Common Insertion Research Articles

A common Alu insertion in the 3'UTR of TMEM106B is associated with risk of dementia.

Sequence variants in TMEM106B have been associated with an increased risk of developing dementia. As part of our efforts to generate a set of mouse lines in which we replaced the mouse Tmem106b gene with a human TMEM106B gene comprised of either a risk or protective haplotype, we conducted an in-depth sequence analysis of these alleles. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. We conclude that this risk haplotype arose early in human development with a single Alu-insertion event within a unique haplotype context. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia. We conducted an in-depth sequence analysis of (1) a risk and (2) a protective haplotype of the human TMEM106B gene. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge Portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia.

High-dose furmonertinib combined with intraventricular chemotherapy as salvage therapy for leptomeningeal metastasis from EGFR exon 20 insertion-mutated lung cancer.

The treatment of leptomeningeal metastasis (LM), a serious complication of advanced non-small cell lung cancer (NSCLC), presents challenges, particularly in patients with EGFR exon 20 insertion (ex20ins) mutations. This study retrospectively analyzed data from 10 EGFR ex20ins-mutated NSCLC patients with LM admitted at our institution from May 2011 to June 2023. Circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) and matched plasma samples was analyzed using next-generation sequencing. All patients received high-dose furmonertinib combined with intraventricular chemotherapy (IVC) as salvage therapy. Data on patient demographics, treatment efficacy, and safety outcomes were collected. The most common insertion mutation identified in this study was p.A767_V769dup (n = 4, 40%), followed by D770-N771insY (n = 2, 20%). Nine patients had EGFR ex20ins occurring in the EGFR loop region following the C-helix, whereas only one patient had an EGFR ex20ins (A763_Y764insFQEA) occurring in the C-helix of the tyrosine kinase domain. LM response assessment using the RANO-LM criteria revealed that 6 patients (60%, 95% CI 26.2-87.8%) achieved a response, 3 had stable disease, and 1 had progressive disease. The median progression-free survival and overall survival were estimated to be 6.5 months and 8.8 months, respectively. The most commonly reported treatment-related adverse events were rash (n = 7) and diarrhea (n = 7), with no treatment-related deaths occurring. The current study demonstrated that high-dose furmonertinib plus IVC as salvage treatment for patients with LM harboring EGFR ex20ins mutations had promising clinical benefits and a manageable safety profile.

Surveillance and Prediction of Risk Factors for Central Line-Associated Bloodstream Infections in Saudi Arabia.

Although central line-associated bloodstream infection (CLABSI) is the most common type of healthcare-associated infection among patients with inserted devices, few studies have comprehensively evaluated the related risk factors. This retrospective study analyzed the risk factors, predictors, causative organisms, and impact of CLABSI on clinical outcomes mortality, and length of stay (LOS) in older adults. We included 36 patients diagnosed with CLABSI according to the Centers for Disease Control and Prevention criteria at King Abdulaziz University Hospital during 2013-2014 casesand 375 control patients controls. Risk factors were evaluated using a multivariate logistic regression analysis. Cases and controls did not differ significantly in age or sex distribution. However, cases had a significantly longer LOS than controls 78 vs. 19 days, p < 0.001. One-third of 12/36 CLABSI cases were admitted to the medical intensive care unit (MICU). Most had renal disease, acute coronary syndrome, and used steroids. Additionally, 34 cases (94.4%) and 2 cases (5.6%) presented with primary and secondary infections, respectively, and hypotension was the most prevalent symptom (12/36).The internal jugular vein was the most common insertion site, and the nasogastric tube and mechanical ventilator were the most common insertion devices. Seven cases died, and three deaths were attributed to bloodstream infection (BSI). The most common cause of blood infection was Staphylococcus epidermidis, followed by Klebsiella pneumoniae. The present study reveals age, LOS, total parenteral nutrition/partial parenteral nutrition (TPN/PPN), and transplantation as the independent risk factors/predictors of CLABSI.

Discovery of Potent and Selective Covalent Inhibitors of HER2WT and HER2YVMA.

HER2 overexpression and amplification have been identified as oncogenic drivers, and the development of therapies to treat tumors harboring these markers has received considerable attention. Activation of HER2 signaling and subsequent cell growth can also be induced by HER2 mutations, including the common YVMA insertion in exon 20 within the kinase domain. Enhertu is currently the only approved treatment for HER2 mutant tumors in NSCLC. TKIs tested in this space have suffered from off-target activity, primarily due to EGFRWT inhibition or attenuated activity against HER2 mutants. The goal of this work was to identify a TKI that would provide robust inhibition of oncogenic HER2WT and HER2 mutants while sparing EGFRWT activity. Herein, we describe the development of a potent, covalent inhibitor of HER2WT and the YVMA insertion mutant while providing oral bioavailability and avoiding the inhibition of EGFRWT.

Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

Abstract PR-011: The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models

Abstract Medulloblastoma (MB) is an embryonal malignant tumor that arises in the cerebellum of infants, children, and adolescents. Despite highly aggressive therapies, including surgery, radiation, and chemotherapy, metastasis remains the primary cause of death in these patients. The tumor microenvironment (TME) has an important role in metastasis and relapse in several cancer types, but it’s role in high-risk MBs (metastatic and relapse) has been understudied. Here, we investigated the process of relapse in an immunocompetent Shh MB mouse model based on somatic mutagenesis induced by Sleeping Beauty (SB)-mediated transposition on a genetically engineered strain carrying a loss-of-function Patched 1 gene (Ptch1) mutation. Lack of one functional copy of the Ptch1 gene predisposes this strain to develop MB. Somatic mutations induced by the transposon increase penetrance of tumor formation in the triple mutant mice carrying the SB transposon system and the Ptch1 mutation. We used this model to study the mechanisms of chemoresistance to cisplatin and cyclophosphamide, that constitute the backbone of the high-dose chemotherapy used to treat patients under the age of 3, who cannot receive craniospinal irradiation. By treating the SB mouse model with surgery followed by high-dose fractionated chemotherapy, we developed a model of MB chemoresistance which is relevant to the infant SHH-MB patient population. While chemotherapy improved mouse survival, only a small number of animals were cured, mirroring the outcome seen in infant patients with Shh MB. Deep sequencing and mapping of transposon-gDNA junctions, along with gene-centric common insertion site analysis (gCIS), revealed that Trp53 transposon insertions are found exclusively at relapse. In the metastatic compartment we observed convergence of gCISes on genes coding for epigenetic regulators, among them a prominent enrichment was found for PRC2 complex genes. RNA-seq analysis on the transcriptome of primary and recurrent tumors indicated that a majority of the differentially expressed genes are associated with inflammatory responses, IL6, and TNF-α/NF-κB signaling. Immunohistochemistry to visualize the expression levels of inflammatory markers: IL-6, CCL2, and F4-80 and spatial targeted multiplexed assays (Geomx) confirmed the increase of immune cells at progression. ChIP-qPCR on cytokine loci revealed binding of PRC2 and deposition of PRC2 mediated repressive histone mark H3K27me3 on the promoter of CCL2 highlighting LOF mutations in PRC2 components as potential epigenetic mechanisms for immune recruitment at progression. Inhibition of immune activation through pharmacological strategies might be a possible therapeutic option for infant SHH-MB. Citation Format: Niusha Khazaei, Ana Castillo Orozco, Wajih Jawhar, Geoffroy Danieau, Kiran Narta, Michael Taylor, Sorana Morrissy, Ana Stuecklin, Livia Garzia. The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-011.

Variations in the cystic duct: frequency and the relationship among insertion sides and heights on the bile duct.

Evaluation of the cystic duct anatomy prior to bile duct or gallbladder surgery is important, to decrease the risk of bile duct injury. This study aimed to clarify the frequency of cystic duct variations and the relationship between them. Data of 205 patients who underwent cholecystectomy after imaging at Sada Hospital, Japan, were analyzed. The Chi-square test was used to analyze the relationships among variations. The lateral and posterior sides of the bile duct were the two most common insertion points (92 patients, 44.9%), and the middle height was the most common insertion height (135 patients, 65.9%). Clinically important variations (spiral courses, parallel courses, low insertions, and right hepatic duct draining) relating to the risk of bile duct injury were observed in 24 patients (11.7%). Regarding the relationship between the insertion sides and heights, we noticed that the posterior insertion frequently existed in low insertions (75.0%, P<0.001) and did not exist in high insertions. In contrast, the anterior insertion coexisted with high and never low insertions. Spiral courses have two courses: anterior and posterior, and anterior ones were only found in high insertion cases. The insertion point of the cystic duct and the spiral courses tended to be anterior or lateral superiorly and posterior inferiorly. Clinically significant variations in cystic duct insertions are common and surgeons should be cautious about these variations to avoid complications.

10000-SPE-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Models of care for intrauterine device provision in Australian general practice.

Intrauterine devices (IUDs) are safe and effective forms of long-acting reversible contraception. However, uptake in Australia is low. Although general practitioners (GPs) manage contraceptive provision, little is known about IUD service delivery. This study aims to describe themodels of care (MoC) used in the provision of IUDs by Australian GPs. Semistructured telephone interviews were conducted with 20 GP IUD providers nationwide. Data were deidentified, transcribed, thematically analysed and evaluated using The RoyalAustralian College of General Practitioners' Quality framework for Australian general practice. Three MoC were described: common, streamlined and same-day insertion. Thecommon model involved three to fourappointments but was of the lowest quality. The streamlined MoC had a maximum of two appointments. Few GPs delivered same-day insertion. Task-sharing and adaptable MoC were identified. The quality of current MoC for IUD provision is non-standardised and largely inadequate. Increased GP awareness about and operation of high-quality MoCwill enhance IUD access.

Association Analysis of Common Structural Variants in the Alzheimer’s Disease Sequencing Project

AbstractBackgroundStructural variants (SV), genomic rearrangements of &gt;50 base pairs, are an important source of genetic variation and have a great impact on gene expression and protein function. However, their contribution to the genetic architecture of Alzheimer’s Disease (AD) has not been comprehensively investigated. Identification of SVs by short‐read alignment can be inaccurate and biased. We used a novel SV calling pipeline that leverages assembly‐based methods and graph‐based representation, to identify SVs with high accuracy in the diverse sample of the Alzheimer’s Disease Sequencing Project (ADSP). We further examined the association of common SVs with AD.MethodsWe applied Biograph for SV calling on the ADSP 17K whole genome sequence data. After filtering out low‐quality SVs and performing sample‐level quality control, we identified 222,956 deletions, 177,139 insertions, and 10,558 inversions in 12,908 individuals (6,304 AD cases; 6,604 controls). We performed association analyses of common SVs (MAF &gt; 1%) with AD using a logistic mixed‐effects model adjusting for sex, technical covariates, principal components, and relatedness. Statistical significance was evaluated using a Bonferroni‐corrected threshold (P&lt; 9.19.x10−7).ResultsThere were 28,634 deletions, 25,522 insertions, and 198 inversions with MAF &gt; 1%. We identified 2 intronic deletions significantly associated with AD. The first deletion is located in CCDC88B, encoding a protein that regulates T‐cell maturation and inflammation. The second deletion is located in CCDC11, predicted to be part of the spliceosomal complex. Among 11 insertions significantly associated with AD, 3 mapped to exonic sequences. A relatively common insertion located in an exon of FOXO6 showed the most significant association with AD (P = 1.41×10−12). FOXO6 is a member of Forkhead transcription factors that have been implicated in aging. Along with its high level of expression in the hippocampus, mice lacking FOXO6 had impaired memory consolidation. We identified one inversion suggestively associated with AD (P = 3.32×10−6) and located in an intron of PDE5A. PDE5A inhibitors have been reported as potential therapeutic targets for AD.ConclusionUsing novel data structures and algorithms to call SV, we discovered 14 SVs significantly associated with AD. Validation and gene prioritization of these findings are warranted.

An In Vivo PiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88 L265P

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Integrated Genomic Analysis Identifies a Downstream Regulatory Element Oncogenically Activating Hematopoietically Expressed Homeobox ( HHEX) in ETP-ALL

Hematopoietically-expressed Homeobox ( HHEX) is required for the maintenance of hematopoietic stem cells (HSPCs) and common lymphoid progenitor cells. HHEX is also the second most frequent integration site in retroviral insertional mutagenesis screens of leukemias and lymphomas arising in AKXD recombinant inbred mouse strains, implying that it is an important oncogene. Enforced expression of HHEX induces T-cell acute lymphoblastic leukemias (T-ALL) in murine bone marrow transplantation models. In human studies, HHEXmRNA expression is upregulated in human T-ALL studies, especially in Early T-cell Precursor (ETP-) ALL subtypes where it is concordantly expressed with LMO2. The HHEX locus is not rearranged in ETP-ALL where it is upregulated so the mechanism of HHEX activation is not clear. We analyzed T-ALL induced by retroviral mutagenesis and found an intergenic site of frequent integration, 65kb 3‘ of the Hhex coding exons. Integrations clustered within a 3 kb genomic area that is also an open chromatin region (OCR) by ATAC-seq analysis and that induced Hhex upregulation. The syntenic human region was highly enriched for H3K27 acetylation and for occupancy by multiple transcription factors, notably GATA1 and TAL1, per ENCODE. Our prior data had shown that HHEX was a downstream target of the LMO2 complex comprised of GATA1/2, TAL1 or LYL1, and nucleated by the scaffolding protein, LIM domain binding protein 1 (LDB1). We had previously shown by ChIP-PCR that the LMO2 complex was bound to the intron 1 enhancer. Thus, we performed ChIP-exonuclease analysis of LDB1 in the human ETP-ALL model cell line, LOUCY. We confirmed its occupancy at the intron 1 enhancer but we also observed LDB1 occupancy 68kb 3‘ of the HHEX coding exons, in the exact region syntenic to the murine common insertion sites. ChIP-exo analysis allowed us to pinpoint a core element with composite GATA/E box sites that was highly conserved across multiple mammalian species. LDB1 occupancy at intron 1 and at +68kb of the HHEX locus was reminiscent of LDB1's occupancy at the beta globin locus where LDB1's occupancy and homodimerization mediates chromatin looping between composite E box/GATA sites in the locus control region (LCR) and the beta globin proximal promoters. To test whether a similar interaction was occurring at the HHEX locus, we analyzed virtual 4C (chromatin conformation capture) data. We confirmed looping between the +68kb element and the proximal promoter of HHEX in human ETP-ALL primary samples and in human HSPCs. The experiments showed that looping occurs in certain developmental contexts in HSPCs and is recapitulated in ETP-ALL and mediated by the LMO2/LDB1 protein complex. HHEX is a downstream oncogene of the LMO2/LDB1 complex and is activated through this distal +68kb regulatory element. Our studies raise the possibility that dissection of this regulatory element and interference with chromatin looping are potential therapeutic mechanisms that could disrupt oncogene expression.

The 2D and 3D MR arthrographic description of aponeurotic expansion of supraspinatus tendon and biceps tendon anomaly in a large patient cohort.

To describe the aponeurotic expansion of supraspinatus tendon (AEST) and biceps tendon abnormalities with magnetic resonance (MR) arthrographic examinations and determine their prevalence in patients, we performed a high-resolution 3D direct MR arthrography. This was a retrospective study of 700 shoulder MR arthrograms performed between May 2010 and January 2022. Extension in the coronal plane of an AEST on 3D fat-suppressed T1-weighted volumetric interpolated breath-hold examination (VIBE) MR arthrography was identified. Based on its morphology, the AEST on MR arthrography was divided into four subtypes: absence of tendinous thickness in the bicipital synovial surface or intra-synovial tendon-like structure in the bicipital groove, thin and flat tendinous thickness ≥1 mm of bicipital synovium, oval tendinous structure less than half the size of the adjacent biceps tendon, oval tendinous structure more than half the size of the adjacent biceps tendon, and oval tendinous structure larger than the adjacent biceps tendon. Based on its origin and termination, aponeurotic expansions can be divided into three subtypes: proximal pulley zone, middle humeral neck zone, and distal myotendinous junction zone. Association with the biceps synovium of the AEST was categorized into three types: intra-synovial, extra-synovial, and trans-synovial. An AEST in the anterior shoulder joint in 3D VIBE MR arthrography images was identified in 63 (9%) of 700 arthrograms. The most common arthrographic type of AEST was type 1-this was detected in 39 of 63 patients. The most common course type of the AEST was anteriorly midline. The most common distal insertion type was at the tenosynovial sheath of the long head of the biceps tendon (LHBT) in the middle humeral neck zone-this was detected in 31 of 63 patients. There were only 10 MR arthrograms biceps tendon abnormality, including 4 biceps agenesis and 6 split ruptures. A 2D and high-resolution 3D MR arthrography can demonstrate the anatomical detail around the bicipital groove and facilitate the differentiation between a biceps tendon anomaly and an AEST. On high-resolution 3D MR arthrographic images, the AEST tends to be in the anterior midline and anteromedial portions of the biceps synovium with intra-synovial, extra-synovial, and trans-synovial courses and its three different insertion types.

Comparison of ultrasound-guided and palpation-inserted peripheral venous cannula in -patients before primary hip or knee arthroplasty: study protocol for a randomized controlled trial

BackgroundMore than 2 billion peripheral vascular cannulas are introduced globally each year. It is the most frequently performed invasive procedure in medicine worldwide. There is a group of patients with difficult intravenous access (DIVA). In experts’ hands, ultrasound-guided vascular access appears to be a significantly better method. Investigators hypothesize that UGVA is superior also in short-term patency of cannula and even for blood draw through cannula. Repeated cannula pricks in the operating room setting not only puts a lot of stress on the patient and medical staff, but they also waste OR time.MethodsThis investigator-initiated prospective randomized monocentric controlled trial is designed to randomly allocate 200 patients undergoing elective primary total joint arthroplasty of hip or knee to one of two groups as follows: Group C (control group) – peripheral venous cannula insertion by palpation or Group USG (intervention) – cannula insertion by ultrasound-guided vascular access. Our primary endpoint is to compare the number of attempts for ultrasound-guided insertion of the peripheral venous cannula with common palpation insertion of the peripheral venous cannula in overweight/obese patients (BMI ≥ 25). The secondary endpoint is a failure rate of the peripheral venous cannula to administer intravenous therapy up to 5 days postoperatively. Tertiary endpoints include a portion of long PVCs that are able to ensure blood draw up to 5 days postoperatively, time needed to insert PVC in each group, number of needle tip redirections in both groups, and reinsertion of PVC needed in both groups for any reason.DiscussionThis study is pragmatic and is looking for clinically relevant data. After completion, it will answer the question of whether it is clinically relevant to use ultrasound-guided vascular access in the context of not only short-term benefit of insertion, but also up to 5 days after insertion. Also, if this method can ensure blood draw through a peripheral vein cannula, it can save resources in the perioperative period — valuable especially considering the ongoing shortage of medical staff worldwide. If this hypothesis is confirmed, this finding could contribute to more widespread implementation of ultrasound-guided peripheral vascular access in the perioperative period.Trial registrationClinicalTrials.gov NCT05156008. Registered on 13.12.2021.

#6898 CATHETER RELATED BLOODSTREAM INFECTION IN NON-TUNNELED HEMODIALYSIS CATHETERS IN A RESOURCE-LIMITED HOSPITAL IN MERIDA, YUCATAN, MEXICO

Abstract Background and Aims There is scarcity in epidemiological data regarding chronic kidney (CKD) disease in low- and medium-income countries, and information about vascular access infectious complications is limited. In Mexico, access to renal replacement therapy (RRT) is burdensome for the uninsured patients. The General Hospital Agustin O'Horán, is a secondary care level unit in Yucatan, which receives uninsured patients from southeast Mexico. In our hospital, patients who are not candidates for peritoneal dialysis start hemodialysis with non-tunneled hemodialysis central venous catheters (NTHC), mainly due to the late nephrology referral and the shortage of vascular access specialists in our unit which prevents for the timely creation of an arteriovenous fistula or the placement of a tunneled hemodialysis catheter (THC). Permanence of NTHC is the main risk factor for catheter related bloodstream infection (CRBI) and according to the literature, Gram positive microorganisms are the most common isolated microbes. The aim of our study was to describe the incidence and risk factors for CRBSI in our hospital, from January to December 2022. Method We conducted a prospective and observational study in patients older than 18 years with CKD who were candidates to start hemodialysis and who had a NTHC inserted in our hospital from January to December 2022. We evaluated the incidence, organisms and risk factors related to CRBSI. Results During the study period, 94 NTHC were placed, 63.8% of them were inserted in women. Median age of the population was 49.4 years, and the most frequent comorbidities were diabetes mellitus (63.8%), hypertension (86.2%) and renal lithiasis (27.7%). 73.4% of the NTHC were Mahurkar® catheters and most of the insertions (73.4%) were not ultrasound guided. The right jugular vein was the most common insertion site (72.3%), followed by the left jugular vein. Mean catheter use duration was 71 ± 66 days. 27 patients had evidence of CRBI, the incidence rate was 0.78 per 1000 catheters-day. CRBI was more frequent in patients with prolonged catheter use (more than 30 days) (p&amp;lt;0.001) (Table 1). In respect to blood cultures reports, 51.83% were Gram positive organisms and 48.12% were Gram negative (Table 2). Conclusion Vascular access survival without infectious complications is of paramount importance in hemodialysis patients. The prolonged used of NTHC with the associated risk for developing CRBI negatively impacts the health outcomes in our population. In accordance to previously published literature, the main isolated microbes in our hospital comprise Gram positive bacteria. One of the biggest challenges in our country is to provide our CKD population with better vascular access options like THC or arteriovenous fistula to improve the outcomes and reduce infectious complications.

Accessory head of the semitendinosus muscle: an unusual variation.

Various studies regarding the morphology of the muscles of the hamstring compartment have exhibited anatomical variations in terms of origin or insertion points. However, variation of the semitendinosus muscle is uncommon. In this case we report an accessory head of the semitendinosus muscle. During routine dissection for medical teaching purposes, a unilateral morphological variant of the semitendinosus muscle was noted on the left side in an 82-year-old female. The accessory head of the semitendinosus muscle originated lateral to the midpoint of the semitendinosus muscle. Fibres of the accessory head originated from the medial border of the long head of biceps femoris as well as the midpoint of the semitendinosus. The accessory head had a common insertion point with the semitendinosus muscle. This case report adds to the existing body of knowledge about the semitendinosus muscle, which is of anatomical and clinical value. Knowledge of the anatomical variants is essential for anatomists and clinicians and may avoid misinterpretation during diagnostic procedures.

Laparoscopic-assisted robotic distal gastrectomy for gastric cancer by Billroth II reconstruction.

There are several reconstructions in distal gastrectomy for gastric cancer, and there is no clear definition regarding the method selection. The optimal reconstruction is likely to vary according to the surgical setting, and the optimal reconstruction for robotic distal gastrectomy is urgently needed. In addition, as robotic gastrectomy is getting popular, cost and operative time are pressing issues of robotic gastrectomy. Gastrojejunostomy was planned with Billroth II reconstruction using a linear stapler arranged specifically for a robotic approach. After firing the stapler, the common insertion orifice of the stapler was closed using a 30 cm long non-absorbable barbed suture, and continuously, the afferent loop of the jejunum was lifted to the stomach with the same barbed suture. In addition, we introduced laparoscopic-assisted robotic gastrectomy, using extracorporeally inserted laparoscopic devices from the assistant port. Scissors, clips, and linear staplers were all laparoscopic tools inserted extracorporeally. Twenty-one gastric cancer patients underwent laparoscopic-assisted robotic distal gastrectomy by Billroth II reconstruction with our modifications. There were no anastomosis-related complications such as leakage, stenosis, or bleeding. There were 2 cases of aspiration pneumonia (Clavien-Dindo grade 2), 1 case of pancreatic juice leakage (grade 3a), and 1 case of delayed gastric emptying (grade 1). We successfully arranged Billroth II reconstruction for robotic distal gastrectomy with fewer operative and postoperative complications. Laparoscopic-assisted robotic gastrectomy using extracorporeally inserted devices, and continuous suturing using a barbed suture will reduce the time and cost of robotic gastrectomy.

Fluoroscopically guided repositioning of peripherally inserted central catheter in patients failing ultrasound-guided-only placement: a retrospective five-year study.

Ultrasound (US)-guided-only insertion at the bedside is safe and improves the success rates of peripherally inserted central catheter (PICC). However, PICC insertion procedures remain challenging for special cases. To show that fluoroscopically guided tip repositioning, for failed US-guided PICC placement, safely led to satisfactory positioning in difficult cases and, importantly, improved success rates of PICC placements. A retrospective study of 1560 patients who underwent US-guided PICC placement were performed. Patients who failed US-guided PICC placement were transferred to the interventional radiology department for fluoroscopically guided tip repositioning. Baseline characteristics as well as insertion-related factors were collected. All data were analyzed using SPSS software. In total, 37 (2.4%) patients who failed US-guided PICC placement accepted fluoroscopically guided adjustment or re-insertion. Of these 37 patients, 32 were enrolled. We observed no significant differences between right and left arm PICC access (P > 0.05), even though a higher percentage of PICCs were inserted into left arms (56.3%). The basilic vein (65.6%) was the most common insertion site. Only four patients experienced slight angiospasm (3.1%) and venous thrombosis (9.4%). US-guided PICC insertion failures were primarily due to line tip malposition (84.4%). All patients successfully underwent fluoroscopically guided tip repositioning, which resulted in optimal catheter tip positioning. PICC lines were adjusted in most patients (n=28, 87.5%). Malposition was the primary issue causing US-guided PICC insertion failure. Fluoroscopically guided tip repositioning safely and efficaciously led to satisfactory positioning in difficult cases; thus, we recommend this method for patients failing US-guided PICC placement.

Abstract 5773: A forward genetic screen identifies SIRT1 as a driver of neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a deadly variant of prostate cancer for which no cure is yet available. Up to 20 percent of the patients with castration resistant prostate cancer (CRPC), will progress to NEPC after treatment with standard-of-care therapies, such as abiraterone and enzalutamide, that target the androgen receptor (AR) pathway. However, the molecular mechanisms that drive the emergence of NEPC are largely unknown, contributing to a lack of treatment options for patients with NEPC. We implemented a forward genetic approach to identify novel drivers of NEPC, by using the Sleeping Beauty (SB) transposon-based forward genetic mutagenesis screening system in our Pten-, Trp53-deficient mouse model that is primed to develop NEPC at a low frequency. We generated a mouse strain (NPp53-SB) in which the Nkx3.1 promoter regulates the expression of tamoxifen-inducible Cre specifically in the mouse prostate epithelium, resulting in transposition of the T2Onc2 transposable element and conditional deletion of Pten and Trp53 in mouse prostate epithelium. We observed an increased NEPC incidence in our NPp53-SB mouse model compared to control mice without the transposon T2Onc2, suggesting that the SB-mediated insertional mutagenesis events are driving the development of NEPC. To elucidate the genetic events that are driving the observed NEPC phenotype, we identified the common insertion sites (CISs) of the transposon insertions, which revealed a total of 330 CIS-associated genes. We further performed an integrative analysis using RNA-seq data from our SB cohort, a second mouse dataset and a metastatic CRPC patient cohort, which allowed us to identify the Master Regulator (MR) proteins that are associated with NEPC, as well as the upstream genomic events that modulate these MRs and the emergence of NEPC. Our integrative analysis identified the deacetylase Sirt1 as a candidate driver of NEPC. Using CRISPR activation (CRISPRa) technology in vivo we observed that SIRT1 activation induced an NEPC-like phenotype in the adenocarcinoma LNCaP cell line, while Sirt1 drug-mediated inhibition in an NEPC cell line resulted in less aggressive tumors.In summary, we have developed a Sleeping Beauty forward genetic screen in our mouse model of CRPC, which not only accelerates disease progression, but also increases the incidence of NEPC. Our integrative analysis identified a panel of CIS-associated genes that are likely to be functionally involved in progression to NEPC. In particular, Sirt1 was shown to be a promising candidate, which may shed light on mechanisms that this aggressive disease uses to evade treatments and may inform the development of novel therapies. Citation Format: Francisca Nunes de Almeida, Alessandro Vasciaveo, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. A forward genetic screen identifies SIRT1 as a driver of neuroendocrine prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5773.

PERCUTANEOUS ENDOSCOPIC GASTROSTOMY RESULTS IN A SECONDARY STATE HOSPITAL

Introduction: The aim of this study was to determine the indications of Percutaneous endoscopic gastrostomy (PEG) patients treated in a secondary state Burdur hospital over a period of three years, the unit where the patient was hospitalized, survival time with PEG therapy, and the complications observed in these patients. Methods: Patients who underwent PEG in the endoscopy unit of our hospital, between January 2019 and January 2022, were retrospectively evaluated. Age, gender, the unit of inpatient, indications for PEG therapy, complications after PEG, and survival status of the patients were recorded in the form of case reports. Patients with coagulation disorders, hemodynamic instability, peritonitis, sepsis, infection at the insertion area, peritonitis carcinomatosis, a history of total gastrectomy and gastric varices were excluded. Results:The median age of the 120 patients included in the study was 79 (17–100) years, and 66 (55%) of the patients were male. Complications were observed in 14 (11.7%) patients, all of which were minor, including seven (5.8%) PEG site infections, three (2.5%) PEG site leakages, three (2.5%) PEG occlusions, and one (0.9%) PEG removal. It was determined that 73 (60.8%) patients survived at least 30 days; the patients were most frequently referred by the neurology unit, with a rate of 52.7%. The most common indications for PEG insertion were cerebrovascular disease (56.7%) and dementia (29.2%). Conclusion: Maintenance of nutritional requirements via PEG allows for the preservation of mucosal integrity and barrier function, intestinal immune response, and normal flora. It is superior to other enteral feeding methods due to the lower risk of aspiration. In conclusion, our study, which presented the experiences of the PEG insertion and therapy in a secondary state hospital, stated that the complication rates, indications, and referring clinics were found to be consistent with available literature.