Looking beyond year 1 in the molecular era of pediatric brain tumor diagnosis: confirmatory testing of germline variants found on tumor sequencing.

Purpose: Somatic molecular profiling of pediatric brain tumors aids with the diagnosis and treatment of patients with a variety of high- and low-grade central nervous system neoplasms. Here, we report follow-up targeted germline evaluation for patients with possible germline variants following tumor only testing in the initial year in which somatic molecular testing was implemented at a single institution. Patients and Methods: Somatic testing was completed for all tumors of the central nervous system (CNS) undergoing diagnostic workup at Seattle Children's Hospital during the study period of November 2015 to November 2016. Sequencing was performed in a College of American Pathologists-accredited, Clinical Laboratory Improvements Amendments-certified laboratory using UW-OncoPlex™ assay (version 5), a DNA-based targeted next generation sequencing panel validated to detect genetic alterations in 262 cancer-related genes. We tracked subsequent clinical evaluation and testing on a subgroup of this cohort found to have potential germline variants of interest. Results: Molecular sequencing of 88 patients' tumors identified 31 patients with variants that warranted consideration of germline testing. To date, 19 (61%) patients have been tested. Testing confirmed germline variants for ten patients (31% of those identified for testing), one with two germline variants (NF1 and mosaic TP53). Eight (26%) patients died before germline testing was sent. One patient (13%) has not yet had testing. Conclusion: Clinically validated molecular profiling of pediatric brain tumors identifies patients who warrant further germline evaluation. Despite this, only a subset of these patients underwent the indicated confirmatory sequencing. Further work is needed to identify barriers and facilitators to this testing, including the role of genetic counseling and consideration of upfront paired somatic-germline testing.

Therapeutic HDAC inhibition in hypermutant diffuse intrinsic pontine glioma.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with the development of hypermutant pediatric high-grade glioma, and confers a poor prognosis. While therapeutic histone deacetylase (HDAC) inhibition of diffuse intrinsic pontine glioma (DIPG) has been reported; here, we use a clinically relevant biopsy-derived hypermutant DIPG model (PBT-24FH) and a CRISPR-Cas9 induced genetic model to evaluate the efficacy of HDAC inhibition against hypermutant DIPG. We screened PBT-24FH cells for sensitivity to a panel of HDAC inhibitors (HDACis) in vitro, identifying two HDACis associated with low nanomolar IC50s, quisinostat (27 nM) and romidepsin (2 nM). In vivo, quisinostat proved more efficacious, inducing near-complete tumor regression in a PBT-24FH flank model. RNA sequencing revealed significant quisinostat-driven changes in gene expression, including upregulation of neural and pro-inflammatory genes. To validate the observed potency of quisinostat in vivo against additional hypermutant DIPG models, we tested quisinostat in genetically-induced mismatch repair (MMR)-deficient DIPG flank tumors, demonstrating that loss of MMR function increases sensitivity to quisinostat in vivo. Here, we establish the preclinical efficacy of quisinostat against hypermutant DIPG, supporting further investigation of epigenetic targeting of hypermutant pediatric cancers with the potential for clinical translation. These findings support further investigation of HDAC inhibitors against pontine high-grade gliomas, beyond only those with histone mutations, as well as against other hypermutant central nervous system tumors.

Current Utilization of Electron Microscopy in the Pediatric Pathology Setting: A Survey by the SPP Practice Committee.

BACKGROUND: Electron microscopy (EM), once an important component in diagnosing pediatric diseases, has experienced a decline in its use. To assess the impact of this, pediatric pathology practices were surveyed regarding EM services. METHODS: The Society of Pediatric Pathology Practice Committee surveyed 113 society members from 74 hospitals. Settings included 36 academic tertiary, 32 free-standing children's, and 6 community hospitals. RESULTS: Over 60% maintained in-house EM services and had more than 2 pathologists interpreting EM while reporting a shortage of EM technologists. Freestanding children's hospitals had the most specimens (100-200 per year) and more diverse specimen types. Hospitals with fewer than 50 yearly specimens often used reference laboratories. Seventeen had terminated all in-house EM services. Challenges included decreasing caseloads due to alternative diagnostic methods, high operating costs, and shortages of EM technologists and EM-proficient pathologists. Kidney, liver, cilia, heart, and muscle biopsies most often required EM. Lung/bronchoalveolar lavage, tumor, skin, gastrointestinal, nerve, platelet, and autopsy samples less commonly needed EM. CONCLUSIONS: The survey revealed challenges in maintaining EM services but demonstrated its sustained value in pediatric pathology. Pediatric pathologists may need to address the centralization of services and training to preserve EM diagnostic proficiency among pathologists who perform ultrastructural interpretations.

Changes to pediatric brain tumors in 2021 World Health Organization classification of tumors of the central nervous system.

New tumor types are continuously being described with advances in molecular testing and genomic analysis resulting in better prognostics, new targeted therapy options and improved patient outcomes. As a result of these advances, pathological classification of tumors is periodically updated with new editions of the World Health Organization (WHO) Classification of Tumors books. In 2021, WHO Classification of Tumors of the Central Nervous System, 5th edition (CNS5), was published with major changes in pediatric brain tumors officially recognized including pediatric gliomas being separated from adult gliomas, ependymomas being categorized based on anatomical compartment and many new tumor types, most of them seen in children. Additional general changes, such as tumor grading now being done within tumor types rather than across entities and changes in definition of glioblastoma, are also relevant to pediatric neuro-oncology practice. The purpose of this manuscript is to highlight the major changes in pediatric brain tumors in CNS5 most relevant to radiologists. Additionally, brief descriptions of newly recognized entities will be presented with a focus on imaging findings.

Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. SIGNIFICANCE: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.

Sensitization of osteosarcoma to irradiation by targeting nuclear FGFR1.

Over the past 25 years, chemotherapy regimens for osteosarcoma have failed to improve the 65-70% long-term survival rate. Radiation therapy is generally ineffective except for palliative care. We here investigated whether osteosarcoma can be sensitized to radiation therapy targeting specific molecules in osteosarcoma. Large-scale RNA sequencing analysis in osteosarcoma tissues and cell lines revealed that FGFR1 is the most frequently expressed receptor tyrosine kinase in osteosarcoma. Nuclear FGFR1 (nFGFR1) was observed by subcellular localization assays. The functional studies using a FGFR1IIIb antibody or small molecule FGFR1 inhibitors showed that nFGFR1, but not membrane-bound FGFR1, induces G2 cell-cycle checkpoint adaptation, cell survival and polyploidy following irradiation in osteosarcoma cells. Further, the activation of nFGFR1 induces Histone H3 phosphorylation at Ser 10 and c-jun/c-fos expression to contribute cell survival rendering radiation resistance. Furthermore, an in vivo mouse study revealed that radiation resistance can be reversed by the inhibition of nFGFR1. Our findings provide insights into the potential role of nFGFR1 to radiation resistance. Thus, we propose nFGFR1 could be a potential therapeutic target or a biomarker to determine which patients might benefit from radiation therapy.

The "SEED" Study: The Feasibility of Selecting Patient-Specific Biologically Targeted Therapy with Sorafenib, Everolimus, Erlotinib or Dasatinib for Pediatric and Young Adult Patients with Recurrent or Refractory Brain Tumors.

BACKGROUND: Pediatric brain tumors are the leading cause of cancer death in children and represent a variety of diseases and molecular subtypes. This study sought to evaluate a rapid immunohistochemistry testing panel to aid in therapy selection at the time of malignant tumor recurrence. METHODS: With IRB approval and appropriate informed consent, we conducted a single-institution prospective clinical trial of selected kinase inhibitor therapy. A laboratory-developed immunohistochemical testing panel was performed on tumor tissue, and therapy with one of four small molecule inhibitors was recommended in combination with oral chemotherapy consisting of temozolomide and etoposide. RESULTS: All 20 enrolled subjects were assigned to Everolimus (n = 4), Erlotinib (n = 6) or Dasatinib (n = 10); 90% (18/20) within the pre-specified 14-day feasibility time period. Only two subjects elected treatment on study, 8 received targeted treatment based on testing results either alone (n = 5) or in combination with chemotherapy (n = 3). Other subjects received chemotherapy alone (n = 7), surgery alone (n = 2) or no further therapy (n = 3). Immunohistochemical targets were associated with correlative genetic changes in 28% (5/18) of those evaluated. CONCLUSIONS: It was feasible to rapidly select targeted therapy in recurrent pediatric brain tumors, but not feasible to treat with a uniform combination treatment regimen.

Chondroblastoma of the Occipital Bone With Atypical Genetic Markers: A Case Report.

Chondroblastoma is a rare bone tumor, most often found in epiphyseal plates of long bones. It has infrequently been reported in the skull, most often in the temporal bone. We present a case of chondroblastoma of the occipital bone in a pediatric patient presenting with a bony protuberance of the occiput and imaging consistent with obstructive hydrocephalus, which persisted even after removal of the obstructing tumor. Pathological examination demonstrated that this unusually placed tumor also lacked the classic genetic markers typically associated with chondroblastoma.

Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor.

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3'-phosphatase and DNA 5'-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.

Two cases of pineal anlage tumor with molecular analysis.

Pineal anlage tumor is a rare pediatric tumor with clinical and histological features overlapping with pineoblastoma. Two patients with pineal anlage tumor, a 13-month-old female and an 11-month-old male, underwent subtotal resection, high-dose chemotherapy with autologous stem cell rescue, and radiation. Neither had tumor progression 50 months after diagnosis. The tumors underwent next-generation sequencing on a panel of 340 genes. Chromosomal copy gains and losses were present and differed between the tumors. No mutations or amplifications, including none specific to pineoblastoma, were identified.

Neuropathology of Pediatric Brain Tumors: A Concise Review.

Pediatric brain tumors are an incredibly diverse group of neoplasms and neuropathological tumor classification is an essential part of patient care. Classification of pediatric brain tumors has changed considerably in recent years as molecular diagnostics have become incorporated with routine histopathology in the diagnostic process. This article will focus on the fundamental major histologic, immunohistochemical, and molecular features that neuropathologists use to make an integrated diagnosis of pediatric brain tumors. This concise review will focus on tumors that are integral to the central nervous system in pediatric patients including: embryonal tumors, low and high grade gliomas, glioneuronal tumors, ependymomas, and choroid plexus tumors.

Predictors of mortality and tumor recurrence in desmoplastic infantile ganglioglioma and astrocytoma-and individual participant data meta-analysis (IPDMA).

PURPOSE: Desmoplastic infantile astrocytoma (DIA) and desmoplastic infantile ganglioglioma (DIG) are classified together as grade I neuronal and mixed neuronal-glial tumor of the central nervous system by the World Health Organization (WHO). These tumors are rare and have not been well characterized in terms of clinical outcomes. We aimed to identify clinical predictors of mortality and tumor recurrence/progression by performing an individual patient data meta-analysis (IPDMA) of the literature. METHODS: A systematic literature review from 1970 to 2020 was performed, and individualized clinical data for patients diagnosed with DIA/DIG were extracted. Aggregated data were excluded from collection. Outcome measures of interest were mortality and tumor recurrence/progression, as well as time-to-event (TTE) for each of these. Participants without information on these outcome measures were excluded. Cox regression survival analyses were performed to determine predictors of mortality and tumor recurrence / progression. RESULTS: We identified 98 articles and extracted individual patient data from 188 patients. The cohort consisted of 58.9% males with a median age of 7 months. The majority (68.1%) were DIGs, while 24.5% were DIAs and 7.5% were non-specific desmoplastic infantile tumors; DIAs presented more commonly in deep locations (p = 0.001), with leptomeningeal metastasis (p = 0.001), and was associated with decreased probability of gross total resection (GTR; p = 0.001). Gender, age, and tumor pathology were not statistically significant predictors of either mortality or tumor recurrence/progression. On multivariate survival analysis, GTR was a predictor of survival (HR = 0.058; p = 0.007) while leptomeningeal metastasis at presentation was a predictor of mortality (HR = 3.27; p = 0.025). Deep tumor location (HR = 2.93; p = 0.001) and chemotherapy administration (HR = 2.02; p = 0.017) were associated with tumor recurrence/progression. CONCLUSION: Our IPDMA of DIA/DIG cases reported in the literature revealed that GTR was a predictor of survival while leptomeningeal metastasis at presentation was associated with mortality. Deep tumor location and chemotherapy were associated with tumor recurrence / progression.

Extramedullary Hematopoiesis in the Dura Mater During Treatment of a CNS Embryonal Tumor.

Extramedullary hematopoiesis (EMH) is hematopoiesis occurring outside of the bone marrow. It has been reported to develop in abdominal organs or lymph nodes after chemotherapy. Here, the authors describe a patient with a localized central nervous system embryonal tumor who, during intensive chemotherapy, developed dural nodules. Biopsy revealed these nodules to be EMH. Without a pathologic diagnosis, this may have been considered disease progression, altering the patient's treatment plan. This report intends to serve as a reminder that EMH should be included in the differential diagnosis of suspicious lesions and highlights the importance of their biopsy because of potential management implications.

Optimal therapeutic targeting by HDAC inhibition in biopsy-derived treatment-naïve diffuse midline glioma models.

BACKGROUND: Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis, with less than 2% surviving 5 years postdiagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified a few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically relevant DMG models to identify and validate other effective HDACi and their biomarkers of response. METHODS: HDAC inhibitors were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically relevant radiation resistance. RNA sequencing was performed to define and compare drug efficacy and to map predictive biomarkers of response. RESULTS: Quisinostat and romidepsin showed efficacy with low nanomolar half-maximal inhibitory concentration (IC50) values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all 3 drugs at similar biologically effective doses, such as overexpression of troponin T1 slow skeletal type (TNNT1) and downregulation of collagen type 20 alpha 1 chain (COL20A1), identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (P < 0.0001) inhibited in vivo tumor growth. CONCLUSIONS: Our data highlight the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain tumor-penetrant versions of potentially efficacious agents.

Histopathologic and Molecular Features of Central Nervous System Embryonal Tumors for Integrated Diagnosis Reporting.

Embryonal tumors of the pediatric central nervous system are challenging clinically and diagnostically. These tumors are aggressive, and patients often have poor outcomes even with intense therapy. Proper tumor classification is essential to patient care, and this process has undergone significant changes with the World Health Organization recommending histopathologic and molecular features be integrated in diagnostic reporting. This has especially impacted the workup of embryonal tumors because molecular testing has resulted in the identification of clinically relevant tumor subgroups and new entities. This review summarizes recent developments and provides a framework to workup embryonal tumors in diagnostic practice.

Heme oxygenase-1 deficiency presenting with interstitial lung disease and hemophagocytic flares.

BACKGROUND: Heme oxygenase-1 (HMOX1) catalyzes the metabolism of heme into carbon monoxide, ferrous iron, and biliverdin. Through biliverdin reductase, biliverdin becomes bilirubin. HMOX1-deficiency is a rare autosomal recessive disorder with hallmark features of direct antibody negative hemolytic anemia with normal bilirubin, hyperinflammation and features similar to macrophage activation syndrome. Clinical findings have included asplenia, nephritis, hepatitis, and vasculitis. Pulmonary features and evaluation of the immune response have been limited. CASE PRESENTATION: We present a young boy who presented with chronic respiratory failure due to nonspecific interstitial pneumonia following a chronic history of infection-triggered recurrent hyperinflammatory flares. Episodes included hemolysis without hyperbilirubinemia, immunodeficiency, hepatomegaly with mild transaminitis, asplenia, leukocytosis, thrombocytosis, joint pain and features of macrophage activation with negative autoimmune serologies. Lung biopsy revealed cholesterol granulomas. He was found post-mortem by whole exome sequencing to have a compound heterozygous paternal frame shift a paternal frame shift HMOX1 c.264_269delCTGG (p.L89Sfs*24) and maternal splice donor HMOX1 (c.636 + 2 T > A) consistent with HMOX1 deficiency. Western blot analysis confirmed lack of HMOX1 protein upon oxidant stimulation of the patient cells. CONCLUSIONS: Here, we describe a phenotype expansion for HMOX1-deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares with hemophagocytosis present in the bone marrow.

Children with DIPG and high-grade glioma treated with temozolomide, irinotecan, and bevacizumab: the Seattle Children's Hospital experience.

INTRODUCTION: Beyond focal radiation, there is no consensus standard therapy for pediatric high-grade glioma (pHGG) and outcomes remain dismal. We describe the largest molecularly-characterized cohort of children with pHGG treated with a 3-drug maintenance regimen of temozolomide, irinotecan, and bevacizumab (TIB) following radiation. METHODS: We retrospectively reviewed 36 pediatric patients treated with TIB at Seattle Children's Hospital from 2009 to 2018 and analyzed survival using the Kaplan-Meier method. Molecular profiling was performed by targeted DNA sequencing and toxicities, steroid use, and palliative care utilization were evaluated. RESULTS: Median age at diagnosis was 10.9 years (18 months-18 years). Genetic alterations were detected in 26 genes and aligned with recognized molecular subgroups including H3 K27M-mutant (12), H3F3A G34-mutant (2), IDH-mutant (4), and hypermutator profiles (4). Fifteen patients (42%) completed 12 planned cycles of maintenance. Side effects associated with chemotherapy delays or modifications included thrombocytopenia (28%) and nausea/vomiting (19%), with temozolomide dosing most frequently modified. Median event-free survival (EFS) and overall survival (OS) was 16.2 and 20.1 months, with shorter survival seen in DIPG (9.3 and 13.3 months, respectively). Survival at 1, 2, and 5 years was 80%, 10% and 0% for DIPG and 85%, 38%, and 16% for other pHGG. CONCLUSION: Our single-center experience demonstrates tolerability of this 3-drug regimen, with prolonged survival in DIPG compared to historical single-agent temozolomide. pHGG survival was comparable to analogous 3-drug regimens and superior to historical agents; however, cure was rare. Children with pHGG remain excellent candidates for the study of novel therapeutics combined with standard therapy.