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Abstract Brain voltage-gated sodium channel NaV1.1 (SCN1A) loss-of-function variants cause the severe epilepsy Dravet syndrome, as well as milder phenotypes associated with genetic epilepsy with febrile seizures plus. Gain of function SCN1A variants are associated with familial hemiplegic migraine type 3. Novel SCN1A-related phenotypes have been described including early infantile developmental and epileptic encephalopathy with movement disorder, and more recently neonatal presentations with arthrogryposis. Here we describe the clinical, genetic and functional evaluation of affected individuals. Thirty-five patients were ascertained via an international collaborative network using a structured clinical questionnaire and from the literature. We performed whole-cell voltage-clamp electrophysiological recordings comparing sodium channels containing wild-type versus variant NaV1.1 subunits. Findings were related to Dravet syndrome and familial hemiplegic migraine type 3 variants. We identified three distinct clinical presentations differing by age at onset and presence of arthrogryposis and/or movement disorder. The most severely affected infants (n = 13) presented with congenital arthrogryposis, neonatal onset epilepsy in the first 3 days of life, tonic seizures and apnoeas, accompanied by a significant movement disorder and profound intellectual disability. Twenty-one patients presented later, between 2 weeks and 3 months of age, with a severe early infantile developmental and epileptic encephalopathy and a movement disorder. One patient presented after 3 months with developmental and epileptic encephalopathy only. Associated SCN1A variants cluster in regions of channel inactivation associated with gain of function, different to Dravet syndrome variants (odds ratio = 17.8; confidence interval = 5.4–69.3; P = 1.3 × 10−7). Functional studies of both epilepsy and familial hemiplegic migraine type 3 variants reveal alterations of gating properties in keeping with neuronal hyperexcitability. While epilepsy variants result in a moderate increase in action current amplitude consistent with mild gain of function, familial hemiplegic migraine type 3 variants induce a larger effect on gating properties, in particular the increase of persistent current, resulting in a large increase of action current amplitude, consistent with stronger gain of function. Clinically, 13 out of 16 (81%) gain of function variants were associated with a reduction in seizures in response to sodium channel blocker treatment (carbamazepine, oxcarbazepine, phenytoin, lamotrigine or lacosamide) without evidence of symptom exacerbation. Our study expands the spectrum of gain of function SCN1A-related epilepsy phenotypes, defines key clinical features, provides novel insights into the underlying disease mechanisms between SCN1A-related epilepsy and familial hemiplegic migraine type 3, and identifies sodium channel blockers as potentially efficacious therapies. Gain of function disease should be considered in early onset epilepsies with a pathogenic SCN1A variant and non-Dravet syndrome phenotype.

Background: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. Methods: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell–derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. Results: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio . Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell–derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. Conclusions: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

5011 Background: Emerging data suggest metastasis-directed therapy (MDT) improves outcomes in patients with oligometastatic castration-sensitive prostate cancer (omCSPC). Prostate-specific membrane antigen positron emission tomography (PSMA-PET/CT) can detect occult metastatic disease and has been proposed as a biomarker for treatment response. Herein we identify and validate a PSMA-PET biomarker for clinical outcomes following MDT in omCSPC. Methods: This was an international multi-institutional retrospective study of two completely independent cohorts of men with omCSPC, defined as ≤3 lesions, treated with metastasis-directed stereotactic ablative radiation therapy (SABR) who underwent PSMA-PET/CT prior to and 3-6 months after treatment. Pre- and post-SABR PSMA-PET/CT standardized uptake value (SUV) was measured for all lesions and PSMA response defined discretely using a cutpoint of ≥ 30% decrease in SUVmax. PSMA-PET response was correlated with lesion local control (LLC), radiographic progression-free survival (rPFS) defined using conventional and PET imaging, and metastasis-free survival (MFS) defined by conventional imaging alone. Results: A total of 131 patients with 261 treated metastases were included in the analysis, with median follow-up of 29 months (IQR 18.5-41.3). Following SABR, 78.4% of lesions experienced a partial or complete PSMA response. Multivariable analysis demonstrated SUV response significantly associated with improved LLC (HR = 9.97, 95%CI 3.92-25.4; p < 0.01). Patients with PSMA response in all lesions experienced significantly better rPFS (HR = 0.49, 95%CI 0.26-0.92; p = 0.03) compared to their counterparts and this maintained significance within both the discovery (p < 0.01) and validation (p = 0.01) cohorts. Within the discovery cohort, patients with PSMA response in all lesions also experienced significantly improved MFS (HR = 0.24, 95%CI 0.07-0.85; p = 0.03); analysis of the independent validation cohort is ongoing. Conclusions: Following SABR, PSMA-PET response is a robust and externally validated radiographic biomarker for rPFS and appears to be associated with MFS pending validation. This approach holds promise for guiding clinical management of omCSPC.

4146 Background: Targeting focal adhesion kinase (FAK) renders checkpoint immunotherapy effective in pancreatic ductal adenocarcinoma (PDAC) mouse models. Defactinib is a highly potent oral FAK inhibitor shown to have a tolerable safety profile. We evaluated the safety and recommended phase 2 dose (RP2D) of defactinib in combination with pembrolizumab and gemcitabine for PDAC patients. Methods: We conducted a multicenter, open-label, phase I study with dose escalation and expansion phases. In 3x3 dose escalation, patients with refractory solid tumors were treated at five escalating dose levels of defactinib and gemcitabine to identify a RP2D. In expansion phase, patients with metastatic PDAC who progressed on frontline treatment (refractory cohort) or had treatment response or stable disease (SD) on standard gemcitabine/nab-paclitaxel (maintenance cohort) were treated at RP2D. Pre- and post-treatment tumor biopsies were performed to evaluate changes in tumor immunity. Results: The triple drug combination was well-tolerated with no dose-limiting toxicities. Among 17 treated patients with refractory PDAC, the disease control rate (DCR) was 58.8% with one partial response (PR) and nine SDs and the median progression-free survival (PFS) and overall survival (OS) were 4.2 months and 9.1 months, respectively. Among the evaluable patients in the maintenance cohort, DCR was 63.6% with one PR and six SD. Three patients with SD came off study due to treatment- or disease-related complications. The median PFS and OS were 5.0 months and 8.3 months, respectively. Conclusions: The combination of defactinib, pembrolizumab, and gemcitabine was well-tolerated, had promising preliminary efficacy, and showed increased infiltrative T lymphocytes in post-treatment tumor biopsies. Incorporation of a more potent chemotherapy backbone should be considered to achieve better clinical response in future trial design. Clinical trial information: NCT02546531.

2068 Background: There is a lack of effective therapy for recurrent high-grade meningiomas who relapsed after prior radiation therapy (RT). ETCTN 10186 is a phase I/II study to evaluate the feasibility and preliminary clinical efficacy of combining reirradiation using fractionated radiosurgery with nivolumab plus or minus ipilimumab for recurrent high-grade meningiomas. The preliminary results from the phase I portion are reported here. Methods: Recurrent grade II-III meningioma patients were treated with radiosurgery plus nivolumab with or without ipilimumab. Key eligibility criteria include age ≥ 18 years; ECOG score ≤ 2; tumor diameter 1-5 cm; prior radiation dose ≤ 70 Gy; normal organ function; no active autoimmunity. During the phase I portion, eligible patients were treated according to treatment-escalation schema following the modified 3+3 design (Table 1). The maximum tolerated combination (MTC) will be the regimen at which ≤ 1/6 patients experience dose-limiting toxicity (DLT) within 8 weeks of the start of study therapy. During the phase II portion, a total of 24 evaluable patients will be enrolled at the MTC using Simon’s MiniMax two-stage design. The primary endpoint of the phase I portion is to determine the MTC. Objective radiological responses (ORRs) are defined as per the Macdonald criteria. One cycle of immunotherapy is defined as 4 weeks of nivolumab with or without concurrent ipilimumab. Results: From 7/2019 to 3/2021, 13 patients were enrolled in the phase I, including 6 with regimen A and 7 with regimen B (Table). The median prior RT dose was 56 Gy (18-70) at a median interval of 2.8 years (1.3-13.3). There was no DLT in either cohort, so regimen B was deemed the MTC. The median cycles of immunotherapy completed were not significantly different between cohort A and B (11 vs. 6, p = 0.41, respectively). Three cohort A patients and 2 cohort B patients completed all planned doses of immunotherapy. Most patients stopped due to progression, and only one cohort B patient stopped due to treatment-related toxicity (grade 3 hypophysitis and encephalitis after 6 cycles of immunotherapy). After a median follow-up of 11.1 months, there have been 5 progressions and 4 deaths. The 6 month-PFS and 12 month-PFS rates are 62% and 54%, respectively. Two ORRs have been reported by institutional assessment, and central radiology review is ongoing and will be reported. Conclusions: Reirradiation using fractionated radiosurgery with nivolumab plus or minus ipilimumab are well tolerated for radiation-relapsed high-grade meningiomas. Phase II study of regimen B is currently enrolling. Clinical trial information: NCT03604978. [Table: see text]

Abstract BackgroundMany studies have evaluated bundled interventions to improve hand hygiene compliance (HHC). However, the compliance rates remain low. We aimed to use Kotter's change model (KCM) to improve HHC and conduct a complete process evaluation for HHC among medical staff in the intensive care unit (ICU). MethodsKCM was administrated in the ICU of Zhongnan Hospital of Wuhan University from March 2018 to August 2021, and 41-month longitudinal monitoring of HHC was carried out. The primary outcome was the absolute change in monthly HHC. The secondary outcomes included the characteristics of HHC in different phases, the different change trends of HHC for different HH opportunities and occupations, the quarterly incidence of the central line-associated bloodstream infection (CLABSI), and the catheter-associated urinary tract infection (CAUTI).Results. 20,222 HH actions and 24,195 HH opportunities were included in this study. The overall HHC was 83.58% (95%CI, 83.11%-84.04%). After implementing the KCM project, the HHC increased from 35.71% (95%CI, 22.99%-50.83%) to 87.75% (95%CI, 85.53%-89.67%), with an increased rate of 145.73%. The fastest-growing rate of HHC was after contact patients, which increased from 35.29% to 89.8%. Quarterly trends of the CLABSI rate (0‰-3.53‰) and CAUTI rate (0.96‰-4.26‰) remained stable at a low level under the background of the number of cases and the treatment complexities of the patients increasing year by year.ConclusionsUsing KCM can systematically change the cognition of HH in healthcare providers and create an atmosphere to promote HH to improve and maintain the stability of the HHC of healthcare providers in ICU.

Abstract Purpose: This phase Ib/2 trial investigated pembrolizumab-containing trimodality therapy in patients with gastroesophageal junction (GEJ) adenocarcinoma. Patients and Methods: Patients with GEJ adenocarcinoma (cT1–3NanyM0) received neoadjuvant pembrolizumab-containing chemoradiation (CROSS regimen) followed by surgical resection and adjuvant pembrolizumab. The primary endpoints were tolerability in the first 16 patients and pathologic complete response [pCR (ypT0N0)]. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). An independent propensity-score-matched cohort (treated with CROSS without immunotherapy) was used for comparison. Exploratory analyses included immune biomarkers in the tumor microenvironment (TME) and plasma. Results: We enrolled 31 eligible patients, of whom 29 received all expected doses of neoadjuvant pembrolizumab and 28 underwent R0 resection. Safety endpoints were met. The primary efficacy endpoint was not met [7/31 (22.6%) achieved pCR]. Patients with high [i.e., combined positive score (CPS) ≥ 10] baseline expression of programmed death (PD)-L1 in the TME had a significantly higher pCR rate than those with low expression [50.0% (4/8) vs. 13.6% (3/22); P = 0.046]. Patients with high PD-L1 expression also experienced longer PFS and OS than propensity-score-matched patients. Among trial patients with PD-L1 CPS &lt; 10, unprespecified analysis explored whether extracellular vesicles (EV) could identify further responders: an elevated plasma level of PD-L1–expressing EVs was significantly associated with higher pCR. Conclusions: Adding pembrolizumab to trimodality therapy showed acceptable tolerability but did not meet the pre-specified pCR endpoint. Exploratory analyses suggested that high PD-L1 expression in the TME and/or on EVs may identify patients most likely to achieve tumor response.

Abstract Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by reexpression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKA), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, cotreatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable. Significance: Metabolic synthetic lethal screening in IDHWT glioblastoma defines a vulnerability to ΑΚG following BCAT1 loss, uncovering a therapeutic strategy to improve glioblastoma treatment. See related commentary by Meurs and Nagrath, p. 2354

AbstractPurposeTo develop an online graphic processing unit (GPU)‐accelerated Monte Carlo‐based adaptive radiation therapy (ART) workflow for pencil beam scanning (PBS) proton therapy to address interfraction anatomical changes in patients treated with PBS.Methods and MaterialsA four‐step workflow was developed using our in‐house developed GPU‐accelerated Monte Carlo‐based treatment planning system to implement online Monte Carlo‐based ART for PBS. The first step conducts diffeomorphic demon‐based deformable image registration (DIR) to propagate contours on the initial planning CT (pCT) to the verification CT (vCT) to form a new structure set. The second step performs forward dose calculation of the initial plan on the vCT with the propagated contours after manual approval (possible modifications involved). The third step triggers a reoptimization of the plan depending on whether the verification dose meets the clinical requirements or not. A robust evaluation will be done for both the verification plan in the second step and the reopotimized plan in the third step. The fourth step involves a two‐stage (before and after delivery) patient‐specific quality assurance (PSQA) of the reoptimized plan. The before‐delivery PSQA is to compare the plan dose to the dose calculated using an independent fast open‐source Monte Carlo code, MCsquare. The after‐delivery PSQA is to compare the plan dose to the dose recalculated using the log file (spot MU, spot position, and spot energy) collected during the delivery. Jaccard index (JI), dice similarity coefficients (DSCs), and Hausdorff distance (HD) were used to assess the quality of the propagated contours in the first step. A commercial plan evaluation software, ClearCheck™, was integrated into the workflow to carry out efficient plan evaluation. 3D Gamma analysis was used during the fourth step to ensure the accuracy of the plan dose from reoptimization. Three patients with three different disease sites were chosen to evaluate the feasibility of the online ART workflow for PBS.ResultsFor all three patients, the propagated contours were found to have good volume conformance [JI (lowest‐highest: 0.833–0.983) and DSC (0.909–0.992)] but suboptimal boundary coincidence [HD (2.37–20.76 mm)] for organs‐at‐risk. The verification dose evaluated by ClearCheck™ showed significant degradation of the target coverage due to the interfractional anatomical changes. Reoptimization on the vCT resulted in great improvement of the plan quality to a clinically acceptable level. 3D Gamma analyses of PSQA confirmed the accuracy of the plan dose before delivery (mean Gamma index = 98.74% with a threshold of 2%/2 mm/10%), and after delivery based on the log files (mean Gamma index = 99.05% with a threshold of 2%/2 mm/10%). The average time cost for the complete execution of the workflow was around 858 s, excluding the time for manual intervention.ConclusionThe proposed online ART workflow for PBS was demonstrated to be efficient and effective by generating a reoptimized plan that significantly improved the plan quality.

BackgroundThe feasibility and reproducibility of multifrequency MR elastography (MRE) for diagnosing pancreatic ductal adenocarcinoma (PDAC) have not been reported.PurposeTo determine the feasibility and reproducibility of multifrequency MRE for assessing pancreatic stiffness in healthy and diseased pancreases.Study typeProspective.SubjectsA total of 40 healthy volunteers and 10 patients with PDAC were prospectively recruited between March 2018 and October 2021.Field strength/sequenceA 3.0‐T pancreatic MRE at frequencies in the order of 30, 40, 60, 80, and 100 Hz.AssessmentBody mass index (BMI) and wave distance of the healthy pancreas and PDAC were measured. Image quality was assessed using the image quality score (IQS: 1–4, ≥3 were considered diagnostic quality). Three readers independently performed the pancreatic stiffness and IQS assessments to evaluate reproducibility.Statistical testsLogistic regression analyses were performed to determine variables that influenced IQS. Statistical significance was set at P <0.05. Levels of inter‐ and intrarater agreement were assessed using intraclass correlation coefficients (ICC) and Cohen's kappa coefficient (κ). Good reproducibility was set at ICC and κ ≥ 0.8.ResultsIn logistic regression analysis, a diagnostic IQS in healthy volunteers was independently associated with a lower BMI (odds ratio [OR] = 0.89 kg/m−2), shorter wave distance (OR = 0.70 cm−1), and lower frequency (30 and 40 Hz: OR = 170.01 and 96.02). In PDAC, frequency was the only independent factor for diagnostic IQS (30–60 Hz: OR = 46.18, 46.18, and 17.20, respectively) with 100 Hz as a reference. In healthy volunteers, good reproducibility was observed at 30 and 40 Hz. In PDAC, good reproducibility was observed at 30–60 Hz.Data conclusionMRE at 30 and 40 Hz provides diagnostic wave images and reliable measurements of pancreatic stiffness in healthy volunteers. MRE at 30–60 Hz is acceptable for PDACs (IQS ≥ 3, ICC and κ ≥ 0.80).Evidence Level1Technical EfficacyStage 2