Background
Guidelines for young children with nutritional iron deficiency anemia (IDA) presenting to the emergency department (ED) are lacking, leading to variability in care. We aimed to standardize ...management of these patients through the development and implementation of an evidence‐based algorithm using quality improvement methodology.
Procedure
Baseline data of the target population (n = 42; 60% male; median age 22.5 months, median hemoglobin 5.3 g/dl) identified variability across four key measures of clinical management: laboratory evaluation, therapy choice, therapy administration, and patient disposition. Literature review and consensus from pediatric hematology providers informed a draft algorithm that was refined in an iterative multidisciplinary process. From September 2020 to June 2021, we aimed to increase IDA management per the algorithm by ≥20% relative to baseline for the four key outcome measures using sequential Plan‐Do‐Study‐Act (PDSA) cycles. Process measures focusing on provider communication/documentation and balancing measures involving efficiency and therapy‐related adverse events were assessed concurrently.
Results
Thirty‐five patients were evaluated among four PDSA cycles and shared similar characteristics as the baseline population. Improvements of ≥20% above baseline adherence levels or 100% adherence were achieved for all outcome measure across four PDSA cycles. Adherence to recommended laboratory evaluation improved from 43 (baseline) to 71%, therapy choice from 78 to 100%, therapy administration from 50 to 83%, and disposition from 85 to 100%. ED length of stay remained stable.
Conclusions
Implementation of a standardized algorithm for young children with nutritional IDA in the ED increased adherence to evidence‐based patient care.
Abstract
Approximately two thirds of supratentorial ependymoma are driven by the ZFTA-RELA gene fusion. ZFTA is a recently described gene with a role in DNA binding and transcriptional regulation, ...while RELA (p65) is a known principal mediator of the canonical NFkB pathway, a pathway activated many human malignancies. The underlying mechanism of NFkB pathway protein interaction with the ZFTA-RELA fusion to drive tumorgenesis has not yet been described. In this study, we utilized our autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain to explore these questions. Using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) targeting HA-tagged ZFTA-RELA fusion in our IUE model, we demonstrate that both canonical and non-canonical NFkB proteins interact with ZFTA-RELA protein in ZFTA-RELA fusion ependymoma. Focusing on select key NFkB proteins, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) targeting p50/NFkB1, a protein that partners with RELA/p65 in the canonical NFkB pathway, revealing that p50/NFkB1 regulatory programs define active genes that are both distinct from and shared with the ZFTA-RELA fusion protein. Functionally, we performed CRISPR-CAS9 knock out (KO) of p50/NFkB1 and identified no phenotypic effect, as KO lines demonstrated no growth impairment. Furthermore, NFkB targeting drugs did not impair tumor growth. Given these findings, suggesting redundant transcriptional programing, we identified that ZFTA and RELA intersect with TEAD transcription factor programs to activate YAP signaling and that p50 localizes to YAP-TEAD pathway members. Targeting TEAD 1 and 4 with ChIP-Seq, we found that TEAD 1 and 4 co-regulate p50/NFkB1 and ZFTA-RELA target genes. Finally, we demonstrate YAP pathway activation across all supratentorial ependymoma, including ZFTA-RELA ependymoma. Our pathway analysis consistently converged on YAP/TEAD pathways, suggesting collaboration of ZFTA-RELA fusion with YAP/TEAD in transcriptional regulation for these tumors. Targeting YAP/TEAD may therefore represent a promising therapeutic strategy for ZFTA-RELA fusion ependymoma.
Abstract
Introduction
Ependymoma is an aggressive type of pediatric brain tumor resistant to chemotherapy, with treatment to date limited to surgical resection and radiation. Thus, identification and ...validation of molecular targets that can translate into clinical trials in ependymoma is desperately needed to improve patient outcomes. Over 70% of supratentorial ependymoma are driven by an oncogenic fusion between C11orf95 and Rela (denoted CRFUS). CRFUS expression initiates ependymoma development in mice by potentially acting as an oncogenic transcription factor and disrupting gene expression programs. We hypothesized that specific CRFUS interacting proteins are required for tumor formation and could represent lead therapeutic targets.
Methods
To study CRFUS ependymoma, a natively-forming tumor model of CRFUS generated by in utero electroporation of the developing mouse brain was utilized. Tumor cells were isolated and then subjected to nuclear Rapid Immunoprecipitation and Mass Spectrometry Analysis of Endogenous Proteins (RIME) of HA-tagged CRFUS protein. Immunoprecipitation and Western Blot (IP-WB) were utilized to probe for leading protein interactions.
Results
We identified NF-kB proteins consistent with canonical Rela mediated transcription (NFKB1 and NFKB2) as well as novel protein interactomes that converged on RNA splicing and translational regulation. In addition, we identified a large series of novel chromatin-binding proteins as candidates potentially required for CRFUS mediated tumorigenesis.
Conclusions
Further study is ongoing to validate key CRFUS protein interaction dependency on tumor development. ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing) and CUT&RUN (cleavage under target and release using nuclease) assays have been employed to further analyze the functional role of canonical Rela pathway members. By interrogating these mechanisms, novel therapeutic targets and pathways may be identified in parallel with dissecting the molecular basis of CRFUS driven ependymoma.
Abstract
BACKGROUND
Treatment of pediatric CNS tumors with T-cells modified with chimeric antigen receptors (CARTs) provides an innovative approach. We employ GD2-directed CART-cells (GD2.CARTs) ...modified to express a constitutively active interleukin-7 receptor (C7R) to increase GD2.CART survival and function independent of external cytokines, thereby augmenting activity against GD2-expressing CNS malignancies.
METHODS
In a Phase I study, we investigated the safety of intravenous GD2.CART therapy for pediatric patients with H3K27-altered diffuse midline glioma (DMG) or other GD2-expressing recurrent CNS tumors. We used a 3 + 3 design in which the first treatment cohort received GD2.CARTs without C7R, while subsequent cohorts received GD2.CARTs co-transduced with C7R (C7R-GD2.CARTs) at two dose levels. As a secondary endpoint we measured disease response 6 weeks after CART infusion.
RESULTS
12 patients were treated without dose limiting toxicity. The first cohort (n=3: thalamic DMG, spinal DMG, recurrent AT/RT) received GD2.CARTs without C7R at 1x107 cells/m2. No toxicity was observed and all 3 patients had clinical neurological improvement lasting approximately 4 weeks before disease progression. The next two cohorts received C7R-GD2.CARTs at 1x107 cells/m2 (n=3) and 3x107 cells/m2 (n=6). Mild tumor inflammation associated neurotoxicity was observed in 7 of 9 (78%) patients, which was controlled with anakinra without need for corticosteroids. Cytokine release syndrome (CRS) was observed in 5 of 9 (56%) cases grade 1 (n=4), grade 3 (n=1), resolving with tocilizumab. Eight of 9 patients receiving C7R-GD2.CARTs had clinical improvement or stability for more than 8 weeks (up to 10 months at last follow-up) and received repeat cell infusions at 6 week intervals (range 2-4 cycles). Partial radiologic responses were confirmed for 2 of 7 (29%) patients with DMG.
CONCLUSION
Intravenous treatment with C7R-modified GD2.CARTs was well tolerated in children with CNS tumors, and the duration of clinical improvement was extended by C7R co-expression.
More than 60% of supratentorial ependymomas harbor a
(ZR
) gene fusion (formerly
). To study the biology of ZR
, we developed an autochthonous mouse tumor model using
electroporation (IUE) of the ...embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZR
tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZR
-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZR
dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZR
activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZR
target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (
- and
-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of
-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.
.
Abstract
BACKGROUND: Despite being the most common central nervous system tumor in children, ≤5% of pediatric low grade gliomas (pLGG) present with metastases. Due to their rarity, there is a paucity ...of clinical and molecular data in metastatic pLGGs. To address the need, we analyzed a cohort of 22 patients with pLGG followed at Texas Children’s Hospital who presented with metastatic disease. RESULTS: The predominant histology was pilocytic astrocytoma (16/22, 73%); average age at diagnosis was 4 years 11 months. The most common sites of primary disease were optic pathway/chiasm (7/22, 32%) and suprasellar (5/22, 23%). Metastatic disease was most commonly noted in the leptomeninges (12/22, 55%). 16/22 patients (73%) were treated with up-front medical therapy following tumor biopsy/resection, the majority with carboplatin-based therapy; the remaining 6 patients received only surgery up-front. Only 2/22 patients (9%) did not progress after their initial treatment with an average follow-up of 42 months. 14 patients (14/22, 64%) had continued disease progression after at least 2 therapeutic interventions; however, only 3 patients (3/22, 14%) eventually received craniospinal radiation. 10 patients (10/22, 45%) received treatment with an agent targeting the mitogen-activated protein kinase (MAPK) pathway. 20/22 patients (91%) were alive at last follow-up (average 72 months). 4/21 patients (19%) harbored a BRAF V600E mutation while 7/20 (35%) had a BRAF::KIAA1549 duplication/fusion. 8/20 patients (40%) were wildtype for both analyzed molecular alterations in BRAF. 8 patients had germline whole exome sequencing performed and all were negative for pathogenic/likely-pathogenic variants related to their clinical phenotype. Methylation analyses are pending on patients with available tumor tissue. CONCLUSION: In our cohort of patients with metastatic pLGG, most tumors progressed despite numerous therapeutic regimens, but the overall survival was >90%. 40% of patients were wild type for the 2 most common MAPK alterations seen in pLGG.
Abstract
RATIONALE
Over 70% of supratentorial (ST) ependymoma are characterized by an oncogenic fusion between C11ORF95 and RELA. C11ORF95-RELA fusion is frequently the sole genetic driver detected ...in ST ependymoma, thus ranking this genomic event as a lead target for therapeutic investigation. RELA is a transcription factor (TF) central to mediating NF-kB pathway activation in processes such as inflammation, cellular metabolism, and chemotaxis. HYPOTHESIS: We posited that C11ORF95-RELA acts as an oncogenic TF that aberrantly shapes the tumor epigenome to drive aberrant transcription. Approach: To this end we developed an in utero electroporation (IUE) mouse model of ependymoma to express C11ORF95-RELA during embryonic development. Our IUE approach allowed us to develop C11ORF95-RELA driven tumor models and cell lines. We comprehensively characterized the epigenome and transcriptome of C11ORF95-RELA fusion driven mouse cells by H3K27ac ChIP-seq, ATAC-seq, and RNA-seq.
RESULTS
This data revealed that: 1) C11ORF95-RELA directly engages ‘open’ chromatin and is enriched at regions with known RELA TF binding sites as well as novel genomic loci/motifs, 2) C11ORF95-RELA preferentially binds to both H3K27ac (active) enhancers and promoters, and 3) Bound C11ORF95-RELA promoter loci are associated with increased transcription of genes shared with human ependymoma.
CONCLUSION
Our findings shed light on the transcriptional mechanisms of C11ORF95-RELA, and reveal downstream targets that may represent cancer dependency genes and molecular targets.
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