. For this model, we used a fixed species tree previously estimated using a large UCE data set20. To test whether some genes show a pattern consistent with positive selection, we compared two sets of site models that allow ω to vary across sites in a protein but not across the phylogeny. The first set of comparisons was between a nearly neutral model that assumes two site categories  and a positive selection model that allows three site categories . The second comparison of site models was between a model that allows ten site categories with ω < 1 along a beta distribution (model M7) and a model that allows 11 site categories, including one with ω > 1 (model M8). Finally, to test the hypothesis that some genes and sites show positive selection in species that plunge dive into water relative to species that do not, we fit a branch-site model that allows for ω > 1 only in predefined foreground  lineages (model BS2) to one that assumes ω = 1 for both plunge-diving and non-plunge-diving species (model BS1). For all genes and models, we used the molecular branch lengths estimated under the neutral model M0 to speed up computational time9. We compared sets of models using likelihood ratio tests and retained ancestral state estimates under the M0 model for convergent evolution tests (see below). Models were run iteratively over all annotated genes using custom R scripts73. To account for multiple comparisons, we adjusted P values using false discovery rates (FDR) and retained all genes with significant evidence (FDR < 0.05) for positive selection. Sets of positively selected genes for each model comparison were retained and used in subsequent gene enrichment tests.Positive selection in DNA sequences is typically tested by estimating ω, the ratio of the rate of nonsynonymous . To account for this effect, we further fit Hyphy RELAX models38 with the same trees used in our branch-site tests. This model is more stringent in testing whether convergent shifts in amino acid dN/dS ratios are sensitive to background lineages. We then filtered branch-site results to include only those genes that also showed significant support for positive selection in foreground lineages under the RELAX model. In total, we confirmed that 308 out of 1426 genes (21.6%) that showed support for positive selection in plunge-divers under the branch-site model were also significant in the more stringent RELAX model.We are aware of recent work suggesting that branch-site models can overestimate the number of significantly converged genes83 in HyPhy. This test conducts significance tests for each branch and gene. To compare the set of positively selected genes associated with plunge-diving behavior among CODEML models and HyPhy models, we retained gene-specific aBSREL tests that showed any significant shifts along a branch  model84. For the latter, we filtered genes to only include those that showed support for adaptive convergence in two or more pairs of species that have convergently evolved plunge-diving. See Supplementary Note We used several approaches to identify sets of genes putatively convergent and positively selected in plunge-diving kingfishers. In addition to the previous analyses, we also used (i) a modified branch-site test that accounts for the issue of positive selection in background species in the codeml BS2 model and (ii) a recent method for detecting Combinatorial SUBSTitutions of codon sequences (CSUBST) that identifies genes in pairs of species that are both convergent and under positive selection40 to analyze the co-occurrence of different gene functions among species. To test whether positive selection is operating more strongly in island-dwelling kingfishers, we calculated the number of PSGs for each species as well as the amount of overlap between pairs of species. Since small body sizes are associated with elevated rates of molecular evolution41, we included body mass (ln grams) as a covariate in the model, along with insularity, plunge-diving behavior, and their interactions with body size. We determined the best-fitting model using bi-directional variable removal based on AIC scores in the phylostep function85. For gene overlap, we used Bayesian mixed models implemented in MCMCglmm80 with number of overlapping PSGs as the response; insularity and plunge-diving behavior as predictors; and species 1, species 2, and the node of the most recent common ancestor in the phylogeny as random effects (see ref. 81 for details).We tabulated the number of positively selected genes for each plunge-diving species branch from the aBSREL results and then used metascape40 with default settings and a custom background of collared kingfisher genes. This analysis links genes with their putative functions  and tests whether a set of target genes with a given function is larger than expected by chance from a random subset of GO terms. We visualized overlapping target genes from different analyses using Venn diagrams and visualized gene networks again with metascape40. Significance of enriched gene functions was estimated using the FDR metric.To understand whether sets of positively selected or convergent genes were associated with certain molecular functions, we used metascape54. To assess whether regulatory regions evolve faster in plunge-diving or island-dwelling kingfishers, we identified a set of avian-specific highly conserved noncoding elements (CNEs)86. Since these elements are relative to the chicken genome, we used LAST70 to align the draft collared kingfisher genome30 to the chicken genome. We then used liftOver87 to convert chicken genome coordinates to collared kingfisher coordinates and extracted DNA sequences with bedtools getfasta88. In total, we located 235559 out of 265983 (88.6%) chicken CNEs in the collared kingfisher genome. After filtering out CNEs smaller than 50 bp and removing badly aligned regions with trimAL89, we fit estimated molecular branch lengths using the BASEML free model37. This model lets each branch assume its own rate of molecular evolution, a requirement of downstream RERconverge analyses. In total, we obtained a final set of 39618 trees. For those CNEs that showed significant support for a convergent rate shift in focal lineages, we used bedtools closest to identify the closest annotated gene in the collared kingfisher genome and input these annotations into GO term enrichment analyses.Recent work has found that regulatory regions of the genome, rather than coding regions, are related to shifts in phenotype90 on sets of gene and CNE trees. Importantly, these analyses utilize overall rates of molecular evolution and do not account for variation in specific sites along a gene or CNE. To assess significance of relationships between rates of molecular evolution and phenotypic changes, we used a phylogenetic permulation approach recently shown to be more rigorous than P values derived from parametric estimates91. This method simulates evolutionary histories of a given phenotype  and then generates a null distribution of correlation values between the phenotype and rates of molecular evolution. Statistical significance is then assessed by determining the proportion of null values that are less extreme than the empirical correlation for a given gene. We did 500 permulations and corrected P values using FDR.To detect significant accelerations or decelerations associated with plunge-diving behavior, we ran RERconvergen = 11938 genes tested). Bayesian phylogenetic linear mixed models were used to analyze genomic convergence and gene overlap (n = 465 species pairs). Phylogenetic generalized least squares analysis was used to analyze positively selected gene counts (n = 30 species). All statistical analyses were performed in R v. 4.2.3. Code was written in the R markdown format to allow for reproducibility of all analyses and generation of figures from raw data sets.Maximum likelihood approaches were used to determine support for significant positively selected genes, with correction for multiple tests using FDR (Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Preclinical studies have suggested that mTOR pathway signaling may be a potential therapeutic target for childhood ependymoma.A phase II clinical trial  of single-agent everolimus was performed to test the hypothesis that mTOR pathway inhibition would result in tumor responses for children with recurrent and/or progressive ependymomas.2/day (each cycle = 28 days) that was titrated to achieve serum trough levels of 5-15 ng/ml. Overall, everolimus was well tolerated; except for a single event of grade 3 pneumonia, all adverse events were grade 1-2. No objective tumor responses were observed. Participating subjects experienced tumor progression and discontinued therapy after a median of 2 cycles of therapy .Eleven subjects  were enrolled on the study. Ten primary tumors were located in the posterior fossa and one primary tumor was located in the spinal cord. Eight of 9 tumors were PF-A subtype epenydmomas. All subjects were treated with oral everolimus 4.5 mg/mEverolimus does not appear to have activity for children with recurrent or progressive PF-A ependymoma. Pediatric ependymoma often has immunohistochemical evidence of mTOR pathway activation.Everolimus does not appear to have activity for recurrent or progressive PF-A ependymoma.Surgery and repeat radiation therapy are often utilized for children with recurrent ependymoma; chemotherapy has not been demonstrated to impact overall survival for this disease. Because preclinical data suggest that the mTOR pathway is activated in ependymoma, a phase 2 study was conducted to evaluate the response rate and tolerability of everolimus against pediatric ependymoma. The drug was well tolerated. Unfortunately, no responses were observed among 11 enrolled patients (8 of 9 tested tumors were PF-A ependymoma).1–4 The prognosis for these children is dismal and the majority of children with recurrent or progressive ependymomas will experience subsequent repeated episodes of tumor recurrence and progression and eventually succumb to their tumor within 8.7-24 months.5–12 Several groups have reported prolonged survival among children with recurrent ependymoma who have been treated with repeat surgery and radiation therapy.13–16 However, treatment options for many children with recurrent ependymomas are frequently limited by an inability to achieve a complete tumor resection at tumor recurrence and prior radiation exposure. Although chemotherapy occasionally demonstrates anti-tumor activity against recurrent pediatric ependymoma, these responses are rarely durable.17Approximately 30%-40% of all children who are diagnosed with a WHO grade II or grade III ependymoma will suffer tumor recurrence or progression.18 Everolimus, an oral protein kinase inhibitor of the mTOR pathway, has been approved by the Food and Drug Administration of the United States and the European Medicines Agency of the European Union for advanced breast cancer, neuroendocrine tumors, renal cell carcinoma, renal angiomyolipoma associated with tuberous sclerosis complex (TSC), and subependymal giant cell astrocytoma (SEGA) associated with TSC.19 Preclinical studies have suggested that inhibition of the mTOR pathway can induce autophagy in mouse ependymoma cell lines, and mice transplanted with ependymoma cell lines and where treated with sirolimus had prolonged survival.20 Other preclinical studies demonstrate that the mTOR pathway is upregulated in posterior fossa ependymoma.22 Published clinical data evaluating the use of mTOR inhibitors for recurrent ependymoma consists of 1 case report and 3 phase I trials, and includes some encouraging durable responses.22–25 Clinical trials of children treated for recurrent solid tumors have demonstrated that everolimus is very well tolerated.25 In Franz’s study of children with subependymal giant astrocytomas treated with everolimus, only grade 1 or 2 adverse events were reported and no patients had to discontinue everolimus because of toxicity.26The mammalian target of rapamycin (mTOR) serine/threonine kinase signal transduction pathway plays a key role in regulation of metabolism, growth, and proliferation of many different cancers.The hypothesis of this study was that children with recurrent and/or progressive ependymomas would respond to mTOR pathway inhibition and experience minimal toxicity. The primary aim of this study was to estimate the objective tumor response rate of everolimus against recurrent or progressive ependymoma. Secondary aims of the study included duration of tumor response; progression-free survival (PFS) and event free survival (EFS); safety and tolerability of everolimus; and identification of biomarkers of tumor response.27This study was a single-arm, open-label study of single-agent, daily oral everolimus for children with recurrent and/or progressive ependymoma conducted at 4 institutions in the United States . The study was reviewed for issues of patient safety and confidentiality and approved by the Institutional Review Board of the University of Texas Southwestern Medical Center and by all collaborating institutions. Informed consent  was obtained prior to all study-related procedures in accordance with federal regulations. Study data were collected and managed using REDCap (Research Electronic Data Capture) tools hosted at UT Southwestern.3, hemoglobin >9.0 g/dl, and platelets ≥100,000/mm3), hepatic , total bilirubin ≤2.0 mg/dl, and INR ≤2), lipids (fasting cholesterol ≤300 mg/dl and triglycerides ≤2.5× ULN) and renal function (serum creatinine ≤1.5× ULN). Subjects must have recovered from the acute effects of all chemotherapy, immunotherapy, biological therapy, radiation therapy, including at least 12 weeks since prior radiation therapy.Children and young adults with recurrent and/or progressive classic ependymoma (WHO grade II) or anaplastic ependymoma (WHO grade III) were eligible for this study if they met all of the following inclusion criteria: Age ≥2 years to ≤21 years of age at study entry; Lansky play-performance scale or Karnofsky performance status of ≥50% for subjects for <16 years or ≥16 years of age, respectively; measurable tumor that could accurately be measured in 2 dimensions; and adequate bone marrow  after initiation of therapy, everolimus trough serum concentrations were obtained immediately prior to the daily dose and subsequent doses was adjusted upward or downward to achieve a target trough concentration between 5 and 15 ng/ml.28Everolimus (Afinitor®) was provided by Novartis Pharmaceuticals Corporation. Participating subjects took everolimus in the form of dispersible tablets  as a starting dose of 4.5 mg/m29 Secondary objectives included duration of tumor response; PFS and EFS; safety and tolerability of everolimus, was collected for all study participants. All subjects who were enrolled on the clinical trial were evaluable for toxicity. Toxicity was assessed according to the NCI Common Toxicity Criteria for Adverse Events, version 4.03.30Response was assessed by magnetic resonance imaging of the brain and spine. Baseline assessments were performed within 14 days of study entry and subsequently every 8 weeks  for 1 year. The primary efficacy variable, tumor objective response rate, was evaluated using the International Society of Pediatric Oncology Brain, Tumor Subcommittee for the Reporting of Trials criteria.This trial was a prospective, single-arm, open-label, multi-center phase II trial utilizing Simon’s two-stage optimal design of an objective response rate  of 10% and 10% alpha. The sample size for the first stage was 11 subjects. If there was no ORR seen in these 11 subjects, stopping rules required the study to be stopped due to futility. Otherwise, an additional 7 subjects were to be accrued for a total of 18 patients. The null hypothesis would be rejected if 4 or more ORRs were observed in 18 patients. This design yielded a type I error rate of 10% and power of 80% when the true ORR is 30%.The primary outcome was maximum tumor response at any timepoint during study participation. Secondary outcomes are limited by small sample sized and are thus reported with descriptive statistics and included: Objective response rate include overall survival (OS); partial response. Descriptive statistics were used to report dose-limiting toxicities and results of correlative immunohistochemistry studies.31 Exploratory biomarkers of mTOR pathway activation and sensitivity to mTOR inhibitors, including phosphorylated S6235/236, phosphorylated S6240/244, phosphorylated PRAS40 (pT246), PTEN expression, and phosphorylated 4EBP1, were performed according to previously reported methods.22 Staining for the biomarkers was either nuclear or cytoplasmic and scored as 1+: weak, focal/multifocal, 2+: weak/diffuse, 3+: strong, focal/multifocal; 4+: strong/diffuse.Hematoxylin and eosin stained slides were reviewed and unstained slides were obtained for additional studies. Immunostaining for H3 K27me3 was performed to determine ependymoma subtype by previously reported methods.Eleven subjects were enrolled in the clinical trial from 2015 to 2018 . Four  (e.g. time of first disease assessment). Median survival after study enrollment was 10.5 months (range = 3.5 months—not reached) .235/236, phosphorylated S6240/244, phosphorylated PRAS40(pT246), PTEN, and phosphorylated 4EBP1, were either weakly (1+/2+) or strongly (3+ or 4+) positive in the majority of tumors examined. Eight of 9 tumors demonstrated loss of nuclear staining for H3 K27me3, indicating that nearly all tumors were of the PF-A subtype.Tissue from nine enrolled subjects was available for biomarker evaluation . Immunos22 Subsequently, a second child with a recurrent ependymoma was treated with sirolimus and oral etoposide and had a near-complete response of 18 months duration. Interestingly, a recent report from Adolph et al. examined a pooled cohort of 138 pediatric patients with recurrent ependymoma from two multi-institutional HIT-REZ trials (HIT-REZ 97 and HIT-REZ 2005) and a registry study (HIT-REZ registry). In this study, treatment with sirolimus had the best response rate (50%) and longest median time to tumor progression, with the strongest impact found when sirolimus was used as a monotherapy. However, treatment with sirolimus did not translate into a survival advantage for these patients. From these reports, it would be reasonable to test the hypothesis of whether a related mTOR pathway inhibitor, everolimus, might have activity against ependymoma. Unfortunately, no tumor responses were observed among patients enrolled on this trial.This study indicates that everolimus, an inhibitor of the mTOR pathway, does not have anti-tumor activity for children with recurrent PF-A ependymoma. The rationale supporting this clinical trial was based upon both preclinical and clinical evidence for activation of the mTOR signaling cascade in pediatric ependymomas. In 2011, our institution published a case report describing a young child with a multiply recurrent ependymoma who had been treated with 4 chemotherapy regimens and two courses of radiation therapy and had an objective tumor response of 18 months duration to sirolimus.235/236, phosphorylated S6240/244, phosphorylated PRAS40(pT246), PTEN, and phosphorylated 4EBP1  pediatric ependymomas were immunoreactive for phosphorylated S6, a biomarker that has been shown to be associated with response to mTOR pathway-targeted therapy.34The majority of tumors from subjects enrolled on this clinical trial had immunohistochemial evidence of mTOR pathway activation, as demonstrated by positive immunostaining for phosphorylated S6ed 4EBP1 . This ob35–37 PF-A ependymomas have less durable responses to surgery and radiation therapy and have dismal prognoses when compared with PF-B ependymomas.38 PF-A tumors overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3 K27me3), similar to the oncohistone H3 K27M.39 A report by Panwalkar et al. demonstrated a strong correlation between loss of nuclear staining of H3 K27me3 and the PF-A ependymoma subtype with high sensitivity and specificity.31 As might be expected, loss of nuclear staining of H3 K27me3 was associated with poor survival among ependymomas.31Eight of 9 tumors from participating subjects enrolled on this study demonstrated loss of nuclear staining for H3 K27me3, demonstrating that nearly all tumors from children enrolled in this study were of the PF-A ependymoma subtype. Recent studies have identified two primary subgroups of childhood posterior fossa ependymomas, referred to as Group A (PF-A) and Group B Ependymomas (PF-B), based upon gene expression profiles and CpG island (CpGi) methylation phenotype (CIMP) status.40 Also, the study population largely consisted of PF-A tumors (which have a by far worse outcome with standard primary therapy) that has been heavily pretreated with multiple regimens of both chemotherapy and radiation therapy (none of which interact directly with the mTOR pathway), although this is rather typical for a population of pediatric patients with recurrent ependymoma who enroll on clinical trials. The study is unable to comment upon whether mTOR pathway-targeted therapy has activity against PF-B ependymoma or newly diagnosed tumors. Finally, the understanding of and development of new agents which target the mTOR pathway is rapidly advancing.41 It is possible that a newer generation mTOR targeting agent may yet have activity against pediatric ependymoma.Limitations of this study are several and include the following: There is poor understanding of the inconsistency between the identified preclinical mTOR pathway activation and the absence of anti-tumor activity of everolimus against pediatric ependymoma. Although multiple genetic and protein predictive biomarkers within both tumors and blood of mTOR activity have been identified across disparate tumor types, an ideal biomarker remains unclear for ependymoma.42 Attempts to define the role of chemotherapy for children with relapsed ependymoma has been disappointing and thus it has been recommended that chemotherapy should be reserved for use in the setting of a clinical trial.5 Repeated surgery and repeat radiation therapy likely prolong survival for children with recurrent ependymoma, but unfortunately may not achieve cure.13–15 Clearly, much more work is needed to better understand the biology of ependymoma including subtypes, as it would seem plausible that different subtypes may respond to therapies differently.43 Thus, future clinical trials for recurrent ependymoma should include molecular subtyping and stratification of tumors. More prospective clinical trials of novel agents and treatment strategies for both the newly diagnosed and recurrent ependymoma will be necessary to improve the outcomes of these patients.In conclusion, although the majority of tumors from subjects in this study had immunohistochemical evidence suggesting mTOR pathway activation, this study suggests that single-agent everolimus has no meaningful activity against pediatric PF-A ependymoma. It remains unknown whether mTOR pathway-targeted therapy may have a role against other subtypes of ependymoma, such as PF-B, supratentorial ependymomas or spinal cord ependymomas. Chemotherapy may have activity for newly diagnosed ependymoma, in either a neo-adjuvant strategy to facilitate a second-surgery attempt at complete resection or as maintenance chemotherapy.


BackgroundIntrathecal morphine (ITM) provides effective postoperative analgesia for patients undergoing total knee arthroplasty (TKA) under spinal anesthesia (SA). The management of pain in patients undergoing TKA has remained a challenge for anesthesiologists, as no single regional anesthesia technique is adequate with regard to balancing effective analgesia with minimal side effects. Severe postoperative pain following TKA has been shown to negatively impact patient outcomes and mortality. This study is aimed to describe the effect of intrathecal morphine in patients undergoing total knee arthroplasty.MethodsThis was a retrospective, descriptive, and single-center study conducted on patients undergoing total knee arthroplasty from June 1, 2022, to June 1, 2023. The sample size consisted of 50 patients who were 18 years and older, American Society of Anesthesiology (ASA) class 1-3, and patients who had received either 150 mcg  or no ITM dose under spinal anesthesia. Oral morphine requirement (OME) and visual analog pain scale (VAS) were used to assess pain in the first 24 hours after surgery.ResultsThe experimental group had significantly lower OME usage in the post-anesthesia care unit (PACU) (p < 0.001) and at six hours (p = 0.040) postoperatively. At 12 hours and 24 hours postoperatively, the two groups had similar OME use . The experimental group had significantly less total OME use over the first 24-hour postoperative period. The experimental group had significantly lower pain scores in the PACU (p < 0.001) and at six hours postop (p = 0.002); there were no significant differences between groups at 12- and 24-hours postop. The ambulation distance was clinically significant and better in the ITM group but was not statistically significant (p = 0.080). There was no difference between groups in the incidence of postoperative nausea and vomiting (PONV).ConclusionThe careful use of ITM with the optimal dose offers an effective addition to regional anesthesia for improved analgesia with minimal side effects. The 150 mcg ITM dose provided good analgesic effects with longer duration and was not associated with respiratory depression. As the population in the United States and other developed countries continues to experience increases in life expectancy and body mass index (BMI), osteoarthritis will continue to be a prevalent and life-altering disease -3. In thEarly ambulation is a crucial endpoint following TKA, as it is well known to contribute to improved functional outcomes, reduced hospital length of stay, and decreased risk of developing deep vein thrombosis (DVT) and pulmonary embolism (PE) -5,7. As According to Memtsoudis et al., in a consensus statement published in 2021, by the International Consensus on Anesthesia-Related Outcomes after Surgery (ICAROS), peripheral nerve blockade (PNB) may be the optimal solution for effective pain control, a minimized side-effect profile, and a significantly lower odds ratio of adverse outcomes compared with total joint replacements performed without PNB . Of noteThe use of intrathecal morphine for total joint replacement shows conflicting data. In 2022, a meta-analysis by the Procedure Specific Postoperative Pain Management (PROSPECT) working group concluded the use of intrathecal morphine for total knee replacement to be better than placebo but equivalent to single-shot and continuous nerve block catheters . For theTo date, no studies have formally compared continuous catheter adductor canal blocks (CCACB) versus single-shot adductor canal blocks (SSACB) plus intrathecal morphine (ITM) for patients undergoing primary knee arthroplasty. In developing a new Enhanced Recovery Program (ERP) for our TKA patient population, our department changed from providing CCACB plus spinal anesthetic (SA) alone to providing SSACB plus SA with ITM. This practice change presented an opportunity to retrospectively study our pre- and post-implementation patient populations. We hypothesized that the combination of SSACB plus SA with ITM would be non-inferior to that of CCACB plus SA alone in regard to postoperative pain, side effects, and patient satisfaction while simultaneously improving practice efficiency. To test our hypothesis we conducted this retrospective study of our TKA patient population, analyzing pre- and post-implementation data for ITM.In this, our institutional review board (IRB)-approved retrospective study, we selected two groups of patients who had undergone primary total knee replacement. One group had undergone primary total knee replacement with continuous adductor canal block (CCACB) under spinal anesthesia without intrathecal morphine (control group). The other group had undergone primary total knee replacement with a single-dose adductor canal block (SSACB) under spinal anesthesia with the addition of intrathecal morphine .The adductor canal block (ACB) was performed preoperatively in our regional block area. Patients were consented for the block and the site confirmed during time-out for the block. Both groups received 50-100 mcg of intravenous fentanyl for sedation during the block. The patient was positioned in the supine position, and a linear high-frequency probe was used to identify the target. Both groups received a bolus of 25 ml of 0.5% ropivacaine for the adductor canal block. The nerve block catheter was placed in the CCACB group after the bolus. Both groups received intraoperative periarticular infiltration of the knee joint by a surgeon. The CCACB group received a continuous infusion of 0.2% ropivacaine at the rate of 6 ml/hour in the postoperative period. We utilized a random numbers generator to select 25 patients from the TKA patient pool from each group. Inclusion criteria consisted of any adult patient 18 years or older, who underwent primary total knee arthroplasty. Rescue pain medication was given orally either as oxycodone or hydrocodone and oral morphine equivalent (OME) was calculated for comparison between the two groups. Exclusion criteria included patients with a past medical history of chronic pain or chronic opioid use greater than 30 days and patients undergoing any surgeries other than primary TKA.Data were summarized using means and standard errors (SE) for continuous outcomes or counts and percentages for categorical outcomes. T-tests and chi-square tests, as appropriate, were used to compare the groups on the study endpoints. A p-value < 0.05 was considered statistically significant. SAS version 9.4  was used to conduct all statistical analyses. Data are expressed as means and standard errors (continuous variables) or counts and percentages . The Wilcoxon rank-sum test was used to compare oral morphine equivalent (OME), pain scores, and ambulation distance.Data were available for 50 subjects. Twenty-five subjects received intrathecal morphine  and 25 received standard of care (control). Demographic information for the subjects is shown in Table The two groups are compared on the study endpoints in Table The experimental group had significantly lower OME usage in the post-anesthesia care unit (PACU) p < 0.001) and at six hours (p = 0.040) postoperatively  and at six hours postop (p = 0.002); there were no significant differences between groups at 12- and 24-hours postop Figure . The ambThere was no difference in the use of antiemetic medications between the two groups. We did not find any incidence of respiratory depression, pruritus, and the use of naloxone in either group.This retrospective study demonstrates that compared to a CCACB plus SA alone, an SSACB plus SA with ITM provides effective analgesia after primary TKA. The decision to use a dose of 150 mcg of morphine for our intrathecal injection was based on several studies that suggest this dose provides maximal analgesia and minimal side effects .For our primary efficacy outcome, our findings suggest that ITM provides both clinically meaningful and statistically significant analgesia in PACU at 6-12 h when compared to control. We used oral morphine equivalent (OME) as a more objective measure of analgesia efficacy. The effect was consistent with lower pain scores in the ITM group at earlier time points. The duration of the effect of ITM is estimated to be up to 16 h . This isWe also demonstrated that postoperative analgesia was more effective in the ITM group even in the presence of a regional anesthesia block when compared to the control group. Both groups received intraoperative periarticular infiltration of the knee with the same volume and concentration of local anesthetic. The superior analgesia provided by ITM questions the utility of continuous adductor canal block compared to single shot block. At our institute, most of our primary TKA patients are going home the day after surgery, and early ambulation is encouraged for better outcomes. The ITM group has shown increased ambulation distance when compared to the control group. This may be attributed to better pain control in the ITM group with less opioid consumption in the first 24 hours.The incidence of PONV, pruritus, respiratory depression, and urinary retention was similar between groups; however, our study was not sufficiently powered to determine whether this finding was statistically significant. Intrathecal morphine has been shown to be associated with an increased risk of PONV, pruritus, and urinary retention, but without any impact on the duration of hospital stay . AlthougBecause of concerns for sedation, hypoxia, and respiratory depression guidelines for the practice of neuraxial opioids have recommended that patients be continuously monitored for 24h after receiving ITM . RespiraThere are some limitations to this case series such as small sample size, retrospective design, and publication bias . The samThere is strong evidence that intrathecal morphine provided effective analgesia after lower extremity arthroplasty. Our study concludes that ITM of 150 mcg for total knee arthroplasty under spinal anesthesia provided improved postoperative analgesia with reduced opioid consumption. Our study warrants no more concerns of PONV, pruritus, or respiratory depression with this dose of ITM and requires standard postoperative care. The ideal dose of ITM for lower extremity arthroplasty under spinal anesthesia is unknown and warrants further investigation.


Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults , microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods.Diversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.The online version contains supplementary material available at 10.1186/s13104-023-06451-7. There has been an increased number of studies exploring the contribution of the gut microbiota to health and disease over the past decade . The potFactors that affect the downstream assessment of microbial composition, such as sample collection techniques, storage conditions, DNA extraction processing methods, selection of 16s rRNA hypervariable regions for library preparation and sequencing , and datThe potential for different DNA extraction methods to contribute variation to assessment of microbial composition is documented in a range of sample types , 10 and This study involved a comparative analysis of four different DNA extraction methods for downstream assessment of faecal microbial composition. Samples  were collected using faecal collection kits which included a 70 mL faecal collection cup with scooped lid  and flushable collection paper  and were to be free of water and urine. Faecal samples were returned to the laboratory within 24 h of collection and stored at -80 °C until analysis. The study was undertaken in accordance with the Declaration of Helsinki. Individuals were otherwise healthy community-dwelling adults and provided written informed consent prior to participation. Ethical approval was provided by the Griffith University Human Research Ethics Committee (ref#: MED/19/15/HREC).Upon thawing, faecal samples were homogenized in phosphate buffered saline, subsampled at equal volumes for extraction, and processed using one of the following DNA extraction methods: (i) an established in-house method involving repeated cycles of chemical and mechanical (bead beating) lysis, salt and alcohol precipitation and subsequent nucleic acid purification extraction as has been previously reported   and Shannon Diversity Index (p = 0.79) did not differ between DNA extraction methods  are considered preferable in achieving a more accurate microbial compositional profile.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Neoadjuvant chemoradiotherapy followed by esophagectomy is the standard of care for locally advanced esophageal squamous cell carcinoma (ESCC). However, approximately 30% of patients still develop distant metastases and have a high incidence of treatment-related adverse events. Immunotherapy, as a new modality for anti-cancer treatment, has shown promising clinical benefits for patients with ESCC. The synergistic effects of immunotherapy and radiotherapy make their combination promising as neoadjuvant treatment for locally advanced ESCC.All participants who meet the inclusion criteria will be enrolled after signing the informed consent form. Patients with thoracic segment esophageal cancer with clinical stage T2–3 N0 M0 or T2–3 N + M0 will be included. A total of 25 patients are to be recruited for the study. Twelve patients will be recruited in phase I, with at least two achieving major pathological response (MPR) before entering phase II. They will be treated with radical surgery within 4–8 weeks after the completion of two cycles of neoadjuvant radiotherapy in combination with camrelizumab according to the study schedule. The primary endpoint is the major pathological remission rate of all per-protocol patients. The secondary endpoints are the R0 resection rate, pathological complete remission rate, and adverse events. The interim analysis will be conducted after 12 patients have been enrolled. The trials will be terminated when more than two treatment-related deaths occur or fewer than five patients have major pathological remission.We designed this prospective single-arm phase II clinical study to evaluate the combination of camrelizumab and standard radiotherapy as preoperative neoadjuvant therapy for patients with resectable ESCC as part of the quest for better treatment options for patients with locally advanced ESCC.www.clinicaltrials.gov/, NCT05176002. Registered on 2022/01/04). The posted information will be updated as needed to reflect protocol amendments and study progress.This trial protocol has been registered on the NIH Clinical Trials database ( Esophageal cancer is the seventh most common cancer and the sixth most common cause of cancer-related death globally . Its mosIn recent years, immunotherapy has demonstrated clinical benefits as a new treatment regimen for patients with locally advanced and metastatic ESCCs , 7. The Some studies have suggested that conventional radiation therapy is effective by promoting systemic anti-tumor immunity, thereby reducing the risk of recurrence and improving patient survival . For othThe primary objective of this study is to assess the safety of camrelizumab combined with standard radiotherapy as preoperative neoadjuvant therapy for patients with locally advanced ESCC, and the secondary objective is to assess the efficacy of the neoadjuvant therapy. The present study also aims to explore the biomarkers and the quality of life in patients with locally advanced ESCC.This prospective study is a single-center, single-arm, prospective phase II clinical study. All participants who meet the inclusion criteria will be enrolled after signing the informed consent form. A total of 25 patients will need to be recruited for the study. Twelve patients will be enrolled in phase I, with at least two patients achieving MPR before entering phase II. The second phase will enroll 13 patients. They will be treated with radical surgery within 4–8 weeks after the completion of two cycles of neoadjuvant radiotherapy in combination with camrelizumab according to the study schedule. The schedule of enrollment, interventions, and assessments is shown in Table All participants will be recruited from the Department of Thoracic Surgery, Fujian Medical University Union Hospital, from September 2021 to September 2023.The primary endpoint is the major pathological remission (MPR) rate in all per-protocol patients. MPR is defined as the remainder of ≤ 10% of viable tumor cells on postoperative pathological examination, and pCR is defined as tumor regression without a residual tumor on pathological examination , 18. TheThe inclusion criteria are as follows: (a) histologically-confirmed squamous cell carcinoma; (b) primary lesions located in the thoracic esophagus; (c) clinical stages of T2–3 N0 M0 or T2–3 N + M0, based on the 8th UICC-TNM classification; (d) having not received neoadjuvant therapy; (e) 18–75 years; (f) ECOG performance status of 0 or 1; (g) no prior chemotherapy, radiotherapy, or immunotherapy for any cancers; (h) adequate organ function; (i) expectation of R0 resection; and (j) provision of written informed consent.The exclusion criteria are as follows: (a) corticosteroid treatment  within 14 days before the first day of drug administration; (b) acquired immunodeficiency syndrome or active hepatitis B (DNA ≥ 104 copies/ml) or C (RNA ≥ 103 copies/ml) viral infections; (c) history of pneumonitis or interstitial lung disease with clinical evidence, such as interstitial pneumonia and pulmonary fibrosis features on baseline CT scans; (d) known or concurrent bleeding disorders or other uncontrolled diseases contraindicating surgical treatment; (e) physical examination or clinical trial findings that can interfere with the results or put the patient at increased risk for treatment complications; (f) comorbidities, including chronic pulmonary disease, poorly controlled hypertension, unstable angina, myocardial infarction within 6 months, and unstable mental disorders requiring therapy; (g) allergy to drugs used in the study; (i) participation in other clinical trials within 30 days before enrollment; and (j) ineligibility for participation based on the decision of investigators.Patients to be considered ineligible for participation by the investigators include the following: (a) patients with second primary malignant disease; (b) patients with hypersensitivity to the investigational drug camrelizumab and chemotherapeutic agents; (c) patients with concurrent autoimmune disease or history of chronic autoimmune disease; (d) participants of other clinical trials within the prior 4 weeks; (e) patients with a history of systemic therapy with immunomodulatory agents for antitumor indications within the prior 2 weeks; (f) patients with an active autoimmune disease requiring systemic therapy within 2 years before the first dose; (g) patients with known allogeneic organ transplantation  or allogeneic hematopoietic stem cell transplantation; (h) patients with severe or uncontrollable systemic disease; (i) patients with medical history or evidence of disease that may interfere with the results of the trial or prevent participation in the study over its entire duration, abnormal treatment outcomes or laboratory test results, or other potential risks deemed by the investigators to be inappropriate for participation in this study.Patients will receive two cycles of neoadjuvant radiotherapy combined with camrelizumab according to the study schedule, and they will undergo radical surgery within 4–8 weeks after neoadjuvant therapy. The details of the neoadjuvant treatment are as follows.The patients will receive two courses of preoperative camrelizumab  repeated every 3 weeks.Three-dimensional conformal or intensity-modulated radiotherapy will be administered at a total radiation dose of 4140 cGy (180 cGy at a time for five times a week). The targeted areas are as follows: CTV will include 3 cm above and below the preoperative primary lesion and the regional lymphatic drainage area, where the metastatic lymph nodes were located; and PTV will include the area targeted by CTV extended by 5 mm.The patients will be re-evaluated by the investigators according to the Response Evaluation Criteria for Solid Tumors version 1.1 approximately 4–8 weeks after neoadjuvant treatment. Esophagectomy will be performed if there is no evidence of metastatic disease. Patients will undergo robotic minimally invasive esophagectomy, minimally invasive esophagectomy, or open right thoracotomy esophagectomy with 2- or 3-field lymph node dissection. Transhiatal or left thoracotomy esophagectomy is not acceptable due to the limited capacity for upper mediastinum lymph node dissection. Surgery-related complications will be recorded on the case report form for up to 90 days after surgery.AEs will be recorded and graded according to the Common Terminology Criteria for Adverse Event (CTCAE) version 5.0 . ThroughWe plan to conduct one interim analysis. The interim analysis will be conducted after 12 patients have been enrolled. The Lan-DeMets method with the O’Brien and Fleming type alpha spending function will be used to adjust the multiplicity of the interim analysis . Trials p-values will be provided for group comparisons. Unless otherwise stated, the data will be summarized using mean ± standard deviation or median . Countable data will be summarized using frequency (percentage).After obtaining written informed consent, the medical records of participants will be continually reviewed and checked by the study staff, and relevant data will be collected and recorded. All statistical analyses will be performed using SPSS 22.0 (or higher), and all statistical tests will be one-sided. 0.05 hypothesis of superiority tests, and 95% confidence intervals and Neither patients nor public will be involved in the design, recruitment, outcome measures, and conduct of the study. Trial results will be disseminated via peer-reviewed scientific journals and conference presentations rather than specifically notified to a single patient.This is a single-arm, single-center prospective phase II study to evaluate the safety and efficacy of camrelizumab immunotherapy combined with radiotherapy as preoperative neoadjuvant therapy for patients with locally advanced ESCC as part of the exploration of better treatment options for locally advanced resectable ESCC.p < 0.001). However, the superimposed toxicity of chemotherapy and radiotherapy may lead to serious acute AEs during radiotherapy, such as severe bone marrow inhibition, pneumonia, and esophageal perforation, which may limit the clinical application of neoadjuvant chemoradiotherapy [The CROSS and NEOCRTEC5010 studies demonstrated the benefits of neoadjuvant radiotherapy for locally advanced ESCC , 5. The otherapy , 22. Morotherapy , 24.Immune checkpoint inhibitors have been proven to be effective for the perioperative treatment of esophageal cancers , 26. TheSeveral studies have demonstrated the efficacy of the preoperative combination of immunotherapy and chemotherapy in the treatment of esophageal cancer. Jun et al. conducted a study of neoadjuvant treatment of locally advanced ESCC with camrelizumab combined with chemotherapy. Of the 60 patients recruited, 51 eventually underwent surgery with a pCR rate of 39.2%. However, the study also reported that 39 (65.0%) patients experienced grade 3 or more severe AEs, with the most common being leukopenia (50.0%) [Previous clinical studies have demonstrated several synergistic effects of immunotherapy and radiotherapy . It was In conclusion, the ideal neoadjuvant therapy is associated with high pCR rates, long-term survival, and fewer treatment-related AEs. As part of the exploration of better neoadjuvant treatment options for esophageal squamous cancer, we designed this prospective single-arm phase II clinical study to evaluate the combination of a PD-1 monoclonal antibody and standard radiotherapy for preoperative neoadjuvant therapy for patients with resectable esophageal squamous cancer to provide practical and better treatment options for ESCCs.Version 1.0, September 7, 2021.Start of inclusion: February 15, 2022.Finish date of recruitment: December 15, 2023.


This study describes 3-year follow-up of 200 infants weighing ≥ 700 grams who underwent transcatheter patent ductus arteriosus (PDA) closure with the Amplatzer Piccolo™ Occluder.Between June 2017 and February 2019, 200 children were enrolled in this U.S. study (NCT03055858). PDA closure, survival, and device- or procedure-related events were evaluated. A total of 156 of the available 182 patients (86%) completed the study.The implant success rate was 95.5% (191/200). At 3 years, PDA closure was observed in 100% (33/33) of patients. Survival was >95% with 9 reported deaths. No deaths were adjudicated as device- or procedure-related. Notable events included aortic obstruction (2) requiring stent placement and tricuspid regurgitation (5), for which no interventions were required.This follow-up study demonstrates high rates of PDA closure, low serious complication rates, and survival > 95% at 3 years. The Amplatzer Piccolo™ Occluder is a safe and effective therapy for PDA treatment in premature infants. ClinicalTrials.gov identifier: NCT0305585. The presence of a hemodynamically significant patent ductus arteriosus (PDA) is common in low birth weight and extremely low birth weight premature infants and has been associated with an increased risk of necrotizing enterocolitis, chronic lung disease, intraventricular hemorrhage, and death . Trials The Amplatzer Piccolo™ Occluder , previously called the Amplatzer™ Ductal Occluder II Additional Sizes (ADO II AS), received the CE Mark for PDA closure in infants ≥ 6 kg in 2011. Based on published literature , 4 demon≥ 3 days of age at the time of implant. The study included an investigational device exemption (IDE) cohort with 50 patients enrolled between 05 June 2017 and 25 January 2018 at 8 clinical sites, and a continued access protocol (CAP) cohort with 150 patients enrolled between 26 March 2018 and 01 February 2019 at 9 sites. Both the IDE and CAP cohorts enrolled patients in the United States and followed the same study protocol. Each study site received approval from the local Institutional Review Board prior to any study conduct or patient enrollment. This study was performed in accordance with the Declaration of Helsinki.The ADO II AS Clinical Study  was a single arm, prospective, multicenter, pre-market clinical investigation designed to characterize the safety and effectiveness of the Amplatzer Piccolo™ Occluder  in children ≥ 700 g and Key inclusion criteria were the presence of a PDA ≤ 4 mm in diameter and ≥ 3 mm in length based on an intra-procedural echocardiogram or angiogram. Key exclusion criteria included weight < 700 g and age < 3 days at time of the procedure, presence of a pre-existing coarctation of the aorta or left pulmonary artery (LPA) stenosis, pulmonary hypertension with cardiac output dependent on a right to left shunt through the PDA, presence of an intracardiac thrombus, or active infection. Demographic information and common morbidities of prematurity were diagnosed prior to enrollment but collected on study case report forms once the patient was enrolled. Patient follow-up occurred prior to hospital discharge and at 30-days and 6-months post-procedure. Additional follow-up visits occurred at 1-, 2-, and 3-years post-implant. All IDE and CAP patients were assessed at each follow-up visit for adverse events.N = 50) was required to undergo transthoracic echocardiography (TTE) at each follow-up visit. The study protocol did not require CAP patients (N = 150) to undergo TTE beyond 6-months post-procedure unless there was a persistent residual shunt observed at 6 months. PDA closure was defined as the presence of either no (Grade 0) or a trivial residual shunt (Grade 1), as assessed by an independent Echocardiography Core Laboratory (core lab) [The IDE cohort N = 0 was reqProcedural success was defined as having the Amplatzer Piccolo Occluder positioned within the PDA by the end of the implant procedure. Patients who did not receive the Amplatzer Piccolo Occluder or had the Amplatzer Piccolo Occluder removed were withdrawn from the study after 30 days of observation. The unique procedural techniques required for this population, as well as the primary outcomes of effectiveness (rate of effective closure at 6 months) and safety (rate of major complications through 180 days) were previously described in the 6-month follow-up publication . SurvivaThis is a single-arm descriptive study. Categorical data are reported as a count and percentage. Continuous data are reported as mean ± SD unless otherwise specified. The range is also provided to present the minimum and maximum values. Patient survival was analyzed using the Kaplan-Meier method. Subjects with a death or unknown survival status were censored on the last known date reported alive. Peak instantaneous Doppler gradients of the LPA and aorta were obtained as part of the study protocol and converted using the simplified Bernoulli equation to calculate peak velocities. Data were analyzed by using SAS software, version 9.4 .n = 100) and > 2 kg (n = 100). A total of 132 infants were referred for PDA closure from the neonatal intensive care unit , bleeding following an unrelated abdominal surgery (n = 1), necrotizing enterocolitis (n = 1), and cerebral infarction (n = 1). The median time from device implant to death was 141 days (range 14–1046 days). After an independent review by the CEC, no deaths were adjudicated as device or procedure related. Among the 9 deaths, there were three patients who underwent an autopsy at 66 days (Patient #9), 141 days (Patient #3), and 593 days (Patient #1) post-implant. Autopsies demonstrated complete coverage of the device with neoendothelium  with echocardiograms Fig. . Echocar≤ 2 kg). Three cases were successfully closed with larger Piccolo devices, while two cases could not be closed. Post procedure migration occurred in 2 additional patients (1 patient ≤ 2 kg). All cases of device embolization and migration were successfully managed by transcatheter device retrieval using a snare. Major device or procedure related complications through 6 months occurred in 4 patients .Intraprocedural device embolization occurred in 5 patients  it was noted that the device was completely covered with a neoendothelium. Once the coarctation was surgically repaired, the aortic velocities were no longer elevated. The second instance of aortic obstruction was adjudicated as device- and procedure-related. That patient died 8 days following stent placement secondary to severe hypoxemic and hypercarbic respiratory failure  following the implant procedure. Two cases of severe TR occurred following retrieval of an embolized device, two cases with moderate TR were due to an unknown cause, and one case of mild TR was associated with difficulty advancing a catheter through the heart. Of the 5 patients, 1 with moderate TR died prior to the 6-month follow-up from necrotizing enterocolitis (Patient #7) and 1 infant with severe TR who had a post-implant device migration was withdrawn from the study following transcatheter device retrieval as a subsequent device was not placed. In the three remaining patients, one had mild TR at the most recent follow-up and the two remaining patients continued to have moderate and severe TR at 3-years post-implant that was unchanged from prior echocardiograms. The patient with severe, persistent TR had a flail septal leaflet. It is unclear if the tricuspid valve injury was sustained during initial device implant or during the extraction of the embolized device. At the 3-year follow-up no patients required an intervention to repair or replace the tricuspid valve.n = 33) demonstrated complete occlusion of the PDA at 3 years and there were no reported instances of the ductus arteriosus reopening. Major complications were reported in 2.1% of cases. Intraprocedural device embolization (2.5%) and increased TR (2.5%) were the most commonly observed adverse events during the 6-month follow-up. Beyond 6-months there were no additional device or procedure related complications reported. None of the three patients with increased TR followed through study completion required an intervention on the valve. Clinically significant aortic obstruction occurred in two patients in the first week post-procedure, requiring stent placement to relieve the obstruction in both cases. There were no late cases of aortic obstruction and no cases of LPA obstruction requiring intervention through 3-years of follow-up. Great attention should be taken to assess the device prior to release to minimize the risk of LPA or aortic obstruction.Clinical outcomes from the IDE and CAP cohorts demonstrate continued safety with low rates of serious complications and high rates of effective transcatheter PDA closure with the Amplatzer Piccolo™ Occluder in pediatric patients through 3-years of follow-up. Survival was > 95% out to 3 years with no deaths attributed to the procedure or device. Successful device implant was achieved in > 95% of patients and 99% of patients ≤ 2 kg. While only the IDE cohort was required to undergo echocardiography through 3 years of follow-up, all IDE patients with available echocardiograms (N = 10) undergoing PDA closure [Other contemporary studies of the Amplatzer Piccolo™ Occluder have demonstrated similar rates of successful closure and adverse events –12. One  closure . This ma closure . Multiva closure .The development of a device specific for PDA closure in preterm infants coincided with new procedural techniques to reduce the risk of vascular and device related complications. Most importantly, this procedure is performed with ultrasound guided venous access only and a greater emphasis on complete intraductal placement of the device in infants ≤ 2 kg to minimize protrusion of the device into the aorta or the LPA compared to larger children ( > 2 kg) where the device size is selected so that the central waist spans the entire length of the ductus with the retention discs placed just outside the ductus to improve positional stability and minimize the potential for device embolization. This approach requires navigation through the cardiac structures in a small, preterm infant and necessitates increased dependence on intraprocedural transthoracic echocardiography to assist with proper device placement. As experience has grown, so too has the understanding about the subtleties of device sizing and device positioning within the ductus to minimize the risk of device embolization and malposition. An increase in TR occurred in 5% of infants ≤ 2 kg (5/100) in the trial. Proposed mechanisms for TR include delivery catheter and wire mismatch, advancement of a catheter underneath chordae, prolonged cases with embolization or malposition necessitating device retrieval, or the stiffness of the delivery catheter and wire causing damage to the valve. Based on some of these proposed mechanisms, guidelines for prevention have been proposed,  and new While this current study was designed to demonstrate the safety and effectiveness of the Amplatzer Piccolo Occluder in treating the PDA in pediatric patients, there is still much work to do in defining what impact this procedure will have on the clinical course of these patients. Defining the appropriate target population with a hemodynamically significant PDA and time window for intervention will enhance the ability to apply these techniques to the right patients. There remains a lack of uniformity in how a hemodynamically significant PDA is defined, which has significantly hampered the outcomes of prior trials of PDA management. A PDA severity score has been developed and demonstrated to predict chronic lung disease or death before discharge in an observational study of infants born < 29 weeks gestational age . Future Ultimately, a randomized controlled trial of transcatheter device closure compared with conventional medical therapy is necessary to establish the clinical benefit of transcatheter closure. The Percutaneous Intervention Versus Observational Trial of Arterial Ductus in Low Weight Infants (PIVOTAL) trial  has been approved and, beginning in 2023, will randomize mechanically ventilated patients between 7 and 32 days of age with a hemodynamically significant PDA to transcatheter PDA device closure or responsive medical management of the PDA. This represents an important step in establishing which patients will derive clinical benefit from this procedure.As the techniques for transcatheter closure improve and the target patient population is refined, the next frontier is bringing this procedure closer to the patients it serves. Transporting these fragile patients is not without risk, and some experienced centers are endeavoring, in a stepwise fashion, to bring this procedure out of the catheterization lab and directly to the bedside in the NICU , 19. ThiThis study was designed to study the safety and effectiveness of transcatheter PDA closure using the Amplatzer Piccolo™ Occluder in pediatric patients. It was not designed to determine whether transcatheter closure offers clinical benefit compared to medical management or conservative therapy. Additionally, only the IDE cohort was followed with serial echocardiography through 3 years of follow-up. The CAP cohort underwent echocardiographic follow-up through 6 months and clinical follow-up for adverse event and mortality tracking through 3 years.Transcatheter closure of the PDA in pediatric patients with the Amplatzer Piccolo™ Occluder is safe and effective with > 99% procedural success in patients ≤ 2 kg, 100% closure documented out to 3-years in the IDE cohort, and overall cohort survival > 95% at 3-years. Major procedure or device related complications were uncommon. These data are an important steppingstone to improving procedural techniques and safety, establishing clinical benefit of transcatheter PDA closure in at-risk preterm infants and extending this procedure beyond the cardiac catheterization lab to the bedside. The availability of a safe and effective intervention for the PDA in preterm infants will help explore important questions about the impact of the PDA on the management of preterm infants and have a significant impact on clinical outcomes for this at-risk population.Supplemental data


Embryonal rhabdomyosarcomas (ERMS) of the uterine cervix and corpus are rare pediatric tumors usually associated with a late age of onset and frequent somatic DICER1 mutation. It may also develop in the context of a familial predisposition such as DICER1 syndrome requiring specific medical care for children and young adults at risk for a broad range of tumors.DICER1 variant. The family history revealed thyroid diseases in the father, aunt and paternal grandmother before the age of 20.This is a case of a prepubescent 9-year-old girl who was presented to our department for metrorrhagias due to a vaginal cervical mass, initially classified as a müllerian endocervical polyp on negative myogenin immunostaining. The patient subsequently manifested growth retardation (-2DS) and learning disabilities leading to genetic explorations and the identification of a germline pathogenic Rare tumors such as cervical ERMS associated with a family history of thyroid disease during infancy could be related to DICER1 syndrome. Identifying at-risk relatives is challenging but necessary to detect early DICER1 spectrum tumors in young patients. DICER1 gene which encodes an endoribonuclease involved in the maturation of microRNAs (miRNAs) and therefore the regulation of gene expression , cystic nephroma, ovarian sex-cord stromal tumor and multinodular goiter develop in the early stages of childhood , 3. ReceDICER1 variant and the low risk for relatives of developing a tumor before the age of 10 complicate care recommendations which have been recently discussed  and neonatal dysmorphia, leading to neonatal urinary glycosaminoglycans (GAGs) analysis for screening of mucopolysaccharidosis with inconclusive results. She is a first child born at 39 weeks of amenorrhoea after normal pregnancy and delivery (mother's age: 24 yrs). At the age of 9 the girl was referred to our pediatric gynecology department for metrorrhagias. Clinical and physical examination revealed no sign of pubertal development but retrieved a cervically protruding vaginal mass corresponding to multiple polyps of 0.5 to 2 cm length. After surgical resection, the histopathological analysis indicated benign müllerian endocervical polyps with variable cellularity, mild atypia, squamous-like epithelium and diffuse Smooth Muscle Actin but negative Myogenin immunostaining. Serum tumor markers alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and human chorionic gonadotropin (hCG) were negative.SHOX deficiency. Complementary hormonal results () before synthetic GH treatment  were all normal  . A physiDICER1 gene (NM_177438.3) designated as c.1716del; p.(Phe572LeufsTer15) was found. This variant leads to a premature stop codon in the P-loop containing the nucleoside triphosphate hydrolase-domain of the protein. The mutation was confirmed on a second blood sample.At the age of 12, due to scholastic difficulties and learning disabilities associated with facial dysmorphism and a history of screening for mucoploysaccharidosis, a more comprehensive genetic exploration was performed using a broad gene panel for rare pediatric disorders combined with next-generation sequencing (NGS) in exon 24  corresponding to the known genetic hotspot for somatic mutations in the ribonuclease III domain  in the catalytic domain of the protein, previously described in DICER1-related tumors . ConcernDICER1-associated ERMS occurs most frequently in pubertal and post-pubertal adolescent girls and young women (mean 10–20 years). A recent study including five cervical and four uterine ERMS showed DICER1 pathogenic variants in all the uterine lesions whereas these variants were absent in the urinary tract ERMS. The authors suggest that DICER1-associated ERMS could be qualified as a distinct subtype in future classifications, especially when related to a specific DNA methylation profile . In addition, screening and surveillance recommendations differ based on age and gender. Therefore, chest imaging is recommended from birth until 12 years of age since PPB is the main lung expression of DICER1 syndrome, whereas thyroid and pelvic ultrasound (US) are only recommended as of 8 years of age throughout life , and four years later any local or distant recurrences were monitored.Family history is an important aspect in evaluating the risk of DICER1 syndrome. In this case, the association of a childhood tumor, although initially considered to be benign, and a familial history of multinodular goiter should alert the clinician to initiate genetic testing for out life , 15. Fornsidered , 17. ThiThis case report highlights that DICER1 syndrome should be suspected in the presence of early typical DICER1-related tumors as well as in case of unusual tumors during childhood, especially when there is a family history of thyroid diseases during infancy or young adulthood.


PNPLA3 rs738409 genotypes as a known non-alcoholic fatty liver disease (NAFLD) genetic risk.Recent reports suggested a different predictive value for TyG index compared to HOMA-IR in coronary artery calcification (CAC) and other atherosclerotic outcomes, despite that both indices are proposed as surrogate markers of insulin resistance. We hypothesized a key role for liver pathology as an explanation and therefore assessed the relationship among the two indices and the intrahepatic lipid content stratified by PNPLA3 rs738409 CC and GG genotypes for metabolic phenotyping and to assess hepatic triglyceride content (HTGC). 75 g OGTT was performed, fasting lipid, glucose, insulin levels and calculated insulin resistance indices (TyG and HOMA2-IR) were used. HTGC was measured by MR based methods. Mann–Whitney-U, χ2 and for the correlation analysis Spearman rank order tests were applied.Thirty-nine women from a prior GDM-genetic study were recalled with PNPLA3 rs738409 genotype had a significant effect on the direct correlation between the HOMA2-IR and TyG index: the correlation  found in the CC group was completely abolished in those with the GG (NAFLD) risk genotype. In addition, the HOMA2-IR correlated with HTGC in the entire study population  and also separately in both genotypes . In contrast, the correlation between TyG index and HTGC was only significant in rs738409 CC genotype group  but not in GG group. A similar pattern was observed in the correlation between TG and HTGC , when the components of the TyG index were separately assessed.The PNPLA3 rs738409 risk genotype completely decoupled the direct correlation between two surrogate markers of insulin resistance: TyG and HOMA2-IR confirming our hypothesis. The liver lipid content increased in parallel with the HOMA2-IR independent of genotype, in contrast to the TyG index where the risk genotype abolished the correlation. This phenomenon seems to be related to the nature of hepatic fat accumulation and to the different concepts establishing the two insulin resistance markers. Insulin resistance (IR) refers to a decreased sensitivity of peripheral tissues to insulin and is a hallmark pathophysiologic feature of type 2 diabetes mellitus (T2DM) that clinically develops when the β-cell compensatory mechanisms might no longer overcome the increased insulin need due to the peripheral IR –3. IR isThe most established method to assess the IR is the euglycemic–hyperinsulinemic clamp  developeNevertheless there are other surrogate markers of IR such as the triglyceride-glucose (TyG) index , 12 thatUnsurprisingly both the HOMA-IR , 16 and Although both the HOMA-IR and TyG indices are proposed as surrogate markers of IR, recently the TyG index was found to be superior compared to the HOMA-IR in predicting coronary artery calcification (CAC) .PNPLA3 rs738409 variant shows the strongest association with NAFLD development , and homeostasis model assessement 2 insulin resistance index (HOMA2-IR) values were calculated , 36. DiaAll MR imaging (MRI) sessions were acquired on a clinical 3 T MRI system  with the subject in a supine position. For MRI protocol, a standard body-array of 18 channel flexible coils was positioned on the liver region and combined with a spine array coil located below the subject. Imaging proton density fat-fraction (PDFF), unenhanced axial images were obtained by using a low–flip-angle, six-echo two-dimensional spoiled gradient-recalled-echo sequence with all array coil elements . The repetition time and flip angle were chosen to avoid T1 weighting: TR: 15 ms, Flip-angle: 11°, FOV = 240 × 400 mm in plane, matrix = 240 × 130, slice thickness = 3.5 mm, space between slices 4.3 mm. Phase and magnitude images were systematically saved. For MR imaging using custom-made MATLAB routines, multi-section liver PDFF maps were generated offline from the source images via joint estimation of water and fat images and field maps [2 tests were applied. TIBCO Statistica  software was used.Due to the limited sample size and the non-normal distribution of the majority of variables we used non-parametric tests. Data are expressed in median and 25th-75th percentiles. For evaluation of the association between two parameters Spearman’s correlation test and for comparing two (genotype) groups Mann–Whitney U/χ2, respectively. We diagnosed thirteen individuals with prediabetes and one with overt T2DM. No patient had established cardiovascular disease at enrolment. No significant difference of anthropometric data, glycaemic or lipid values was detected between genotype groups, except of the HTGC values . The characteristic of the study population is indicated in Table Age and BMI values of study participants were 37.0 (34–40) years and 26.2 (22.8–32.5) kg/mCC genotype  but not with GG . The scatterplot is indicated in Fig. Between the two insulin resistance indices we found an association , however a significant correlation was only detectable in individuals with the rs738409 HTGC values significantly correlated with both markers of insulin resistance: , and their components .CC genotype group indicating different associations between the parameters according to the genotypes. Similarly, the correlation between HOMA2-IR and fasting TG was disrupted in individuals with GG genotype. Correlations between HTGC and TyG, HTGC and HOMA2-IR, HTGC and TG, HOMA2-IR and TG are indicated in Fig. After genotype stratification In contrast to HOMA2-IR and its components, where the correlations were nearly parallel in the two genotype groups after genotype stratification, the correlations of TyG index and fasting TG were only significant in the rs738409 CC genotype group indicating different associations between the parameters according to the genotypes to HOMA2-IR and its components, where the correlations were nearly parallel in the two genotype groups, the correlations of TyG index and fasting TG were only significant in the rs738409 PNPLA3 NAFLD risk genotype to decrease the number of patients needed to be enrolled [GG vs CC) were included. Despite the two IR indices were suggested to have a linear relationship [PNPLA3 genotypes. The profound genetic effect on the correlations between TyG, TG and HTGC and also between TG and HOMA2-IR was observed.We assessed intrahepatic lipid content measured by MR method and two indices proposed as surrogate markers of insulin resistance indices (TyG and HOMA2-IR) in 39 middle-aged women prone to T2DM development. Genotype based recall study design was applied on the basis of enrolled , 33. Thetionship , 41, we Insulin resistance and hyperinsulinemia are classified as major factors contributing to pathology of T2DM development and its cardiovascular complications. Many concepts and methods were developed to measure the “insulin resistance” (IR), however no single parameter could meet the need of a precise pathophysiological assessment combined with an easy use for the everyday clinics. Out of the many IR indices HOMA-IR index appears to be the most widely used in the clinical practice, including the recent recommendation to establish the diagnosis of metabolic (dysfunction) associated fatty liver disease (MAFLD) . The HOMTwo markers of insulin resistance, the HOMA-IR and TyG were compared in a recent study on the prognostic value of T2DM development and atherosclerosis and the authors concluded that TyG index performed better in both endpoints .Nevertheless there is still no clear-cut opinion in the literature regarding the predictive powers of the TyG and HOMA-IR index in CVD, and studies suggested only a moderate coronary artery disease (CAD) predictive power for TyG, although its inclusion into predictive models of MACEs could improve the predictive accuracy in patients with ACS .PNPLA3 rs738409 gene variant on the correlation between TyG and HOMA2-IR. This gene variant was identified in fatty liver genome-wide association studies (GWAS) [Due to that hepatic insulin resistance is significantly increased in NAFLD that is the most prevalent liver disease and strongly related to obesity, T2DM, dyslipidaemia and metabolic syndrome, the pathophysiology of the liver lipid accumulation could be a key factor in (hepatic) IR associated to the common metabolic diseases. Therefore we assessed the effect of s (GWAS)  with pats (GWAS) , 27, 28.GG risk genotype (M148M) in contrast to those with the CC (I148I) genotype where the strong positive correlation was conserved.We astonishingly found that TyG and HOMA2-IR indices were completely dissociated in individuals with the rs738409 PNPLA3 rs738409 genotype effect.This finding should mean that despite both the TyG and the HOMA2-IR are considered as surrogate markers of insulin resistance they display fundamentally different characteristics according to the genetic background of the liver lipid accumulation confirming the hypothesis of the G allele carriers (M148M and I148M) and it was hypothetically suggested that the G risk allele carriers might be protected from insulin resistance [PNPLA3 risk genotypes [PNPLA3 NAFLD risk genotype based on the reported hepatic DAG species content critical in disturbing the insulin signal [Although prior studies with human liver tissue samples reported that the hepatic content of diacylglycerol (DAG) species  remained unaltered in rs738409 homozygous and heterozygous sistance  we reporenotypes . In contn signal .GG risk genotype. The explanation could be attributed to that the input parameters and their PNPLA3 risk genotype associated changes are largely different. The insulin concentrations were increasing in parallel with HTGC, in contrast to the glucose levels that were not different. Consistently, the fasting insulin levels and the HOMA-IR indices were significantly higher and the glucose levels were unchanged in a severely obese large European cohort in the rs738409 GG genotype group [GG genotype was found to be lower compared to CC genotype group [PNPLA3 risk genotype group could be due to the lower hepatic TG efflux in the fasting state associated to the genetic effect on intrahepatocellular lipolysis [G allele frequencies in the East Asian populations are substantially higher compared to populations with European or African origin [We observed that the correlation between TyG and HTGC was diminished in those with the rs738409 pe group  that waspe group  and confpe group , 48. Theipolysis , 48, 49.ipolysis  these obipolysis , 51–53. ipolysis . It couln origin  which isn origin  and coulPNPLA3 rs738409 GG risk genotype and partially behaves like a biomarker of intrahepatic steatosis that is dissociated from the other surrogate marker of IR, the TyG index composed of only metabolic parameters but does not directly include serum insulin level.The current state of the art is that HOMA-IR primarily reflects hepatic insulin resistance . The inc G NAFLD risk allele is the major PNPLA3 allele [As a consequence, it may also be raised, that HOMA-IR could be significantly dissociated from the TyG index in those populations where the rs7384093 allele , 54.The authors suggest that the genetic background of the pathologic liver lipid accumulation and its relation to HOMA-IR and TyG index are important insights towards a better understanding, more precise interpretation and perhaps towards a better clinical use of these IR estimates.Our results were limited by the sample size and the lack of supplementary methods to study the underlying pathology. Further studies are needed to replicate our findings and to confirm our results in other populations.