Abstract Background: Our ability to interpret pediatric cancer genomes to identify targeted therapeutic strategies is bottlenecked by limited structured knowledge about the clinical significance of detected somatic mutations. Clinical genomics laboratories routinely keep internal records of previously classified variants in ad hoc storage systems (e.g., spreadsheets, custom databases), but significant technical barriers exist that prevent the broad dissemination of such knowledge for use across institutions. Recently developed genomic knowledge standards from the Global Alliance for Genomics and Health (GA4GH) provide the foundation to remove these barriers through a shared community framework for disseminating genomic knowledge. Methods and Results: We implemented these GA4GH standards to produce over 1,500 publicly available somatic variant classification records. These were curated as part of routine pediatric cancer genome assessment in a research hospital setting, 94% (1415/1504) of which were assessed under the Molecular Characterization Initiative of the National Cancer Institute’s Childhood Cancer Data Initiative. To broadly disseminate these data, we extended the open-source “ClinVar This!” community software to ingest GA4GH-standardized records for submission to the NIH ClinVar knowledgebase. As a result, we have more than doubled the total number of somatic cancer records submitted to ClinVar (previously 1,325 records across all other community submissions). We anticipate submitting an additional 2,000 records by April 2026. To support this effort, we developed the Variation Categorizer (VarCat) web platform to simplify the structured application of community standards for clinical variant classification in a pediatric setting. The software currently supports community somatic variant classification guidelines for clinical significance (the “AMP/ASCO/CAP guidelines”) and oncogenicity (the “ClinGen/CGC/VICC guidelines”). VarCat is an open-source, clinically validated web tool that provides a streamlined mechanism for automating GA4GH-compliant dissemination of pediatric somatic cancer knowledge as part of routine clinical workflows. Conclusions: This work demonstrates how these novel GA4GH standards enable us to disseminate somatic cancer variant knowledge as part of routine clinical operations. Our presentation will highlight the use of the VarCat platform and the associated standardized variant classification data we have made publicly available. We will also share insights from our applications of these standards to capture nuanced adaptation of community guidelines for clinical variant interpretation. We will conclude with practical guidance about how to best leverage these resources as a scalable approach to addressing the variant interpretation bottleneck in the clinical setting. Citation Format: Alex H. Wagner, Kori Kuzma, Kathleen M. Schieffer, Wesley Goar, Don Corsmeier, Michael McCarrick, Kathryn Perry, Jennifer Bowser, James Stevenson, Mohammad Marhabaie, Matthew Cannon, Liana Hernandez, Doug Depoorter, Hongtao Jia, Amy Everest, Jessica Howard, Swetha Ramadesikan, Vijayakumar Jayaraman, Ying-Chen C. Hou, Mariam T. Mathew, Marco L. Leung, Yassmine M. Akkari, Daniel Puthawala, Anastasia Bratulin, Ben Kelly, Elaine R. Mardis, Catherine E. Cottrell, . A large-scale resource of standardized pediatric somatic cancer variant classifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1508.

DNA mismatch repair (MMR) is critical for maintaining genome integrity through correction of single-base mismatches and insertion-deletion loops arising from DNA replication. Heterozygous germline alteration of MMR genes (MSH2, MSH6, MLH1, PMS2) cause autosomal dominant Lynch syndrome (LS), most commonly manifesting as colonic or endometrial cancers, although brain, ovarian, and other organ systems may be involved. Neoplasia in LS usually arises after the age of 30 years. Constitutional mismatch repair deficiency (CMMRD) is inherited in an autosomal recessive manner due to biallelic germline alteration in one of the four MMR genes. Individuals with CMMRD typically develop cancer in the first decade of life, although some may present during the second decade. We present a series of five children who developed cancer prior to the age of 20 years (range: 2–12 years) with malignancies including colonic adenocarcinoma (N = 1), T-lymphoblastic lymphoma (N = 3), and high-grade glioma (N = 4). Two patients with MSH6 alterations developed a constellation of three primary tumors: high-grade glioma, T-lymphoblastic lymphoma, and colonic neoplasia including colonic adenocarcinoma in one patient and a tubular adenoma in the other.

The Molecular Characterization Initiative (MCI), a key effort of the National Cancer Institute's Childhood Cancer Data Initiative (CCDI), was launched in 2022 in collaboration with the Children's Oncology Group (COG) to bring comprehensive genomic and molecular profiling to children, adolescents, and young adults diagnosed with cancer. The MCI provides paired tumor and germline molecular testing, with results returned to clinicians to inform care. Deidentified data are made available to the research community through the CCDI Data Ecosystem to facilitate the discovery of new treatment strategies. This commentary outlines the MCI's development, key accomplishments to date, and its role in laying the foundation for standardized clinical diagnostics in pediatric oncology.

10013 Background: The Molecular Characterization Initiative (MCI), a collaboration between the Children's Oncology Group (COG) and the NCIs Childhood Cancer Data Initiative (CCDI) which is intended to define a standardized genomic characterization of pediatric cancer, provides rapid, clinical sequencing for newly diagnosed central nervous system (CNS) tumors, rare tumors, soft tissue sarcomas (STS), or advanced stage neuroblastoma (NB) to guide diagnosis and treatment for these children. Methods: Patients enrolled on APEC14B1-MCI who are ≤25 years of age with an eligible tumor type treated at national or international COG sites with available snap frozen or FFPE tissue and paired germline samples undergo RNA/DNA extraction. Whole exome sequencing (paired tumor and germline), targeted RNA fusion (excluding NB) and methylation array (CNS clinical / STS, NB and rare tumor research only) analyses are performed, with clinical results returned in 2-3 weeks. Results: Between 3/31/22 and 11/1/24, MCI provided results for 3,972 patients, including 2,666 with CNS tumors, 781 with STS, 372 with rare tumors and 153 with NB, across 188 institutions. 89% of the > 10,000 individual tests resulted within 2 weeks of receiving nucleic acids for sequencing. Tier I/II germline single nucleotide (SNV) or copy number (CNV) variants were identified in 528 (14.1%) patients [10.4% (NB) to 25.1% (rare tumors)]. The most common germline alterations included SNVs in TP53 (n = 52,1.4%), CHEK2 (n = 50,1.3%), DICER1 (n = 35,0.93%), NF1 (n = 29,0.78%) and ATM (n = 24, 0.64%). Somatic SNVs or CNVs were identified in 85% of samples overall. Somatic SNVs most commonly involved TP53 (n = 309,8.3%), BRAF (n = 222,6.9%), or CTNNB1 (n = 166,4.4%). Targeted RNA sequencing identified gene fusions in 30% overall (23% rare, 27% CNS, 40% STS). Methylation array resulted in positive subclassification of CNS tumors in 90% of patients, including 522 patients with medulloblastoma. Additional characterization of residual nucleic acid samples is planned, with data available through the database of Genotypes and Phenotypes (dbGaP). Follow up data has been collected for 1236 patients (NCI-CCDI Hub), including frontline treatment (chemotherapy and/or radiation), response to therapy, and vital status. Additionally, 749 reported on the utility of MCI testing six months following enrollment. MCI results were used for: enrollment on a clinical trial (n = 86,11.5%), treatment with a targeted therapy (n = 8,10.7%), and/or refining the pathologic diagnosis (n = 223,29.5%). Conclusions: The MCI has resulted > 10,000 sequencing assays from 3,972 children with cancer in 31 months. This has directly impacted the diagnosis and/or management of patients with newly diagnosed tumors, providing access to timely molecular testing (including methylation in CNS tumors and fusion testing in STS), and guiding therapy and clinical trial enrollment for many patients.

PURPOSE:We describe findings from genomic profiling of tumors among infantile pediatric patients studied within a translational research protocol established at our pediatric tertiary care center. Comprehensive genomic profiling was initiated to aid in diagnosis, prognostication, treatment, and detection of germline disease predisposition in this patient cohort. METHODS:Enhanced exome sequencing of disease and comparator tissue was coupled with RNA sequencing of the disease-involved specimen to assess for single nucleotide variation, insertion/deletions, copy number alteration, structural variation, fusions, and methylation profiling-based tumor classification scores. RESULTS:Among 317 patients who consented to the protocol, 39 (12%) had infantile cancers diagnosed at ≤1 year of age. Germline genetic alteration was frequent with 11 of 39 patients (28%) harboring a pathogenic change. Clinically relevant findings affecting diagnosis, prognosis, therapy, or surveillance were identified in 37 of 39 (95%) patients. CONCLUSION:Our data support that a pediatric cohort gains significant benefit from a comprehensive profiling approach, with a high yield of clinically significant findings. Nearly half of the infants in this cancer cohort harbored tumors potentially susceptible to therapeutic targets on the basis of genomic profile, and among these, another half sought benefit from therapeutic implementation.

10012 Background: Through collaboration with the National Cancer Institute as part of the Childhood Cancer Data Initiative, the Children’s Oncology Group offers prompt paired tissue and germline sequencing for newly diagnosed rare tumor subtypes. Methods: Individuals are eligible for paired germline and somatic blood/tissue sequencing if they are age 25 or younger, have been diagnosed with a rare tumor in the past 6 months and have both germline and tissue samples available. The paired samples undergo DNA and RNA extraction, followed by whole exome sequencing (paired tumor and germline) of cancer associated genes and RNA targeted fusion analysis. Results are returned to the primary institution within 2-3 weeks of receipt of both samples. Results: Between 09/12/2022 and 11/01/2024, 490 individuals from 123 institutions were enrolled with a total of 98 distinct diagnoses reported. The most common diagnosis groups were thyroid carcinoma (n = 120), neuroendocrine tumors (n = 53), sex cord stromal tumors (n = 41), and other carcinomas (n = 83). Of the 490 patients enrolled, 438 had submitted samples with successful return of exome results in 351/438 (80.1%) and fusion results in 302/438 (69.1%) by the data cut. Tier I/II germline single nucleotide (SNV) or copy number (CNV) variants were identified in 88 (25.1%) of patients that completed sequencing. The most prevalent germline alterations included SNVs in DICER1 (n = 20, 5.7%), TP53 (n = 7, 2.0%), RB1 (n = 7, 2.0%), CHEK2 (n = 6, 17%), SDHB (n = 5, 1.4%) and VHL (n = 5, 1.4%). 98% of samples demonstrated a Tier I/II somatic variant across 26 genes, most commonly found in DICER1 (n = 39, 11.1%), BRAF (n = 30, 8.5%), TP53 (n = 22, 6.3%) and CTNNB1 (n = 14, 4.0%). RNA fusion analysis identified positive results in 22.8% of samples. Testing identified over 33 distinct fusions. Fusions were most commonly associated with thyroid cancer; RET in 16 and NTRK in 12, or desmoplastic small round cell tumor; EWSR1::WT1 in 8. In 16.5% (n = 58) of the samples, the final diagnosis was refined based on the results of the molecular testing and 6.8% (n = 24) of the centers reported using a commercially available treatment targeting an identified molecular alternation. Conclusions: The MCI has enabled access to genetic sequencing to patients across the Children’s Oncology Group across a wide range of rare tumor diagnoses. Information about the available data can be accessed through the CCDI Hub Explore. Germline cancer predisposition was identified in a quarter of these samples, highlighting the importance of tumor-normal profiling to allow genetic counselling in these patients and appropriate surveillance. These results have the potential for lasting impact on understanding and treating individuals with rare cancers and the development of targeted future clinical trials.

10025 Background: The Molecular Characterization Initiative (MCI), a partnership between the Children’s Oncology Group (COG) and the NCI’s Childhood Cancer Data Initiative (CCDI), provides standardized genomic profiling of tumors and germline for subjects with newly diagnosed pediatric soft tissue sarcomas (STS). Here, we report on STS patients <25 years enrolled in MCI from July 2022 to July 2023. Methods: MCI enrollment was offered to COG institutions through APEC14B1 (Project: EveryChild), enabling patient consent, collection of clinical data, and submission of tissue/blood samples. Bio-pathology Center centrally managed sample processing, quality control, and nucleotide extraction, while molecular assays were performed at Nationwide Children’s Hospital’s Institute for Genomic Medicine. Whole-exome sequencing (WES) of tumor/normal, DNA methylation arrays and RNA fusion analysis were conducted in a CLIA-certified environment. Clinical reports, except methylation results, were returned to treating institutions within 21 days, and clinical, sequencing and methylation data were deposited in NCI’s Cancer Data Service. Results: In total, 226 rhabdomyosarcoma (RMS),158 non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), and 36 non-malignant soft tissue tumors (21 desmoid tumors) from 129 institutions were enrolled. Of 172 RMS patients, 56 were fusion-positive (FP) (46 with FOXO1 fusion and 10 with fusions of other genes). WES of 179 RMS patients identified somatic mutations in 33 genes in 110 patients (61.4%). Most frequently mutated genes included FGFR4 [25/179,14%; FN (fusion-negative) RMS:21%, FP RMS:2%), TP53 (21/179,12%; FN RMS:15%, FP RMS: 7%), and NRAS (18/179, 10%; FN RMS:14%, FP RMS: 3%). Somatic copy number variants (CNVs) were detected in 164/179 (92%) of RMS patients. Germline variants were identified in 18 of 179 RMS (10%; FN RMS:15%, FP RMS: 2%); and most commonly germline altered genes included TP53 (4/179, 2%), APC (2/18, 1%), and ATM (2/179,1%). Among 158 NRSTS > 20 histologies were enrolled, most common being synovial sarcoma (n = 16, 8%). Of 49 patients with an initial diagnosis of undifferentiated sarcoma, round cell sarcoma, spindle cell sarcoma and sarcoma NOS, 32 underwent fusion testing, and 28 had WES: in 13 (40%) sequencing resulted in specific diagnosis [CIC::DUX4 in 5, BCOR::CCNB3 in 4, NTRK rearrangement in 2, SS18::SSX2 in 1 and EWSR1::ETV1 in 1], and 5 (15%) had rare fusions involving NUTM1 , NSD3 , EGFR and COL1A1 genes;16 exhibited somatic CNVs; 5(18%) had somatic mutations; and 2 (7%) carried germline variants in TP53 and RET genes. Overall, MCI results, as reported by institutions, facilitated clinical trial enrollment in 15%, receipt of targeted therapy outside trials in 17%, and diagnostic refinement in 25% of tested patients, respectively. Conclusions: CCDI’s MCI program provides comprehensive genomic profiling of pediatric and adolescent STS, uncovering distinct somatic genetic alterations, rare fusions, actionable genomic targets and germline variants.

BACKGROUND:Liquid biopsy assays using cerebrospinal fluid (CSF) can revolutionize care for children with central nervous system (CNS) tumors by enabling precise monitoring of therapeutic responses and detecting recurrence or measurable residual disease (MRD). These assays can detect cell-free, circulating tumor DNA (ctDNA) via somatic alterations, though accurately measuring low-abundance ctDNA in CSF is challenging. METHODS:Our research focused on the optimization of next-generation sequencing library preparation from cell-free DNA (cfDNA), evaluating four commercial kits to address the low nucleic acid yield in CSF-derived cfDNA. The selected kit minimized false positives and detected somatic variants at 5% variant allele frequency using 0.1 ng input of synthetic cfDNA, suitable for low-volume CSF samples. RESULTS:We then applied our optimized workflow to six children with CNS tumors using a personalized hybrid-capture sequencing strategy ("MRD4U"), in which individualized panels were designed based on each patient's tumor sequencing. Using MRD4U, we identified ctDNA in two samples, even though neither patient had radiographic or clinical evidence of disease at the time of liquid biopsy. Notably, one ctDNA-positive patient developed radiographic recurrence four months later, demonstrating the assay's potential to detect molecular relapse ahead of conventional clinical measures. CONCLUSIONS:These findings demonstrate applicability of our personalized MRD4U assay in early detection of disease recurrence. Unlike non-targeted or tumor-agnostic CSF liquid biopsy approaches, MRD4U leverages patient-specific genomic information to enable sensitive, tumor-informed monitoring that can be deployed across a wide range of pediatric CNS tumors. Our approach is broadly applicable to any tumor type with existing genomic data, enabling ctDNA detection across diverse diagnoses. Ultimately, this strategy may inform clinical decision-making and enable earlier therapeutic intervention.

Introduction Somatic overgrowth and vascular anomalies are often present at birth but sometimes are only clinically diagnosed at a later stage. Commonly affected tissues include veins and arteries, skin, fat tissue, and even bones and the brain, resulting in phenotypic presentations ranging from vascular malformations and lipomatous and melanocytic nevus syndromes to skeletal anomalies and brain malformations. They are typically caused by variants that occur in only a subset of cells, attributed to mosaicism. The time at which these variants occur, as well as the involved cell lineage(s) determine downstream organ system and tissue involvement. Due to the mosaic occurrence of these variants, they are poorly detected in blood, necessitating sensitive assays (NGS, ddPCR) to detect them in affected tissues. Additionally, there is significant variability in testing strategies employed by different diagnostic laboratories, along with variability in the size and content of gene panels, and restrictive unclear sample requirements, resulting in a lack of consistency in genetic testing decisions and clinical management for these anomalies. Methods To address these challenges, the CGC working group on Somatic Overgrowth and Vascular Anomalies was formed. It includes 15 members comprising clinical laboratory geneticists, and molecular pathologists (with expertise in molecular testing of vascular anomalies), and medical geneticists and oncologists (with experience in treatment and management of individuals with these disorders). The group met monthly via Zoom from January 2024-Dec 2024. Each meeting centered on a sub-topic with discussion led by a team of primary and secondary reviewers, overseen by the working group co-chairs. Results As conceived from monthly discussions, this working group has under development a comprehensive overview of appropriate sample types for testing, causative genes (with both germline and somatic variants), current clinical testing methodologies, variant classification standards, and reporting guidelines that could be employed for these disorders. Major classes of genomic aberrations and recurrent hot spot variants will also be described. Lastly, knowledge gaps in the field will be addressed and recommendations for best practices for genetic testing for these disorders from the perspectives of providers, pathologists, and clinical laboratory geneticists will be outlined. Discussion and Conclusion Several genes associated with these conditions have been extensively characterized, including activating variants in the PI3K-MTOR and RAS-MAPK pathways The application of highly sensitive molecular techniques has allowed for the reliable identification of low-level mosaic variants, with use of high depth sequencing often a core step in the genetic diagnosis of these disorders. The genetic testing of individuals with vascular anomalies provides diagnostic clarity, informs prognosis, guides recurrence risk assessment, and, in some cases, opens doors to personalized treatment options, such as alpelisib for PIK3CA associated disease.

Background:Identifying germline predisposition in CNS malignancies is of increasing clinical importance, as it contributes to diagnosis and prognosis, and determines aspects of treatment. The inclusion of germline testing has historically been limited due to challenges surrounding access to genetic counseling, complexity in acquiring a germline comparator specimen, concerns about the impact of findings, or cost considerations. These limitations were further defined by the breadth and scope of clinical testing to precisely identify complex variants as well as concerns regarding the clinical interpretation of variants including those of uncertain significance. Methods:In the course of conducting an IRB-approved protocol that performed genomic, transcriptomic and methylation-based characterization of pediatric CNS malignancies, we cataloged germline predisposition to cancer based on paired exome capture sequencing, coupled with computational analyses to identify variants in known cancer predisposition genes and interpret them relative to established clinical guidelines. Results:In certain cases, these findings refined diagnosis or prognosis or provided important information for treatment planning. Conclusions:We outline our aggregate findings on cancer predisposition within this cohort which identified 16% of individuals (27 of 168) harboring a variant predicting cancer susceptibility and contextualize the impact of these results in terms of treatment-related aspects of precision oncology.

The ClinGen/CGC/VICC Oncogenicity guidelines were published in 2022 as a standardized approach for assessing a variant's capacity to promote cancer formation. We describe an assessment tool, the Variation Categorizer (VarCat), that creates highly-structured oncogenicity classifications following these guidelines suitable for a clinical laboratory setting through an intuitive user-facing web application. The resulting classification is stored in a standardized genomic knowledge format developed by the Global Alliance for Genomics and Health (GA4GH) to promote interoperability and data sharing.Here we report our assessment of 100 somatic variants from pediatric cancer cases studied by NGS-based exome sequencing in comparison to prior assessments characterized under the AMP/ASCO/CAP clinical actionability guidelines. Our study assesses application of these complementary guidelines in a high-throughput clinical setting. We describe the frequency and impact of specific oncogenicity codes used in these variant classification assessments. Our findings highlight specific codes that would benefit from further clarification for application to clinical classification workflows pursuant to clinical testing. Of particular note, we discuss applications of the OP2 (single genetic etiology) code and the need for objective criteria that classify cited studies as providing well-established, reproducible, and robust evidence. We present the challenges created due to these subjective guidelines and present our internal framework for systematically applying these codes. We conclude with an overview of the impact of our revised assessment criteria on interpretation turn-around time and reproducibility.

Retinoblastoma is an ocular cancer associated with genomic variation in the RB1 gene. In individuals with bilateral retinoblastoma, a germline variant in RB1 is identified in virtually all cases. We describe herein an individual with bilateral retinoblastoma for whom multiple clinical lab assays performed by outside commercial laboratories failed to identify a germline RB1 variant. Paired tumor/normal exome sequencing, long-read whole genome sequencing, and long-read isoform sequencing performed on a translational research basis ultimately identified a germline likely de novo LINE-1 mediated deletion resulting in a premature stop of translation of RB1 as the underlying genetic cause of retinoblastoma in this individual. Based on these research findings, the LINE-1 mediated deletion was confirmed via Sanger sequencing in our clinical laboratory and results reported into the patient medical record to allow for appropriate genetic counseling.

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants—for example how they affect gene expression profiles—remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

Introduction In the setting of pediatric and adolescent young adult cancer, increased access to genomic profiling has enhanced the detection of genetic variation associated with cancer predisposition, including germline syndromic conditions. Noonan syndrome (NS) is associated with the germline RAS pathway activating alterations and increased risk of cancer. Herein, we describe our comprehensive molecular profiling approach, the association of NS with glioma and glioneuronal tumors, and the clinical and histopathologic characteristics associated with the disease.Methods Within an institutional pediatric cancer cohort (n = 314), molecular profiling comprised of paired somatic disease-germline comparator exome analysis, RNA sequencing, and tumor classification by DNA methylation analysis was performed.Results Through the implementation of paired analysis, this study identified 4 of 314 (1.3%) individuals who harbored a germline PTPN11 variant associated with NS, of which 3 individuals were diagnosed with a glioma or glioneuronal tumor. Furthermore, we extend this study through collaboration with a peer institution to identify two additional individuals with NS and a glioma or glioneuronal tumor. Notably, in three of five (60%) individuals, paired genomic profiling led to a previously unrecognized diagnosis of Noonan syndrome despite an average age of cancer diagnosis of 16.8 years. The study of the disease-involved tissue identified signaling pathway dysregulation through somatic alteration of genes involved in cellular proliferation, survival, and differentiation.Discussion Comparative pathologic findings are presented to enable an in-depth examination of disease characteristics. This comprehensive analysis highlights the association of gliomas and glioneuronal tumors with RASopathies and the potential therapeutic challenges and importantly demonstrates the utility of genomic profiling for the identification of germline cancer predisposition.

Abstract BACKGROUND HGAP is a recently defined rare glial neoplasm, whose classification requires pathognomonic epigenetic signatures. HGAP can arise anywhere within the central nervous system but most have been reported in the cerebellum (74%). Rarely seen in children, the diagnosis of HGAP can be challenging due to morphologic overlap with other glial neoplasms, especially pilocytic astrocytoma (PA). CASE We present an 11-year male with history of poor weight gain and bilateral sensorineural hearing loss detected on routine screening. Examination showed ataxia, with nystagmus on lateral gaze. MRI brain showed a solid/cystic, dorsally exophytic, cervicomedullary tumor with extension into the 4th ventricle, and hydrocephalus. The tumor showed heterogeneous enhancement with no restricted diffusion, nor intracranial or spinal metastases. Histologic sections from the biopsy suggested classic biphasic PA, with abundant Rosenthal fibers, vascular sclerosis, and no obvious mitoses. Ki67 index was low (<5%; hotspots up to 5-10%+). ATRX mostly retained, even within the most atypical nuclei. The histological impression was classic PA. Comprehensive molecular analysis with DNA methylation showed a high confidence match (0.9883) for HGAP. Paired exome sequencing revealed CDKN2A/2B biallelic loss, missense variant and copy number loss involving NF1, loss of function splice variant in ATRX with no germline alterations detected. Interestingly, fusion panel revealed a BCR::NTRK2 fusion. He received focal photon radiation (54Gy) and adjuvant therapy with Larotrectinib. He remains well with stable disease (EFS 7months). CONCLUSION A comprehensive 2023 review of literature revealed only 14 pediatric cases (range 4-18yo) out of 144 reported cases. Including our patient, 9 were male; 8 posterior fossa, 4 supratentorial, 1 spinal cord, and 2 of unknown location. Molecular analysis of the previously reported 14 pediatric cases showed only one case with a different NTRK fusion. We herein present a unique pediatric HGAP, with entirely classic biphasic and benign PA-like features, brainstem location, and targetable NTRK2 fusion.

Neuropathology and Applied NeurobiologyVolume 50, Issue 1 e12959 FEATURED COVERFree Access Cover Image, Volume 50, Issue 1 Sage Green, Sage GreenSearch for more papers by this authorTravis Hoover, Travis HooverSearch for more papers by this authorDavid Doss, David DossSearch for more papers by this authorKimberly Davidow, Kimberly DavidowSearch for more papers by this authorAndrew W. Walter, Andrew W. WalterSearch for more papers by this authorCatherine E. Cottrell, Catherine E. CottrellSearch for more papers by this authorSidharth Mahapatra, Sidharth MahapatraSearch for more papers by this author Sage Green, Sage GreenSearch for more papers by this authorTravis Hoover, Travis HooverSearch for more papers by this authorDavid Doss, David DossSearch for more papers by this authorKimberly Davidow, Kimberly DavidowSearch for more papers by this authorAndrew W. Walter, Andrew W. WalterSearch for more papers by this authorCatherine E. Cottrell, Catherine E. CottrellSearch for more papers by this authorSidharth Mahapatra, Sidharth MahapatraSearch for more papers by this author First published: 24 January 2024 https://doi.org/10.1111/nan.12959AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract The cover image is based on the Short Communication WNT-activated, MYC-amplifi ed medulloblastoma displaying intratumoural heterogeneity by S. Green et al., https://doi.org/10.1111/nan.12945. Volume50, Issue1February 2024e12959 RelatedInformation

Background Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms. Results In sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich ( SPARC ) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms. Conclusion This work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

The PI3K enzymes modify phospholipids to regulate cell growth and differentiation. Somatic variants in PI3K are recurrent in cancer and drive a proliferative phenotype. Somatic mosaicism of PIK3R1 and PIK3CA are associated with vascular anomalies and overgrowth syndromes. Germline PIK3R1 variants are associated with varying phenotypes, including immunodeficiency or facial dysmorphism with growth delay, lipoatrophy, and insulin resistance associated with SHORT syndrome. There has been limited study of the molecular mechanism to unify our understanding of how variants in PIK3R1 drive both undergrowth and overgrowth phenotypes. Thus, we compiled genomic variants from cancer and rare vascular anomalies and sought to interpret their effects using an unbiased physics-based simulation approach for the protein complex. We applied molecular dynamics simulations to mechanistically understand how genetic variants affect PIK3R1 and its interactions with PIK3CA. Notably, iSH2 genetic variants associated with undergrowth destabilize molecular interactions with the PIK3CA receptor binding domain in simulations, which is expected to decrease activity. On the other hand, overgrowth and cancer variants lead to loss of inhibitory interactions in simulations, which is expected to increase activity. We find that all disease variants display dysfunctions on either structural characteristics or intermolecular interaction energy. Thus, this comprehensive characterization of novel mosaic somatic variants associated with two opposing phenotypes has mechanistic importance and biomedical relevance and may aid in future therapeutic developments.