Hippocampal sclerosis is a frequent finding in pediatric epilepsy surgery and has traditionally been regarded as an acquired lesion. It commonly co-occurs with focal cortical dysplasia (FCD IIIa), yet whether hippocampal injury is secondary to seizures or reflects a shared underlying etiology remains unresolved. Here we identified somatic variants activating the RAS-MAPK pathway in 40% of patients with hippocampal sclerosis, but in none with non-sclerotic hippocampus. Gain-of-function variants in PTPN11 were the most common finding, with mutations present in both cortex and hippocampus and enriched in hippocampal neurons, consistent with a shared developmental origin. In mice, Ptpn11 D61Y mutants developed profound hippocampal degeneration and gliosis following subthreshold kainic acid exposure, whereas wild-type controls were unaffected. p38-dependent stress pathways were upregulated in patients and mice, suggesting a mechanism through which ERK-p38 crosstalk lowers the threshold for seizure-induced injury. These results provide a genetic explanation for FCD IIIa, elucidate the role of somatic mutations within the RAS-MAPK pathway in driving hippocampal sclerosis, and provide a target for pathway-specific interventions for intractable seizures.

Somatic variants are a prominent cause of epilepsy-associated cortical malformations, but about half of patients undergoing genetic testing have no finding due partly to limitations in variant detection. Most studies have focused on single-nucleotide variants or small indels that are accessible to short-read sequencing technologies, but somatic structural variants are also emerging as important contributors despite their unique detection challenges. Optical genome mapping (OGM) is a promising methodology for the detection of structural variants, but requires high quality, high molecular weight DNA from clinical specimens. Here we successfully optimize a protocol for OGM of surgically-resected patient brain tissue which yields ~450x effective coverage - suitable for detecting somatic variants at low allele fractions. We apply this approach to brain specimens from four patients with epilepsy. OGM identifies large and complex mosaic structural variants ranging from 7-40% variant allele fraction, most of which are not captured by short-read exome sequencing of the same specimen. In one patient with a known germline DEPDC5 variant, OGM reveals a somatic variant - a 13.2kb deletion in DEPDC5 at approximately 20% VAF - consistent with the established two-hit model in DEPDC5-associated lesional epilepsies. By resolving the breakpoints in PacBio HiFi sequencing data, we identify a mechanism for this somatic deletion, mediated by recombination of two Alu elements flanking the region. Our findings demonstrate that OGM is a robust and complementary tool for detecting somatic structural variation in human brain tissue, with potential to improve diagnostic yield and refine genotype-phenotype correlations in neurological disorders.

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.

Dysembryoplastic neuroepithelial tumors (DNTs) are low-grade glioneuronal tumors with FGFR1 alterations. They show significant histologic and molecular overlap with other glioneuronal tumors, complicating diagnosis. We analyzed 44 tumors that were either classified as DNT by DNA methylation (n = 37), or were diagnosed histologically as DNT but did not classify as DNT by DNA methylation (n = 7). 13/37 (35%) DNT-classifying tumors were histologically diagnosed as DNTs. High-confidence DNTs (score >0.9, 23 cases, 62%) demonstrated variable histology, most frequently DNT (39%), oligodendroglioma, and ganglioglioma and most frequently harbored FGFR1 alterations. Lower-confidence DNTs (score < 0.9, 14 cases, 38%) showed greater heterogeneity; their histologic diagnoses included papillary glioneuronal tumor, extraventricular neurocytoma, and pilocytic astrocytoma. Tumors with low confidence score exhibited diverse molecular alterations including BRAF V600E mutations, PDGFRA amplification, or multiple gene fusions. Among 7 histologically diagnosed DNTs that did not classify as DNT by methylation, most grouped with the myxoid glioneuronal PDGFRA-mutant class despite lacking canonical PDGFRA mutations. Thus, DNTs with high confidence scores are relatively homogenous but DNTs with low methylation confidence scores are heterogenous, highlighting the importance of integrated molecular profiling. Our findings also suggest that the myxoid glioneuronal tumor methylation class may require further classification of underlying drivers.

Diffuse leptomeningeal glioneuronal tumors (DLGNTs) are rare, and optimal treatment remains undefined. We aim to comprehensively characterize their clinical and molecular features, offering granular insights into presentations and therapies to elucidate prognostic factors and therapeutic targets. Histologic, molecular, and clinical data of 30 patients with DLGNT were analyzed. Median age at diagnosis was 7.5 years (range: 0.9–20 years). Disease was localized at diagnosis in 16 patients (53.3

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.

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disease caused by the deficiency of the glycogen branching enzyme encoded by GBE1. GSD IV can present with variable age of onset and severity of disease processes involving liver, central and peripheral nerves, muscles, and heart. Adult Polyglucosan Body Disease (APBD) is now increasingly recognized as a continuum of the GSDIV spectrum. If the clinical disease presentation includes progressive liver failure, treatment may require liver transplant to prevent morbidity and mortality. The variable presentation of GSD IV, including the hepatic phenotypes, creates diagnostic and treatment challenges. Here we describe a girl presenting with hypotonia and hepatomegaly at age 4 years; genetic analysis revealed compound heterozygosity in GBE1: c.1621A>G p.(Asn541Asp) and c.1655C>T p.(Pro552Leu). Based on her presentation and genotypes, her phenotypic prognosis was not immediately clear. She was monitored closely for liver disease progression including, synthetic dysfunction, cholestasis, or cirrhosis, but her liver function proved stable over time.Recent analysis suggested that liver disease progression is a spectrum and some develop a progressive/severe hepatic form and others stabilize with an attenuated hepatic form. Previous reviews of GSD IV genotype-phenotype correlations have not adequately addressed the prediction of hepatic phenotype based on GBE1 genotypes. We performed an updated comprehensive literature search and genotype-phenotype analysis, while updating the GBE1 genotypes according to the HGVS nomenclature.Our detailed and comprehensive review of GSDIV adds to the previously published literature available on GSD IV genotypes (Li et al. 2010, Iijima 2018, Souza et al. 2021).

BACKGROUND:The outcome for pediatric patients with high-grade glioma (HGG) remains poor. Veliparib, a potent oral poly(adenosine diphosphate-ribose) polymerase (PARP) 1/2 inhibitor, enhances the activity of radiotherapy and DNA-damaging chemotherapy. METHODS:We conducted a single-arm, non-randomized phase 2 clinical trial to determine whether treatment with veliparib and radiotherapy, followed by veliparib and temozolomide, improves progression-free survival in pediatric patients with newly diagnosed HGG without H3 K27M or BRAF mutations, compared to patient-level data from historical cohorts with closely matching clinical and molecular features. Following surgical resection, newly diagnosed children with non-metastatic HGG were screened by rapid central pathology review and molecular testing. Eligible patients were enrolled on Stratum 1 (IDH wild-type) or Stratum 2 (IDH mutant). RESULTS:Both strata were closed to accrual for futility after planned interim analyses. Among the 23 eligible patients who enrolled on Stratum 1 and received protocol therapy, the 1-year event-free survival (EFS) was 23% (standard error, SE = 9%) and the 1-year overall survival (OS) was 64% (SE = 10%). Among the 14 eligible patients who enrolled on Stratum 2 and received protocol therapy, the 1-year EFS was 57% (SE = 13%) and 1-year OS was 93% (SE = 0.7%). CONCLUSIONS:Rapid central pathology review and molecular testing for eligibility were feasible. The protocol therapy including radiation, veliparib, and temozolomide was well tolerated but failed to improve outcomes compared to clinically and molecularly matched historical control cohorts treated with higher doses of alkylator chemotherapy. CLINICALTRIALS.GOV IDENTIFIER:NCT03581292 (first posted: July 10, 2018).

Abstract ATRT is notorious for its aggressive behavior and propensity to relapse despite multimodal treatment, with most recurrences reported within the first year from upfront therapy. Although most common in younger children (< 3 years), ATRT represents a clinically and biologically heterogeneous disease which can present in older adolescents, with similar recurrence patterns. In unique instances, ATRT can recur at later time points, potentially providing additional treatment opportunities. Herein we present a case of a late relapse in an 18-year-old male, originally diagnosed at 12 years with localized right frontal ATRT (SHH molecular subgroup) treated with gross total resection, focal proton radiation (50.4Gy), and 6 cycles of alternating cisplatin/etoposide and cyclophosphamide/etoposide. Surveillance imaging remained reassuring until 6 years later, when the patient became symptomatic, with MRI demonstrating a new large left temporal mass, for which he underwent resection; pathology was consistent with recurrent ATRT (conserved SHH subgroup) and germline whole exome sequencing was negative for tumor predisposition variants, confirming a late intracranial metastatic relapse (not second primary tumor). Within 3 weeks post resection, the tumor rapidly regrew. The patient received proton craniospinal radiation (CSI; 36 Gy with 19.8Gy boost), followed by second look surgery for residual tumor, with pathology confirming viable (albeit less proliferative) ATRT. Chemotherapy per ACNS0332 was administered for 3 cycles, though discontinued due to nephrotoxicity and mucositis. He subsequently completed 12 cycles of maintenance tazemetostat (oral EZH2 inhibitor) with minimal toxicity, aside from fatigue. This patient is now more than 1 year post-completion of tazemetostat therapy with no radiographic evidence of residual or recurrent disease. This case importantly illustrates the rare possibility of a very late metastatic intracranial recurrence in an adolescent patient who did not receive upfront CSI, and highlights potential utility of maintenance therapy with tazemetostat on the backbone of conventional re-resection, re-irradiation, and chemotherapy.

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumors (DLGNT) with 1q gain (1q+) display aggressive clinical behavior. We sought to better describe the clinical characteristics of patients with DLGNT and identify molecular drivers and therapeutic vulnerabilities of DLGNT with 1q+. METHODS DLGNTs were collected from multiple institutions, sent for methylome array and if quantity-sufficient, were subjected to RNA-seq, proteome, and phosphoproteome profiling. Wherever available, clinical data was collected. RESULTS Twenty DLGNTs (13 with 1q+, 7 without 1q+) were collected. Clinical data was obtained on 15 patients (8 with 1q+, 7 without 1q+). The median progression-free survival (PFS) was 17.5 months for those with 1q+ tumors, compared to 51 months for those without (p=0.058). All with 1q+ tumors (n=4) that received CSI radiotherapy progressed (median 12 months), compared to none without (n=3) (p=0.12). There were no responses (complete or partial) in 1q+ tumors that received chemotherapy (n=8), with 2 progressions on therapy. In tumors without 1q+ that received chemotherapy (n=6), 2 responded (1 complete, 1 partial) and 1 progressed on therapy. For 1q+ tumors that received targeted therapy (n=6), there were no responses and 2 progressions on therapy, compared to 1 response and 1 progression for tumors without (n=3). Gene Set Enrichment Analysis of all differentially methylated regions demonstrated enrichment of genes acting in mRNA processing, transcriptional regulation, p53 signaling, TGF-β receptor signaling, and T cell receptor signaling in 1q+ tumors. It identified genes with transcription factor (TF) binding sites for TFs involved in MAPK, PI3K/AKT/mTOR, WNT, and p53 pathways. Consistent with these results, hypomethylated genes at chromosome 1q in 1q+ tumors are positive regulators of MAPK, PI3K/AKT/mTOR, WNT, and p53 signaling. CONCLUSIONS DLGNT with 1q+ were more refractory to current treatment options. We identified putative molecular drivers in 1q+ tumors. Our findings suggest therapy targeting the multiple affected pathways should be considered.

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Genetically, MB can be divided into four subgroups of which the SHH subtype histologically shows nodular architecture. Within this nodular architecture, there are islands of mature cells, with more abundant neuropil and a low proliferation rate, scattered among sheets of primitive cells. METHODS: We performed laser capture microdissection followed by whole transcriptome analysis, spatial transcriptomics using Digital Spatial Profiling, whole genome DNA methylation and ChIP-Seq analysis of mature and primitive areas from 8 medulloblastomas. We developed a genetically-engineered mouse model of SHH MB showing spontaneous maturation and lack of maturation with a conditional EZH2 genetic ablation or EZH2 overactivation respectively. Finally, we developed a fucoidan-based nanoparticle drug delivery across the blood brain barrier (BBB) for targeted molecular inhibition. RESULTS: Using whole transcriptome and DNA methylation analysis, we identified ∼120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3. ChIP-Seq analysis showed striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Medulloblastoma specific EZH2 genetic ablation resulted in diffuse tumor cell differentiation and prolonged survival in mice (n = 10 per group, log-rank p = 0.01). Conversely, conditional EZH2 (Y641F) activation prevented medulloblastoma differentiation. A fucoidan-based nanoparticle successfully delivered the EZH2 inhibitor (EPZ-6438) across the murine BBB to achieve significant extension of mouse survival (median 70 days compared to 21 days in control mice; *p = 0.01, Mantel-Cox). CONCLUSIONS: Spontaneous maturation of medulloblastoma cells can be induced by inhibition of EZH2. Fucoidan-based nanoparticle delivery systems allows tumor specific targeted delivery across the BBB extending survival in mice.

Abstract BACKGROUND “Head Start-4” (HS-4) is a prospective, randomized clinic trial that tailors treatment based on medulloblastoma molecular subgroups (WNT, SHH, Group 3, and Group 4) and response to induction chemotherapy, and compares the efficacy of one versus three (tandem) cycles of myeloablative therapy. Here we compare different methodologies used to distinguish between WNT/SHH medulloblastoma (low-risk arm) and non-SHH/non-WNT medulloblastoma (high-risk arm) during the course of the trial. METHODS When HS-4 trial began enrolling patients in 2015, in the absence of a CAP-CLIA certified test for methylation and gene expression profile, we utilized histopathology/immunochemistry (HP/IHC) and chromosomal microarray (CMA) via OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either WNT, SHH, or non-WNT/non-SHH (Subgroups 3 and 4) at the time of diagnosis. Retrospectively, we performed both NanoString based 22-gene assay and DNA methylation profiling on all patient samples. RESULTS We have HP/IHC, CMA, NanoString and methylation profiling for 54 infants and young children with medulloblastoma enrolled on HS-4. While indeterminate result occurred with CMA in three cases and NanoString in two cases, HP/IHC successfully assigned samples to SHH/WNT and non-SHH/non-WNT arms of the study in all 54 cases. We have HP/IHC, CMA and DNA methylation profiling for additional 33 patients. While pathology/IHC was indeterminate in two cases (one WNT and one group 3 MB), remaining cases accurately categorized medulloblastoma methylation subtype as WNT/SHH and non-WNT/non-SHH. CONCLUSION Due to the long turnaround time, waiting for medulloblastoma molecular subtype confirmation by DNA methylation array may delay treatment initiation. HS-4 data displays robust prediction of WNT/SHH versus non-WNT/non-SHH molecular subtypes by HP/IHC in comparison to DNA methylation profiling and provides a platform to initiate treatment based on HP/IHC while awaiting molecular information. In addition, these data support relying on histopathology for medulloblastoma subtypes in resource poor countries where methylation profiling is not readily available.

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.

Abstract BACKGROUND Infants and young children with SHH medulloblastoma were demonstrated to have a favorable outcome on “Head Start (HS)” III clinical trial utilizing five cycles of induction followed by one myeloablative high-dose chemotherapy (HDCT) cycle. We present the results of “Head Start” 4 (HS-4) trial where SHH subgroup patients received either three or five cycles of induction based on response followed by one HDCT cycle (similar to HS III). METHODS Eligibility included children <6 years of age at diagnosis of localized medulloblastoma and <10 years for patients with disseminated disease. Eligible patients with SHH medulloblastoma were considered “low-risk” and non-randomly assigned to receive three cycles (five cycles if RESULTS Thirty-nine children with SHH medulloblastoma were enrolled on HS-4 with median age of 2.18 years (range: 0.28-6.88 years). Median follow-up for this cohort is 41 months (range: 18-67 months). Patients with localized SHH medulloblastoma (n=28) had significantly better 3-year event-free (EFS) compared to disseminated patients (n=11): 96.4% (95% CI: 90-100%) and 36.4% (95% CI: 16.6-79.5%), respectively (p<0.0001); however, there was no significant difference in 3-year overall survival (OS) between the two groups: 100% and 90.0% (95% CI: 73.2-100%), respectively (p=0.10). The estimated 3-year EFS for localized SHH subtype 1 and 2 patients was 100% and 95%, respectively (p=0.63). None of trial patients received irradiation prior to progression. All patients, except for four, underwent three cycles of induction. Germline variants were detected in 27% of patients tested (8/30). CONCLUSION We report excellent results for young children with localized SHH medulloblastoma when treated with only three cycles (reduced) of induction and single HDCT cycle on HS-4 trial without irradiation. Molecular data will be presented.

Background Outcomes for children with high-grade gliomas (HGG) remain poor. This multicenter phase II trial evaluated whether concurrent use of vorinostat or bevacizumab with focal radiotherapy (RT) improved 1-year event-free survival (EFS) compared to temozolomide in children with newly diagnosed HGG who received maintenance temozolomide and bevacizumab. Methods Patients ≥ 3 and < 22 years with localized, non-brainstem HGG were randomized to receive RT (dose 54–59.4Gy) with vorinostat, temozolomide, or bevacizumab followed by 12 cycles of bevacizumab and temozolomide maintenance therapy. Results Among 90 patients randomized, the 1-year EFS for concurrent bevacizumab, vorinostat, or temozolomide with RT was 43.8% (±8.8%), 41.4% (±9.2%), and 59.3% (±9.5%), respectively, with no significant difference among treatment arms. Three- and five-year EFS for the entire cohort was 14.8% and 13.4%, respectively, with no significant EFS difference among the chemoradiotherapy arms. IDH mutations were associated with more favorable EFS (P = .03), whereas H3.3 K27M mutations (P = .0045) and alterations in PIK3CA or PTEN (P = .025) were associated with worse outcomes. Patients with telomerase- and alternative lengthening of telomeres (ALT)-negative tumors (n = 4) had an EFS of 100%, significantly greater than those with ALT or telomerase, or both (P = .002). While there was no difference in outcomes based on TERT expression, high TERC expression was associated with inferior survival independent of the telomere maintenance mechanism (P = .0012). Conclusions Chemoradiotherapy with vorinostat or bevacizumab is not superior to temozolomide in children with newly diagnosed HGG. Patients with telomerase- and ALT-negative tumors had higher EFS suggesting that, if reproduced, mechanism of telomere maintenance should be considered in molecular-risk stratification in future studies.

Acute promyelocytic leukemia (APL) with variant RARA translocation is linked to over 15 partner genes. Recent publications encompassing 6 cases have expanded the spectrum of RARA partners to torque teno mini virus (TTMV). This entity is likely underrecognized due to the lack of clinician and pathologist familiarity, inability to detect the fusion using routine testing modalities, and informatic challenges in its recognition within next-generation sequencing (NGS) data. We describe a clinicopathologic approach and provide the necessary tools to screen and diagnose APL with TTMV::RARA using existing clinical DNA- or RNA-based NGS assays, which led to the identification of 4 cases, all without other known cytogenetic/molecular drivers. One was identified prospectively and 3 retrospectively, including 2 from custom automated screening of multiple data sets (50,257 cases of hematopoietic malignancy, including 4809 acute myeloid leukemia/myeloid sarcoma/APL cases). Two cases presented as myeloid sarcoma, including 1 with multiple relapses after acute myeloid leukemia-type chemotherapy and hematopoietic stem cell transplant. Two cases presented as leukemia, had a poor response to induction chemotherapy, but achieved remission upon reinduction (including all-trans retinoic acid in 1 case) and subsequent hematopoietic stem cell transplant. Neoplastic cells demonstrated features of APL including frequent azurophilic granules and dim/absent CD34 and HLA-DR expression. RARA rearrangement was not detected by karyotype or fluorescent in situ hybridization. Custom analysis of NGS fusion panel data identified TTMV::RARA rearrangements and, in the prospectively identified case, facilitated monitoring in sequential bone marrow samples. APL with TTMV::RARA is a rare leukemia with a high rate of treatment failure in described cases. The diagnosis should be considered in leukemias with features of APL that lack detectable RARA fusions and other drivers, and may be confirmed by appropriate NGS tests with custom informatics. Incorporation of all-trans retinoic acid may have a role in treatment but requires accurate recognition of the fusion for appropriate classification as APL.