Citations

Abstract Marginal zone lymphoma (MZL) is an uncommon non-Hodgkin B-cell lymphoma accounting for 7-10% of lymphoma diagnoses. Extranodal MZL (EMZL) of mucosa-associated lymphoid tissue (MALT) is the most common subtype (61%) of MZL. While the mutational landscape, chromosomal aberrations and transcriptome of nodal and splenic MZL have been extensively evaluated, the mutational landscape of EMZL has only been evaluated in a small number of patients and mainly by targeted sequencing. Consequently, the comprehensive genomic landscape of EMZL remains largely uncharacterized. Herein, we performed whole exome sequencing followed by targeted sequencing of potential tumor driver genes, copy number and/or RNA transcriptome analyses in 165 patients with pretreatment EMZL tumors involving 16 anatomic locations. All samples were collected and underwent expert review as part of the Atlas of Blood Cancer Genomes consortium. The most common anatomic locations were ocular adnexa (OAMZL) (n=35), gastric (n=25, including 6 positive for Helicobacter pylori), salivary glands (n=25), and lungs (n=24). Using common criteria for variant mutation calling and significant focal copy number (CN), we identified 1218 variants (1030 missense and 188 truncation mutations) across 174 genes and 2837 CN focal alterations (1614 copy gains and 1223 copy number losses). Of these genes, 44 and 11 were mutated in >5% and >10% of specimens, respectively. We detected variants identified previously by targeted sequencing in EMZL from different locations (e.g. TNFAIP3 (A20) (12%), TBL1XR1 (13%), SPEN (10%), CARD11 (7%), TET2 (7%) and CREBBP (6%)). We detected mutations in KLF2 (10 mutations in 8 EMZL patients) and PTPRD (7 mutations in 7 EMZL patients) that were previously suggested to be specific for splenic and nodal MZL, respectively. We also detected variants not previously reported in EMZL, including a transcriptional factor ZEB2, LRP1B,and others. The most frequently mutated gene in our study (25% of tumors) was IGLL5. Some mutations were limited to specific locations (e.g. ZEB2 mutations seen exclusively in gastric and salivary gland EMZL), while most were observed across all anatomic locations. There was no association between specific mutations and clonal IGHV, in contrast to previous reports. Using Enrichr tool, we observed enrichment of mutations in genes belonging to the B cell receptor activation, NOTCH signaling, Wnt-beta catenin signaling, NF-kB signaling , PI3K AKT signaling, DNA repair and other pathways. Chromosomal and copy number changes were also commonly observed including previously reported trisomy of chromosomes 3 and 18. Combining DNA mutations and CN alterations, the number of genetic lesions per tumor sample in the 174 mutated genes ranged from 0 to 95, with an average load of 17 lesions per case. RNA sequencing was successful in 159 EMZL tumors from 16 distinct anatomic locations. Unsupervised clustering using all genes with a median log2-normalized gene expression value greater than 5 and a standard deviation greater than 1 (1323 genes) demonstrated that the majority of gastric EMZL with and without H. pylori infections clustered together on 2 dendrograms as did most of the samples of OAMZL. Since the clustering could be forced by organ-specific and not tumor-specific gene expression, we next focused on the expression of the 345 genes from the LM22 matrix that passed our transcriptomic row filtering. Unsupervised clustering resulted in clustering of most gastric EMZL irrespective of H. pylori positivity together in the same dendrogram branch, accompanied by few additional EMZL from other anatomic locations. Most of the EMZL samples from other locations did not show preferential co-clustering together. On comparison of gene expression between gastric and OAMZL, there was enrichment of the B-cell receptor signaling pathway, B-cell activation, B-cell naïve and B-cell memory cells, B-cell proliferation and NF-kB signaling in the latter. Immune microenvironment analysis showed a statistically significant increase in resting mast cells and decrease in M1 macrophages in gastric EMZL and increase in regulatory T cells in salivary EMZL. The broad genomic studies reported here underscore the biological similarity of EMZL across anatomical sites with the potential exceptions of gastric EMZL (regardless of H. Pylori status). These data provide valuable genomic and transcriptomic resources to inform future diagnostic and therapeutic strategies.

Abstract Diffuse Large B Cell Lymphoma (DLBCL), the most common subtype of aggressive B-cell Non-Hodgkin Lymphoma (NHL), accounts for 30-40% of newly diagnosed NHL cases. Despite recent progress improving our understanding of molecular features of DLBCL, there is still a high relapse rate (~45%) among patients who undergo standard treatment (R-CHOP). Several groups have identified reproducible molecular clusters of DLBCL although, the biologic significance of many of the specific gene mutations within these clusters is poorly understood. DTX1 mutations are a feature of the BN2/C1 DLBCL classifiers and are among the mutations with unknown significance. BN2/C1 DLBCL are a mixture of ABC, GCB, and Unclassified cell of origin (COO) with a common signature being the presence of genetic alterations in genes known to regulate the NOTCH pathway; yet, these tumors lack transcriptional signatures of traditional NOTCH activation, suggesting a unique and undefined biology driven through non-canonical NOTCH pathway activation. DTX1 is an E3 ubiquitin ligase which directly binds the intracellular domain of the NOTCH receptor (NICD), targeting it for degradation, resulting in suppression of NOTCH pathway activity. We therefore hypothesize that DTX1 mutations lead to NOTCH pathway dysregulation and are a critical driver for a subset of BN2/C1 DLBCLs. Using whole exome sequencing (WES) data from 404 newly diagnosed DLBCL patients, we found 8% of patients with mutations in DTX1. Alterations in DTX1 were localized to the NICD binding domain, WWE1. In silico Alphafold modeling of DTX1 WWE1-interaction with NICD revealed that patient mutations clustered within predicted interacting surfaces with NICD. These findings lead to the hypothesis that mutations in the DTX1 WWE1 domain suppress its ability to bind to and degrade NICD, enhancing NOTCH pathway activation in DLBCL. To address this question, we first compared DTX1 protein expression by immunoblot in DTX1 WWE1 mutant (OCI-Ly3 and SUDHL2) and wildtype (WT) (OCI-Ly7, OCI-Ly19, SUDHL6, and U2932) DLBCL cell lines. We found higher expression of NICD1 (p=0.03) in OCI-Ly3 and NICD2 (p<0.0001) in SUDHL2 cell lines compared to WT. This increase was accompanied by an increase in NOTCH surface expression with higher NOTCH1 (p<0.0001) and NOTCH2 (p<0.0001) expression on OCI-Ly3 and SUDHL2 cells, respectively, compared to WT. To remove variability across cell lines we next used CRISPR to generate DTX1 knock out (KO) models using WT DTX1 DLBCL cell lines U2932 and OCI-Ly19. Analysis of NICD and NOTCH receptor protein expression confirmed our initial finding, showing an 8.5 and 4.8-fold increase of NICD1 and a 6 and 2.5-fold increase of NICD2 expression in the U2932 and OCI-Ly19 DTX1 KO cell lines, respectively, compared to empty vector (EV) controls. An increase in surface NOTCH1, and to a lesser extent NOTCH2, was seen in U2932 (p=0.01 and p=0.07, respectively) and OCI-Ly19 (p=0.001 and p=0.2, respectively) KO cells compared to EV controls. To identify pathways affected by DTX1 mutations, we next performed RNAseq on U2932 EV and KO cell lines. Of interest, we did not detect any significant changes in classical NOTCH driven genes (Hes1, Hey1, etc.), supporting prior studies on BN2/C1 tumors. However, we did identify potential non-canonical NOTCH-target genes that may contribute to lymphomagenesis. Gene expression in KO cells showed upregulation of genes involved in germinal center (GC) processes (AICDA, IRF4; p<0.001) and cellular growth and survival (IL6R, CCND2, STAT5B, BCL2; p<0.0001). Measurement of IL6R by flow cytometry and CyclinD2 by immunoblot further verified DTX1-mediated upregulation of IL6R (p<0.01) and CyclinD2 (p<0.01) for U2932 KO cells compared to EV control. We also observed downregulation of genes contributing to cell death (ex. BAX, CASP8, FAS; p<0.05) in KO cells compared to EV. Together, these novel findings support our hypothesis that DTX1 WWE1 mutations lead to dysregulation of the NOTCH pathway by driving overexpression of NICD and NOTCH, resulting in dysregulated expression of proteins involved in cell cycle, GC reaction, and apoptosis. We further provide new biologic insight into BN2/C1 DLBCL, ultimately highlighting a path toward novel therapeutic targets and the advancement of personalized medicine, as patients harboring these mutations may benefit from new therapies targeting inflammatory pathways, such as CELMoDs.

Goals: Describe the establishment and expansion of a global gastrointestinal (GI) partnership and its impact on the advancement of GI care in Rwanda. Background: GI disease disproportionately affects low- and middle-income countries. In Rwanda, governmental and nonprofit efforts have made significant progress in expanding access to health care in the wake of the 1994 genocide; however, specialized care remains largely unrecognized or inaccessible. In response, the Rwanda Ministry of Health established a partnership with an international gastroenterology NGO to address this critical need. Study: We present summative data generated by this partnership. To inform future global GI programs, we provide lessons learned and details about Rwanda’s successful establishment of an in-country, self-sustainable, and locally governed gastroenterology training program. Results: From 2017 to 2023, the partnership expanded the number of clinical sites receiving care from 4 to 11. Total procedural volume increased from 244 to 1069. Most common presenting upper GI symptoms included dyspepsia, reflux, and emesis, while most common lower GI symptoms included hematochezia and constipation. Since 2017, this partnership has increased the number of faculty volunteering in Rwanda (and Rwandan clinicians studying in US academic medical centers), expanded its geographical reach, assisted in the creation of a GI fellowship, and enabled Rwanda to become an East African hub for medical education. Conclusions: Lessons learned across ethical, financial, leadership, cultural, and medical domains from the successful establishment and expansion of an international GI partnership in Rwanda provides invaluable insights to guide development of future models of health care in emerging economies.

Abstract Despite concerns about the toxicity of allogeneic hematopoietic cell transplantation (alloHCT) in older patients, prospective data characterizing prevalence or risk stratification for geriatric morbidity such as disability or frailty are limited. We prospectively assessed the prognostic impact of the novel composite health assessment risk model (CHARM), a score established to predict 1-year nonrelapse mortality (NRM), among 1105 patients aged ≥60 years enrolled on the Bone Marrow Transplant Clinical Trials Network Study 1704. Secondary end points were assessed post-alloHCT at day 100 (D100), D180, and D365 in multivariable models adjusted with predetermined clinical variables. Among alloHCT survivors, the prevalence of disability by instrumental activities of daily living (IADL), frailty by the Physical Frailty Phenotype, and physical function impairment by Patient Reported Measurement Information System (PROMIS) was highest at D100 and lower on D180 and D365. Higher CHARM scores were independently associated with greater disability (coefficient, −0.64; 95% confidence interval [CI], −0.85 to −0.43; P< .001), increased frailty (coefficient, 0.19; CI, 0.081-0.31; P< .001), worse PROMIS physical function, greater PROMIS depression, increased serious organ toxicity by D100, more cognitive decline at D100, and higher mortality after acute graft-versus-host disease (GVHD) but not significantly associated with PROMIS anxiety or acute GVHD. Higher CHARM scores predicted worse disability-free survival (odds ratio [OR], 2.03; CI, 1.66-2.48; P< .001) and lower frailty-free survival (OR, 2.00; CI, 1.61-2.49). In summary, CHARM is an independent prognostic scoring system not only for NRM but also for geriatric morbidity and functional limitation–free survival through 1 year after alloHCT. Pre-alloHCT CHARM is a novel tool to aid shared decision-making for older patients. This trial was registered at www.clinicaltrials.gov as #NCT03992352.

Abstract Description Broad spectrum antibiotics are being increasingly used in obstetric patients without long term follow up on neonatal risks. Previous data have suggested antibiotics for preterm premature rupture of membranes increases neonatal late onset sepsis and nectrozing enterocolitis (NEC), but the mechanism is unknown. We analyzed samples from the Mechanisms of Maternal Immunoglobulin A control over the neonatal microbiota and the development of NEC (MAMI) trial. MAMI enrolled infants born under 32 weeks and collected maternal-infant biospecimens. We performed a detailed chart review for maternal antibiotic exposure and analyzed breast milk samples collected 10-45 days post delivery by ELISA. Data were compared by t test and one way ANOVA. Breast milk IgA was higher than IgG and IgM (p < 0.0001, p < 0.005). When compared by antibiotic exposure, IgA and IgM were decreased with any antibiotic exposure (p = 0.02, p = 0.01). IgA was decreased by length of antibiotic exposure (p = 0.02). We stratified by the antibiotic spectrum of activity into narrow (penicillin, ampicillin) and broad (2nd and 3rd generation cephalosporin, gentamicin, etc) and found that the spectrum of antimicrobial activity was associated with a decrease in maternal IgA with broad spectrum antibiotics with the least amount of IgA (p < 0.004). Our data demonstrate mammary IgA and IgM are decreased with antenatal antimicrobial exposure. These data suggest a mechanism by which antenatal antibiotics may lead to neonatal sepsis and NEC. Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Objective Dravet syndrome (DS) is a developmental and epileptic encephalopathy characterized by drug‐resistant seizures and developmental slowing. Although cognitive and executive function deficits have been described, their early trajectory is not well understood. Methods The prospective ENVISION natural history study (NCT04537832) assessed cognitive, executive, and adaptive function in children younger than 5 years of age with SCN1A + DS every 6 months for up to 2 years using Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID‐III), Wechsler Preschool & Primary Scale of Intelligence, 4th Edition (WPPSI‐IV), Vineland Adaptive Behavior Scales, 3rd Edition (VABS‐3), Behavior Rating Inventory of Executive Function – Preschool Version (BRIEF‐P), and Pediatric Evaluation of Disability Inventory (PEDI). Results Fifty‐eight children were enrolled, with 47% younger than age 2 years. At least 80% of children did not achieve age‐appropriate milestones. Mean BSID‐III Cognitive raw scores increased minimally, with age‐equivalent gains of only 3 months over 1.5 years. Mean Cognitive Composite scores declined significantly by Month 12 (from 81.6 to 72.2; change: –11.0, 95% confidence interval [CI]: −15.3 to −6.8), signaling a widening gap compared with neurotypical development. Executive function worsened, with mean BRIEF‐P Global Executive Composite T‐scores increasing by 3.2 points/year. For some participants, scores were 5 standard deviations (SD) above the normative mean, reflecting abilities profoundly below age expectations (bottom .00003% of the population). Adaptive functioning worsened, with mean VABS‐3 Adaptive Behavior Composite decreasing from 78.7 to 68.1 over 1.5 years (change: –9.0, 95% CI: −11.9 to −6.1) and greater decline among children <2 years at enrollment, with scores decreasing by ~15 points (1 SD). Over half of children >3 years could not remove clothing independently; and when placed on a toilet, 48% could not use it. Significance Infants and young children with SCN1A + DS show significant and progressive developmental slowing across several domains, highlighting urgent need for therapies to mitigate the devastating impact on individuals and families.

Abstract Background Computational phenotyping from electronic health records (EHRs) is essential for clinical research, decision support, and quality/population health assessment, but the proliferation of algorithms for the same conditions makes it difficult to identify which algorithm is most appropriate for reuse. Objective To develop a framework for assessing phenotyping algorithm fitness for purpose and reuse. Fitness for Purpose Phenotyping algorithms are fit for purpose when they identify the intended population with performance characteristics appropriate for the intended application. Fitness for Reuse Phenotyping algorithms are fit for reuse when the algorithm is implementable and generalizable—that is, it identifies the same intended population with similar performance characteristics when applied to a new setting. Conclusions The PhenoFit framework provides a structured approach to evaluate and adapt phenotyping algorithms for new contexts increasing efficiency and consistency of identifying patient populations from EHRs.

Abstract Case Description: A 73-year-old female with history of stage IIIC high-grade mixed endometroid and serous ovarian cancer in remission presented with lower extremity weakness and paresthesias for several months ultimately wheelchair bound by presentation. Her exam was notable for severe proximal greater than distal lower extremity weakness, allodynia, absent patellar and Achilles reflexes, and diminished vibration and pinprick sensation. MRI demonstrated symmetric enhancement of the spinocerebellar tracts in the lateral cord from T6-T9. EMG showed a length-dependent axonal predominant sensorimotor neuropathy. The differential included metabolic, paraneoplastic, or inflammatory etiologies of her myelopathy. CSF studies revealed TNCs 30, elevated kappa free light chain, and negative cytology. Autoimmune/paraneoplastic myelopathy panels were negative. With her past history of cancer, FDG PET performed showed a right hypermetabolic chest wall and upper abdominal mass. Core needle biopsy demonstrated recurrence of high-grade carcinoma of mullerian origin consistent with recurrence of ovarian cancer. Her in-hospital treatment included 5 days of 1g IV methylprednisolone and seven sessions of PLEX. Her exam improved following PLEX with improvements in strength, sensation, and neurogenic bowel and bladder. She also started Carboplatin and Doxorubicin chemotherapy prior to discharge. She continued 60mg prednisone after discharge. She unfortunately had many hospitalizations for infections and anemia impacting her neurological recovery and strength has declined. Discussion: This case illustrates an interesting case of paraneoplastic myelopathy as the presenting symptom for relapsed ovarian cancer previously in remission for more than 10 years. The case shows the importance of keeping a paraneoplastic process on the differential when imaging findings and clinical syndrome are consistent with a paraneoplastic myelopathy even though the patient did not have a known, active cancer or positive antibodies identified. The work-up for malignancy is crucial as cancer-specific therapy is needed for hopeful symptom improvement.

Abstract CIC-rearranged sarcoma (CRS) is a rare disease driven by a specific fusion protein involving the CIC gene. The occurrence in the brain is 3% in all CRS patients, and these tumors frequently metastasize to the brain. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. Preliminary data show that CIC-DUX4/NUTM1 fusions activate JAK and its downstream effector STAT1/3. We hypothesize that JAK/STAT1/3 signaling cooperates with CIC-fusions to drive CIC-sarcomas by inducing ETV1/4/5 expression. Patient-derived CRS cell lines showed elevated levels of JAK1/STAT1/3 activation compared to fusion-negative sarcoma lines. Inhibition of JAK1 using Ruxolitinib and Solicitinib reduced STAT1/3 phosphorylation, downregulated ETV1/4/5 expression at both mRNA and protein levels, and diminished ETV5 promoter activity, cell proliferation, and tumorigenicity. Although the mechanism by which CIC-fusions activate oncogenic targets is still under investigation, histone acetylation appears to play a central role. STAT1/3 interacts with p300/CBP to enhance transcription, and STAT1 is necessary for p300 acetyltransferase activity. Ruxolitinib significantly reduced histone acetylation at ETV1/4/5 promoters in hMSC cells expressing CIC-DUX4/NUTM1, as well as in CRS cell lines. Unlike the p300 inhibitor C646, which causes global hypoacetylation, Ruxolitinib’s effects were promoter-specific, indicating its potential as a more targeted and less toxic therapeutic option. Importantly, we found that STAT1/3 binds to ETV1/4/5 promoters only in the presence of CIC-fusions. Luciferase assays confirmed that STAT1/3 alone cannot activate ETV5 transcription without CIC-fusions, revealing a novel cooperative mechanism. In vivo, Ruxolitinib treatment of CRS xenografts led to significant reductions in tumor volume, STAT1/3 activation, and ETV1/4/5 expression. These findings support JAK1/STAT1/3 inhibition as a promising therapeutic strategy for CRS and warrant further preclinical investigation.

Importance Treatment for cognitive dysfunction due to postacute sequelae of long COVID (ie, symptoms of fatigue, malaise, weakness, confusion that persist beyond 12 weeks after an initial COVID infection) remains a significant unmet need. Objective To test evidence-based rehabilitation strategies for improving cognitive symptoms in persons with long COVID. Design, Setting, and Participants This was a 5-arm, multicenter, randomized clinical trial of 3 remotely delivered interventions conducted between August 17, 2023, and June 10, 2024. The study took place at 22 trial sites and included the screening of individuals with cognitive long COVID. Interventions Participants were randomized to 1 of 5 arms: adaptive computerized cognitive training (BrainHQ [Posit Science]), cognitive-behavioral rehabilitation involving both group and individual counseling sessions (PASC-Cognitive Recovery [PASC-CoRE]) paired with BrainHQ, and transcranial direct current stimulation (tDCS) paired with BrainHQ. Two comparator arms were included as follows: unstructured computer puzzles and games (active comparator) and sham tDCS paired with BrainHQ. The interventions occurred 5 times per week over 10 weeks. Main Outcomes and Measures Cognitive and behavioral in-person assessments were performed at baseline, midintervention, at the end of intervention, and 3 months after the end of the intervention. The primary outcome measure was the modified Everyday Cognition Scale 2 (ECog2) completed at the end of the intervention compared to the baseline visit based on participant self-report looking back over the prior 7 days. Results A total of 378 individuals were screened, from which there were 328 participants (median [IQR] age, 48.0 [37.0-58.0] years; 241 female [73.5%]; race: 15 Asian [4.6%], 47 Black [14.3%], and 235 White [71.6%]; ethnicity: 52 Hispanic [15.9%]). None of the 3 active interventions demonstrated benefits on the modified ECog2 in the intention-to-treat population by the end of the intervention period. The adjusted differences in mean change were 0.0 (95% CI, −0.2 to 0.2) for BrainHQ vs active comparator, 0.1 (95% CI, −0.1 to 0.3) for PASC-CoRE + BrainHQ vs active comparator, 0.0 (95% CI, −0.2 to 0.2) for tDCS-active + BrainHQ vs tDCS-sham + BrainHQ, and 0.1 (95% CI, −0.1 to 0.3) for PASC-CoRE + BrainHQ vs BrainHQ alone. Secondary participant-reported outcomes and neuropsychological tests showed no differential benefits for any treatment arm. All 5 arms demonstrated some improvements over time on the modified ECog2 and on secondary outcomes. There were no serious adverse events attributable to the interventions. Conclusions and Relevance This phase 2 randomized clinical trial failed to demonstrate differential benefits for online cognitive training, a structured cognitive rehabilitation program, and tDCS for cognitive long COVID. Trial Registration ClinicalTrials.gov Identifier: NCT05965739 .