Investigation of white blood cell characteristics in cerebrospinal fluid samples at pediatric brain tumor diagnosis.

PURPOSE: The role of white blood cells (WBC) in the pediatric central nervous system (CNS) tumor microenvironment is incompletely defined. We hypothesized that the WBC profile in cerebrospinal fluid (CSF) correlates with the presence of tumor cells and prognosis in pediatric CNS tumors, as well as other patient and disease characteristics, and differs by tumor type, thus giving insight into the tumor immune response. METHODS: We conducted a retrospective analysis of CSF WBC profiles at CNS tumor diagnosis in 269 patients at our institution. We examined total nucleated cell count, absolute counts, and percentages by WBC subtype. We compared CSF WBC values by tumor cell presence, patient vital status, tumor location, and the most common tumor types. RESULTS: Patients who died of their tumor had a lower CSF lymphocyte percentage and a higher absolute monocyte count in CSF at diagnosis. The presence of tumor cells in CSF was associated with fewer lymphocytes and monocytes. Ventricular tumors had higher CSF lymphocyte, monocyte, macrophage, and total nucleated cell counts than extraventricular tumors. Germ cell tumors, low-grade glioma, high-grade glioma, and ependymoma had lower macrophage counts or percentages compared to other tumor types. CONCLUSIONS: WBC profile in CSF at pediatric CNS tumor diagnosis correlates with patient prognosis and presence of metastatic cells, along with tumor type and other tumor characteristics like relationship to the ventricles. Prospective CSF profiling and study may be useful to future immunotherapy and other pediatric CNS tumor clinical trials.

Retrospective analysis of combination carboplatin and vinblastine for pediatric low-grade glioma.

INTRODUCTION: Low-grade glioma (LGG) represent the most common pediatric central nervous system tumor. When total surgical resection is not feasible, chemotherapy is first-line therapy in children. Multiple pediatric LGG chemotherapy regimens have been investigated with variable 2-year event free survival (EFS) rates of 39-69%. To date, treatment of pediatric LGG with a carboplatin and vinblastine (C/VBL) chemotherapy regimen has only been evaluated in a phase 1 dose-finding study. METHODS: A retrospective review of pediatric patients with LGG who were treated with C/VBL at Children's Hospital of Colorado or Akron Children's Hospital from 2011 to 2017 was conducted. Data collected included patient demographics, tumor location, disease response, neurofibromatosis 1 (NF1) status, therapy duration and toxicities. Response to therapy was determined by objective findings on imaging and treating physicians' evaluation. RESULTS: Forty-six patients were identified for analysis, all of whom were chemotherapy-naive. Only five patients treated in this cohort had NF1. BRAF fusion was identified in 65% (22/34) of tested tumors. Best therapy response was partial response in nine patients and stable disease in twenty-five patients. Twelve patients had progressive disease. One-year, 3-year, and 5-year EFS probabilities for all patients were 69.6%, 39.4%, and 34.5%, respectively. Nine patients had admissions for febrile neutropenia and seven patients experienced one delay in chemotherapy due to neutropenia. Only two patients had to discontinue this chemotherapy regimen because of treatment-related toxicities [carboplatin allergy (n = 1) and vinblastine neuropathy (n = 1)]. CONCLUSION: C/VBL achieves similar EFS rates to other single-agent and combination cytotoxic chemotherapy regimens for pediatric LGG with manageable toxicities.

Targeted fusion analysis can aid in the classification and treatment of pediatric glioma, ependymoma, and glioneuronal tumors.

BACKGROUND: The use of next-generation sequencing for fusion identification is being increasingly applied and aids our understanding of tumor biology. Some fusions are responsive to approved targeted agents, while others have future potential for therapeutic targeting. Although some pediatric central nervous system tumors may be cured with surgery alone, many require adjuvant therapy associated with acute and long-term toxicities. Identification of targetable fusions can shift the treatment paradigm toward earlier integration of molecularly targeted agents. METHODS: Patients diagnosed with glial, glioneuronal, and ependymal tumors between 2002 and 2019 were retrospectively reviewed for fusion testing. Testing was done primarily using the ArcherDx FusionPlex Solid Tumor panel, which assesses fusions in 53 genes. In contrast to many previously published series chronicling fusions in pediatric patients, we compared histological features and the tumor classification subtype with the specific fusion identified. RESULTS: We report 24 cases of glial, glioneuronal, or ependymal tumors from pediatric patients with identified fusions. With the exception of BRAF:KIAA1549 and pilocytic/pilomyxoid astrocytoma morphology, and possibly QKI-MYB and angiocentric glioma, there was not a strong correlation between histological features/tumor subtype and the specific fusion. We report the unusual fusions of PPP1CB-ALK, CIC-LEUTX, FGFR2-KIAA159, and MN1-CXXC5 and detail their morphological features. CONCLUSIONS: Fusion testing proved to be informative in a high percentage of cases. A large majority of fusion events in pediatric glial, glioneuronal, and ependymal tumors can be identified by relatively small gene panels.

Specific expression of PD-L1 in RELA-fusion supratentorial ependymoma: Implications for PD-1-targeted therapy.

BACKGROUND: A desperate need for novel therapies in pediatric ependymoma (EPN) exists, as chemotherapy remains ineffective and radiotherapy often fails. EPN have significant infiltration of immune cells, which correlates with outcome. Immune checkpoint inhibitors provide an avenue for new treatments. This study characterizes tumor-infiltrating immune cells in EPN and aims at predicting candidates for clinical trials using checkpoint inhibitors targeting PD-L1/PD-1 (programmed death ligand 1/programmed death 1). METHODS: The transcriptomic profiles of the primary study cohort of EPN and other pediatric brain tumors were interrogated to identify PD-L1 expression levels. Transcriptomic findings were validated using the western blotting, immunohistochemistry and flow cytometry. RESULTS: We evaluated PD-L1 mRNA expression across four intracranial subtypes of EPN in two independent cohorts and found supratentorial RELA fusion (ST-RELA) tumors to have significantly higher levels. There was a correlation between high gene expression and protein PD-L1 levels in ST-RELA tumors by both the western blot and immunohistochemisty. The investigation of EPN cell populations revealed PD-L1 was expressed on both tumor and myeloid cells in ST-RELA. Other subtypes had little PD-L1 in either tumor or myeloid cell compartments. Lastly, we measured PD-1 levels on tumor-infiltrating T cells and found ST-RELA tumors express PD-1 in both CD4 and CD8 T cells. A functional T-cell exhaustion assay found ST-RELA T cells to be exhausted and unable to secrete IFNγ on stimulation. CONCLUSIONS: These findings in ST-RELA suggest tumor evasion and immunsuppression due to PD-L1/PD-1-mediated T-cell exhaustion. Trials of checkpoint inhibitors in EPN should be enriched for ST-RELA tumors.

Paving the road ahead for CD19 CAR T-cell therapy.

PURPOSE OF REVIEW: Modern immunotherapies, most notably in the form of anti-CD19 chimeric antigen receptor (CAR) T cells, have produced significant clinical responses in otherwise refractory pre-B-cell acute lymphoblastic leukemia patients. Several groups have simultaneously reported robust response rates in children and adults alike. These early studies indicate an impending shift in paradigm for the treatment of acute lymphoblastic leukemia. Incorporating CD19 CAR T-cell therapy into upfront or salvage regimens has its challenges and opportunities. RECENT FINDINGS: Most CD19 CAR T-cell products in trial today are excellent at inducing minimal residual disease negative remissions, and most responding patients experience cytokine release syndrome and/or neurotoxicity. The challenges facing the CAR community involve how best to minimize the severity of cytokine release syndrome and neurotoxicity while maximizing antitumor efficacy, determining what role this therapy will play for the prophylaxis and treatment of central nervous system leukemia, and its implications on subsequent hematopoietic stem cell transplant given the emergence of CD19-negative relapses. SUMMARY: CD19 CAR T-cell therapy is a powerful new tool in the oncologist's arsenal. How it is incorporated into standard practice and how it will shift survival curves are the exciting questions that are waiting to be answered.

Immunotherapy Associated Neurotoxicity in Pediatric Oncology.

Novel immunotherapies are increasingly being employed in pediatric oncology, both in the upfront and relapsed/refractory settings. Through various mechanisms of action, engagement and activation of the immune system can cause both generalized and disease site-specific inflammation, leading to immune-related adverse events (irAEs). One of the most worrisome irAEs is that of neurotoxicity. This can present as a large spectrum of neurological toxicities, including confusion, aphasia, neuropathies, seizures, and/or death, with variable onset and severity. Earlier identification and treatment, generally with corticosteroids, remains the mainstay of neurotoxicity management to optimize patient outcomes. The pathophysiology of neurotoxicity varies across the different therapeutic strategies and remains to be elucidated in most cases. Furthermore, little is known about long-term neurologic sequelae. This review will focus on neurotoxicity seen with the most common immunotherapies used in pediatric oncology, including CAR T cell therapy, alternative forms of adoptive cell therapy, antibody therapies, immune checkpoint inhibitors, and tumor vaccines. Herein we will discuss the incidence, pathophysiology, symptomatology, diagnosis, and management strategies currently being utilized for immunotherapy-associated neurotoxicity with a focus on pediatric specific considerations.

Neoplastic and immune single-cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. METHODS: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. RESULTS: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. CONCLUSIONS: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.

Optimizing CARs for ocular delivery.

Effective methods for treating retinoblastoma while preserving vision are an unmet clinical need. Subretinal delivery of a hydrogel containing T cells that secrete the cytokine IL-15 and express a chimeric antigen receptor directed at the ganglioside protein GD2 completely controls retinoblastoma in immunocompromised mice, with no obvious damage to the surrounding retina.

ddPCR Analysis Reveals BRAF V600E Mutations Are Infrequent in Isolated Pituitary Langerhans Cell Histiocytosis Patients.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with a highly variable clinical presentation affecting people of all ages. Mutations in BRAF V600E are the most identifiable molecular alteration in LCH although its incidence in pediatric patients with isolated pituitary stalk involvement is not well described. Pediatric patients with LCH and isolated pituitary stalk involvement typically present with central diabetes insipidus. Diagnosis requires a transcranial biopsy which often yields scant tissue. We sought to determine the prevalence of BRAF V600E mutations in patients with isolated pituitary stalk LCH using digital droplet polymerase chain reaction because this method requires minimal tumor DNA. We identified 8 patients with isolated pituitary stalk thickening who underwent a biopsy at Children's Hospital Colorado from January 2001 to December 2019, as well as 6 patients with systemic LCH diagnosed by biopsy in the same period as a comparison. Only one out of the 8 patients with isolated thickened pituitary stalk was found to have a detectable BRAF V600E mutation. Five out of the 6 patients with systemic LCH had a detectable BRAF V600E mutation. In our series, BRAF V600E mutations are rare in pediatric patients with LCH and isolated pituitary stalk involvement.

Clinical and molecular characterization of a multi-institutional cohort of pediatric spinal cord low-grade gliomas.

BACKGROUND: The mitogen-activated protein kinases/extracelluar signal-regulated kinases pathway is involved in cell growth and proliferation, and mutations in BRAF have made it an oncogene of interest in pediatric cancer. Previous studies found that BRAF mutations as well as KIAA1549-BRAF fusions are common in intracranial low-grade gliomas (LGGs). Fewer studies have tested for the presence of these genetic changes in spinal LGGs. The aim of this study was to better understand the prevalence of BRAF and other genetic aberrations in spinal LGG. METHODS: We retrospectively analyzed 46 spinal gliomas from patients aged 1-25 years from Children's Hospital Colorado (CHCO) and The Hospital for Sick Children (SickKids). CHCO utilized a 67-gene panel that assessed BRAF and additionally screened for other possible genetic abnormalities of interest. At SickKids, BRAF V600E was assessed by droplet digital polymerase chain reaction and immunohistochemistry. BRAF fusions were detected by fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or NanoString platform. Data were correlated with clinical information. RESULTS: Of 31 samples with complete fusion analysis, 13 (42%) harbored KIAA1549-BRAF. All 13 (100%) patients with confirmed KIAA1549-BRAF survived the entirety of the study period (median [interquartile range] follow-up time: 47 months [27-85 months]) and 15 (83.3%) fusion-negative patients survived (follow-up time: 37.5 months [19.8-69.5 months]). Other mutations of interest were also identified in this patient cohort including BRAF V600E , PTPN11, H3F3A, TP53, FGFR1, and CDKN2A deletion. CONCLUSION: KIAA1549-BRAF was seen in higher frequency than BRAF V600E or other genetic aberrations in pediatric spinal LGGs and experienced lower death rates compared to KIAA1549-BRAF negative patients, although this was not statistically significant.

Effect of early-stage autophagy inhibition in BRAFV600E autophagy-dependent brain tumor cells.

Autophagy is a multistage process. Progress within the field has led to the development of agents targeting both early (initiation) and late (fusion) stages of this process. The specific stage of autophagy targeted may influence cancer treatment outcomes. We have previously shown that central nervous system (CNS) tumors with the BRAFV600E mutation are autophagy dependent, and late-stage autophagy inhibition improves the response to targeted BRAF inhibitors (BRAFi) in sensitive and resistant cells. Drugs directed toward initiation of autophagy have been shown to reduce tumor cell death in some cancers, but have not been assessed in CNS tumors. We investigated early-stage inhibition for autophagy-dependent CNS tumors. BRAFi-sensitive and resistant AM38 and MAF794 cell lines were evaluated for the response to pharmacologic and genetic inhibition of ULK1 and VPS34, two crucial subunits of the autophagy initiation complexes. Changes in autophagy were monitored by western blot and flow cytometry. Survival was evaluated in short- and long-term growth assays. Tumor cells exhibited a reduced autophagic flux with pharmacologic and genetic inhibition of ULK1 or VPS34. Pharmacologic inhibition reduced cell survival in a dose-dependent manner for both targets. Genetic inhibition reduced cell survival and confirmed that it was an autophagy-specific effect. Pharmacologic and genetic inhibition were also synergistic with BRAFi, irrespective of RAFi sensitivity. Inhibition of ULK1 and VPS34 are potentially viable clinical targets in autophagy-dependent CNS tumors. Further evaluation is needed to determine if early-stage autophagy inhibition is equal to late-stage inhibition to determine the optimal clinical target for patients.

CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.

PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. RESULTS: B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.

Durable regression of Medulloblastoma after regional and intravenous delivery of anti-HER2 chimeric antigen receptor T cells.

BACKGROUND: Standard-of-care therapies for treating pediatric medulloblastoma have long-term side effects, even in children who are cured. One emerging modality of cancer therapy that could be equally effective without such side effects would be chimeric antigen receptor (CAR) T cells. Knowing that human epidermal growth factor receptor 2 (HER2) is overexpressed in many medulloblastomas and has been used as a CAR T target before, we sought to evaluate the efficacy of more sophisticated anti-HER2 CAR T cells, as well as the feasibility and efficacy of different routes of delivering these cells, for the treatment of pediatric medulloblastoma. METHODS: Daoy, D283 and D425 medulloblastoma cell lines were characterized by flow cytometry to evaluate HER2 expression. Anti-tumor efficacy of HER2-BBz-CAR T cells in vitro was performed using cytokine release and immune cytotoxicity assays compared to control CD19 CAR T cells. In vivo, Daoy and D283 tumor cells were orthotopically implanted in the posterior fossa of NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice and treated with regional or intravenous HER2-BBz-CAR T cells or control CD19 CAR T cells. Non-human primates (NHPs) bearing ventricular and lumbar reservoirs were treated with target autologous cells bearing extracellular HER2 followed by autologous HER2-CAR T cells intraventricularly. Cerebrospinal fluid and blood were collected serially to measure the persistence of delivered cells and cytokines. RESULTS: HER2-BBz-CAR T cells effectively clear medulloblastoma orthotopically implanted in the posterior fossa of NSG mice via both regional and intravenous delivery in xenograft models. Intravenous delivery requires a log higher dose compared to regional delivery. NHPs tolerated intraventricular delivery of autologous cells bearing extracellular HER2 followed by HER2-BBz-CAR T cells without experiencing any systemic toxicity. CONCLUSIONS: HER2-BBz-CAR T cells show excellent pre-clinical efficacy in vitro and in mouse medulloblastoma models, and their intraventricular delivery is feasible and safe in NHPs. A clinical trial of HER2-BBz-CAR T cells directly delivered into cerebrospinal fluid should be designed for patients with relapsed medulloblastoma.

Hoyeraal-Hreidarsson Syndrome due to PARN Mutations: Fourteen Years of Follow-Up.

BACKGROUND: Hoyeraal-Hreidarsson syndrome is a dyskeratosis congenita-related telomere biology disorder that presents in infancy with intrauterine growth retardation, immunodeficiency, and cerebellar hypoplasia in addition to the triad of nail dysplasia, skin pigmentation, and oral leukoplakia. Individuals with Hoyeraal-Hreidarsson syndrome often develop bone marrow failure in early childhood. Germline mutations in DKC1, TERT, TINF2, RTEL1, ACD, or PARN cause about 60% of individuals with Hoyeraal-Hreidarsson syndrome. PATIENT DESCRIPTION: We describe 14 years of follow-up of an individual with Hoyeraal-Hreidarsson syndrome who initially presented as an infant with intrauterine growth retardation, microcephaly, and central nervous system calcifications. He was diagnosed with Hoyeraal-Hreidarsson syndrome at age 6 years and had a complicated medical history including severe developmental delay, cerebellar hypoplasia, esophageal and urethral stenosis, hip avascular necrosis, immunodeficiency, and bone marrow failure evolving to myelodysplastic syndrome requiring hematopoietic cell transplantation at age 14 years. He had progressive skin pigmentation, oral leukoplakia, and nail dysplasia leading to anonychia. Whole exome sequencing identified novel biallelic variants in PARN. CONCLUSIONS: This patient illustrates that the constellation of intrauterine growth retardation, central nervous system calcifications, and cerebellar hypoplasia, esophageal or urethral stenosis, and cytopenias, in the absence of congenital infection, may be due to Hoyeraal-Hreidarsson syndrome. Early diagnosis of Hoyeraal-Hreidarsson syndrome is important to optimize medical management and provide genetic counseling.