Citations

AbstractObjectivesAlthough hemispheric surgeries are among the most effective procedures for drug‐resistant epilepsy (DRE) in the pediatric population, there is a large variability in seizure outcomes at the group level. A recently developed HOPS score provides individualized estimation of likelihood of seizure freedom to complement clinical judgement. The objective of this study was to develop a freely accessible online calculator that accurately predicts the probability of seizure freedom for any patient at 1‐, 2‐, and 5‐years post‐hemispherectomy.MethodsRetrospective data of all pediatric patients with DRE and seizure outcome data from the original Hemispherectomy Outcome Prediction Scale (HOPS) study were included. The primary outcome of interest was time‐to‐seizure recurrence. A multivariate Cox proportional‐hazards regression model was developed to predict the likelihood of post‐hemispheric surgery seizure freedom at three time points (1‐, 2‐ and 5‐ years) based on a combination of variables identified by clinical judgment and inferential statistics predictive of the primary outcome. The final model from this study was encoded in a publicly accessible online calculator on the International Network for Epilepsy Surgery and Treatment (iNEST) website (https://hops‐calculator.com/).ResultsThe selected variables for inclusion in the final model included the five original HOPS variables (age at seizure onset, etiologic substrate, seizure semiology, prior non‐hemispheric resective surgery, and contralateral fluorodeoxyglucose–positron emission tomography [FDG‐PET] hypometabolism) and three additional variables (age at surgery, history of infantile spasms, and magnetic resonance imaging [MRI] lesion). Predictors of shorter time‐to‐seizure recurrence included younger age at seizure onset, prior resective surgery, generalized seizure semiology, FDG‐PET hypometabolism contralateral to the side of surgery, contralateral MRI lesion, non‐lesional MRI, non‐stroke etiologies, and a history of infantile spasms. The area under the curve (AUC) of the final model was 73.0%.SignificanceOnline calculators are useful, cost‐free tools that can assist physicians in risk estimation and inform joint decision‐making processes with patients and families, potentially leading to greater satisfaction. Although the HOPS data was validated in the original analysis, the authors encourage external validation of this new calculator.

PurposeTo show that the acoustic noise of spiral MRI can be reduced by derating the gradients with minimal penalty to image quality and scan time, and to illustrate an algorithm for optimal choice of derating parameters.Theory and MethodsAcoustic noise level was measured and compared for various values of maximum gradient amplitude and slew rate for T1‐weighted spin‐echo spiral scans while maintaining image contrast, FOV and resolution, and readout time. A full gradient trajectory and a derated gradient (undersampled) trajectory were chosen for a volunteer scan followed by parallel imaging‐aided reconstruction to illustrate comparable image SNR. Two auto‐derating methods, which prioritize slew rate and gradient amplitude, respectively, were derived using analytical results from the WHIRLED PEAS variant of spiral waveforms and compared in their acoustic noise level under test use cases.ResultsDerating the gradients made the scan quieter by 16.6 dB(A) on average than a full gradient trajectory and required an undersampling factor R = 2 in order to maintain scan time, with no appreciable penalty in image SNR. Prioritizing reduced slew rate resulted in maximal loudness reduction.ConclusionScanner gradients can often be derated to reduce the acoustic noise for spiral MRI with minimal penalty in scan time and image quality with the help of parallel imaging. An automatic slew‐priority derating method that maximizes loudness reduction is given.

AbstractBackgroundUnderstanding mortality risk across the Alzheimer’s disease (AD) clinical stages is essential for resource planning and estimating the value of disease modifying therapies (DMTs), which may slow progression. Such studies are lacking.MethodThe study used the Uniform Data Set of the National Alzheimer’s Coordinating Center (NACC) from September 1, 2005 to March 1, 2021 to assess mortality risk across the AD clinical stages. It included 12,414 participants aged ≥ 50 years, who contributed to the following stages: cognitively unimpaired (CU n = 11,458), incident prodromal AD (n = 937), and AD dementia (n = 745). AD etiology and stage were established based on clinical evaluation and cognitive, behavioral, and functional scores. Study centers report mortality for actively followed participants. Participants contributed observation time until all‐cause death, disease progression, discontinuation, loss to follow‐up, data cutoff or the last visit before a longer than 36‐month gap in visits or recorded scores.Hazard ratios for each AD stage versus CU were estimated using Cox proportional hazards models. Least absolute shrinkage and selection operator facilitated final predictor selection.ResultParticipants’ mean age (SD) at study entry was 71.0 years (SD 9.2) (34.9% males). Participants with incident mild, moderate, and severe AD had increased mortality risk relative to CU after controlling for age, sex, education, apolipoprotein E ε4 genotype status, BMI, and residence type. Relative all‐cause mortality risk by AD stage decreased with increasing age; the HRs (95% CI) for mortality at 65 years of age were 6.7 (2.7‐16.9), 14.8 (8.8‐24.8), and 30.1 (16.7‐56.8) for mild, moderate and severe AD respectively; at 80 years of age, the corresponding HR (95% CI) were 2.4 (1.7‐3.3), 3.1 (2.4‐3.9), and 6.6 (4.8‐9.1). Male sex (HR = 1.75, 95% CI 1.52‐2.04) and residing in an institution (vs. private residence; HR = 3.11, 95% CI 2.28‐4.25) significantly increased relative all‐cause mortality risk.ConclusionRelative all‐cause mortality risk increases with AD severity, and more so at younger ages. Despite data limitations, findings are significant as efforts to delay disease, for instance with DMTs, could not only improve patient and caregiver quality of life and reduce costs, but also extend the lives of patients with AD.NIA/NIH Grant U24 AG072122 funds the NACC database.

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdeveloped left ventricle. Early serial postmortem examinations revealed a high rate of coronary artery abnormalities in HLHS fetal hearts (e.g., thickened wall and kinking arteries). However, the intrinsic defect in HLHS coronary vessels and its genetic basis remain unclear. Methods: We profiled human fetal heart with an underdeveloped left ventricle (ULV) and induced pluripotent stem cells derived endothelial cells (iPSC-ECs) from HLHS patients at single-cell resolution. CD144 + / NPR3 - vascular ECs were selected and classified as venous, arterial, and late arterial populations. To study the arterial EC phenotypes, we generated iPSC-arterial ECs (AECs, CDH5 + CXCR4 + NT5E -/low ) derived from 3 HLHS patients and 3 age-matched controls, and evaluated their functionalities including cell cycle regulation, angiogenesis, and inflammatory response. Results: Revealed by single cell RNA-seq and subsequent gene ontology analysis, ULV late arterial EC population showed significant defects in EC development, proliferation, angiogenesis, and Notch signaling compared to the control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation judged by the reduced CXCR4 + NT5E -/low AEC progenitors. Mature HLHS iPSC-AECs showed reduced angiogenesis and enhanced G0/G1 cell cycle arrest with downregulated cell cycle-related genes (e.g., Ki67, CCND1/2 ). Healthy human aortic smooth muscle cells exhibited abnormal proliferation and synthetic phenotypes when co-cultured with HLHS iPSC-AECs. Additionally, NOTCH pathway genes (e.g., DLL4, HEY1, GJA5 ) were suppressed in both ULV AECs and HLHS iPSC-AECs. HLHS de novo variant KMT2D directly regulated the transcription of NOTCH targeted genes involved in arterial development and proliferation via H3K4me2. Intriguingly, the treatment of NOTCH ligands (Jag1, Dll1) significantly improved the proliferation of HLHS AECs. Conclusions: Our study revealed that HLHS coronary AECs were dysfunctional in angiogenesis, proliferation, and EC-SMC interaction. KMT2D-NOTCH signaling may contribute to the impaired development and proliferation of HLHS AECs.

Introduction: Multifocal ectopic Purkinje-related premature contractions (MEPPC), the most recent addition to the spectrum of SCN5A -mediated cardiac channelopathies, is characterized by frequent premature ventricular complexes (PVCs) originating from the Purkinje system, atrial arrhythmias, and a predilection for PVC-mediated dilated cardiomyopathy. Hypothesis: To investigate the electrophysiology phenotype of MEPPC in a novel, patient-specific, re-engineered cardiomyocyte model. Methods: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from a 27-year-old male with a history of frequent, multifocal PVCs and atrial tachycardia, positive for a putative MEPPC-causative SCN5A variant (p.Q419P). A CRISPR-Cas9 Q419P variant-corrected isogenic control (IC) was created to establish the normalized electrophysiology recordings for that MEPPC model. The standard whole-cell patch clamp technique was used to measure Q419P-SCN5A- and IC-derived NaV1.5 sodium currents using voltage clamp configuration. In addition, action potential duration (APD) of the iPSC-CMs were measured using FluoVolt optical voltage-sensing dye. Results: Analysis of the current-voltage relationship revealed that neither peak sodium current densities nor late inward sodium current were impacted by Q419P-SCN5A. However, Q419P-SCN5A significantly right-shifted the V 1/2 in inactivation by 8.3 mV from -81.1±1.7 mV (IC, n=5) to -72.8±0.2 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) and left-shifted the V 1/2 in activation by -4.1 mV from -39.5±0.3 mV (IC, n=5) to -43.6±0.9 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) leading to a larger hyperpolarized window current. Optical APD90 measurement revealed no difference between the Q419P-containing (476 ± 23 ms) and the IC (474 ± 46 ms, p=0.867) cardiomyocytes. Conclusions: This is the first in-vitro disease model of MEPPC using patient-specific, re-engineered heart cells. Consistent with the findings in the non-cardiac HEK cell system, this MEPPC-causative variant contributes to increased window current in cardiomyocytes without affecting the cardiac APD. This novel cardiac cell model for MEPPC may facilitate new insights into the pathobiology of MEPPC and enable the testing of novel therapies for it.

Background: Long QT syndrome (LQTS) stems from cardiac action potential duration (APD) prolonging pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3). Sodium/glucose co-transporter 2 inhibitor (SGLT2i), a class of drugs used to treat type II diabetes and all forms of heart failure, reduces cardiac late sodium channel current. Objective: To test the ability of three commercially available SGLT2i to reduce APD in patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) models of LQT1, LQT2, and LQT3. Method: Patient-specific iPSC-CMs and a CRISPR/Cas9 gene-edited variant corrected, isogenic control (IC) iPSC-CMs were generated. The variant-corrected IC was used to determine the curative APD in order to establish the degree of APD attenuation. The SCN5A-P1332L (LQT3) and IC iPSC-CMs were tested with 10 μM of either canagliflozin (CANA), dapagliflozin (DAPA), or empagliflozin (EMPA). An ATXII (5μM)-induced APD prolongation iPSC-CM model, mimicking drug-induced LQT3, was also treated. The APD was measured 30 minutes, 1 hour, and 2 hours after treatment using local extracellular action potential recordings on multielectrode array. The efficacy of these SGLT2i were also tested in KCNQ1-V254M (LQT1) and KCNH2-G604S (LQT2) iPSC-CMs. Results: The APD90 was prolonged in SCN5A-P1332L iPSC-CMs compared to its IC (469 ± 4 ms vs 305 ± 2 ms, p<0.0001). While 10 μM of SGLT2i had no APD shortening effect on the KCNQ1-V254M or KCNH2-G604S iPSC-CMs, all three SGLT2i compounds shortened the APD90 of the SCN5A-P1332L iPSC-CMs after 1 hour of treatment from 463±5 ms to 432±6 ms (EMPA, 18.8% attenuation, p=0.0002), 459±8 ms to 430±5 ms (CANA, 17.5%, p=0.004), and 453±6 ms to 434±4 ms (DAPA, 11.4%, p=0.01). Additionally, IC iPSC-CMs treated with 5 μM of ATXII induced significant APD prolongation from 318±9 ms to 379±12 ms (p=0.0003) after 30 min incubation. However, co-incubation of ATXII and SGLT2i abolished the APD prolonging effect of ATXII. Conclusions: Therapeutically inhibiting SGLT2 effectively shortens the cardiomyocyte APD in human heart cell models of the LQT3, but not LQT1 or LQT2. This pre-clinical data compels further assessment of SGLT2i as a novel therapy for patients with congenital LQT3.

Introduction: Gain-of-function mutations in SCN5A- encoded cardiac sodium channel (Na V 1.5) cause type 3 long QT syndrome (LQT3). Patients with SCN5A variants localizing to the local anesthetic binding domain might not be sensitive to lidocaine or mexiletine treatment. Hypothesis: Phenytoin, a sodium channel blocker used to treat epilepsy, may be a novel LQT3-directed therapy for a variant involving a critical residue of the Nav1.5’s local anesthetic binding domain. Methods: SCN5A-F1760C was identified in a now deceased infant with severe LQT3 (QTc of 680-1200 ms) who was refractory to left cardiac sympathetic denervation and pharmacological treatments with either lidocaine or mexiletine. SCN5A-F1760C was engineered by site directed mutagenesis and expressed in TSA201 cells. Patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the SCN5A-F1760C-positive infant and gene-edited/variant-corrected isogenic control (IC) iPSC-CMs were generated. Na V 1.5 currents were recorded in TSA201 cells transfected with SCN5A-WT/ F1760C and action potentials (APs) from iPSC-CMs were recorded before and after treatment with 5μM Phenytoin using the whole cell patch-clamp technique. Results: SCN5A-F1760C did not change the NaV1.5 current density. However, the V1/2 of inactivation was right-shifted significantly by +14.1 mV from -86.3±0.9 mV (WT) to -72.2±0.7 mV (F1760C, p<0.0001) resulting in a marked increase in window current. F1760C increased sodium late current 2-fold from 0.18±0.04% in WT to 0.49±0.07% in F1760C (p=0.0005). Baseline APD to 90% repolarization (APD90) was prolonged significantly in F1760C-derived iPSC-CMs (641±40 ms) compared to IC iPSC-CMs (412±22 ms, p<0.0001). Treatment with 5μM phenytoin normalized the APD90 in F1760C-derived iPSC-CMs to 423+34 ms (p<0.0001), values approximating the ‘gene/variant-edited’ cure. Conclusions: Na V 1.5 gain-of-function caused by a novel SCN5A-F1760C variant results in marked APD90 prolongation which were rescued by phenytoin indicating the antiepileptic drug, phenytoin, may be a mutation-specific, LQT3-directed therapy for those LQT3-causative variants that disrupt the local anesthetic/lidocaine-binding domain.

Abstract Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase known to regulate immune cell function, cell adhesion, and vascular development. Here, we report that Syk can be expressed in high grade serous ovarian cancer and triple negative breast cancers and promotes DNA double strand break resection, homologous recombination (HR) and therapeutic resistance. We found that Syk is activated by ATM following DNA damage and is recruited to DNA double strand breaks by NBS1. Once at the break site, Syk phosphorylates CtIP, a key mediator of resection and HR, at Thr-847 to promote repair activity, specifically in Syk expressing cancer cells. Syk inhibition or genetic deletion abolished CtIP Thr-847 phosphorylation and overcame the resistant phenotype. Collectively, our findings suggest that Syk drives therapeutic resistance by promoting DNA resection and HR through a novel ATM-Syk-CtIP pathway, and that Syk is a new tumor-specific target to sensitize Syk-expressing tumors to PARPi and other DNA targeted therapy.

4028 Background: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) inhibitor in combination with an anti-programmed cell death protein 1 (PD-1) antibody has shown antitumor activity in solid tumors. AdvanTIG-105 (NCT04047862) is a phase 1/1b open-label study designed to assess the safety and preliminary antitumor activity of OCI, an anti-TIGIT monoclonal antibody (mAb), + TIS, an anti-PD-1 mAb, in pts with unresectable, locally advanced or metastatic solid tumors. In the dose-escalation part, OCI + TIS was well tolerated with preliminary antitumor activity observed, and the recommended phase 2 dose (RP2D) of OCI 900 mg intravenously (IV) every three weeks (Q3W) + TIS 200 mg IV Q3W was established. We report results from the dose-expansion part (GC/GEJC Cohort 9) of the AdvanTIG-105 study. Methods: Eligible pts had histologically/cytologically confirmed stage IV GC/GEJC. Pts were excluded if they had squamous cell, undifferentiated or other histological types of GC, had GC/GEJC with positive HER2 expression, or if they had received any prior therapy for metastatic disease. Pts received either the RP2D of OCI + TIS with oxaliplatin + capecitabine Q3W for 6 cycles (C), followed by maintenance therapy with the RP2D of OCI + TIS, + capecitabine Q3W, or the RP2D of OCI + TIS with cisplatin + 5-fluorouracil Q3W for 6 C. Treatment continued until disease progression, intolerable toxicity, or withdrawal of consent. The primary endpoint was investigator-assessed overall response rate (ORR) per RECIST v1.1. Secondary endpoints included progression-free survival (PFS), duration of response (DoR), disease control rate (DCR) per RECIST v1.1, and safety. Results: As of September 29, 2022, 60 pts with a median age of 61.5 years (range 35-82) were enrolled; 59 were efficacy evaluable. Median study follow-up was 31.1 weeks (range 1.4-78.4). ORR was 50.8% (95% CI: 37.5, 64.1); DCR was 84.7% (95% CI: 73.0, 92.8) with a median DoR of 8.1 months (95% CI: 4.5, not evaluable [NE]). Median PFS was 8.2 months (95% CI: 5.8, NE). In a subgroup analysis, ORR in pts with PD-L1 tumor area positivity (TAP) score ≥5% (n = 27) was 59.3% (95% CI: 38.8, 77.6), and 50.0% (95% CI: 30.7, 69.4) in pts with PD-L1 TAP < 5% (n = 28). All 60 pts reported ≥1 treatment-emergent adverse event (TEAE), the most common being anemia (43.3%) and platelet count decreased (41.7%). In total, 43 pts (71.7%) had ≥grade 3 TEAEs and 28 (46.7%) had serious adverse events. TEAEs leading to discontinuation of TIS and OCI occurred in 5 (8.3%) pts. TEAEs led to death in 2 (3.3%) pts; one event (neutropenic sepsis) was related to chemo, while the other (pulmonary embolism) was not treatment related. Conclusions: OCI 900 mg + TIS 200 mg + chemo was generally well tolerated and showed encouraging antitumor activity in pts with stage IV GC/GEJC. Clinical trial information: NCT04047862 .

Understanding of the evolution of metazoans from their unicellular ancestors is a fundamental question in biology. In contrast to fungi which utilize the Mon1–Ccz1 dimeric complex to activate the small GTPase RAB7A, metazoans rely on the Mon1–Ccz1–RMC1 trimeric complex. Here, we report a near-atomic resolution cryogenic-electron microscopy structure of the Drosophila Mon1–Ccz1–RMC1 complex. RMC1 acts as a scaffolding subunit and binds to both Mon1 and Ccz1 on the surface opposite to the RAB7A-binding site, with many of the RMC1-contacting residues from Mon1 and Ccz1 unique to metazoans, explaining the binding specificity. Significantly, the assembly of RMC1 with Mon1–Ccz1 is required for cellular RAB7A activation, autophagic functions and organismal development in zebrafish. Our studies offer a molecular explanation for the different degree of subunit conservation across species, and provide an excellent example of how metazoan-specific proteins take over existing functions in unicellular organisms.