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.
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.
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
Il2rg
/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.
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.
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.
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.
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.
Abstract 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 Hoyeraal-Hreidarsson syndrome cases. 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.
Abstract
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, BRAFV600E 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 BRAFV600E, PTPN11, H3F3A, TP53, FGFR1, and CDKN2A deletion.
Conclusion
KIAA1549–BRAF was seen in higher frequency than BRAFV600E 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.
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.
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.
B7-H3 CAR T cells mediate significant antitumor activity
, 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.
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.
Abstract Atypical teratoid rhabdoid tumor (ATRT) is an uncommon, aggressive tumor affecting the brain and spinal cord primarily in children. Despite significant strides in understanding cancer ...biology through DNA and RNA sequencing, nearly 90% of ATRTs exhibit a crucial deletion that triggers rampant tumor growth. The absence of easily targetable genetic mutations in ATRT complicates the development of effective treatments, necessitating innovative strategies for therapy. Evading immune system detection is a key feature of cancer cells. In our study, we conducted an exhaustive, unbiased examination of 350 surface receptors through high-throughput multicolor flow cytometry on samples from ATRT surgeries, cell lines, and xenograft models derived from patients. By using a combination of antibodies targeting CD31, CD45, CD11b, CCR2, Cx3CR1, CD4, and CD8, we could identify immune cell populations within the tumors. Our findings revealed a heightened presence of CD44, CD146, CD59, CD151, and CD276 on the surface. Using qPCR we identified CD44V6 a variant isoform of the CD44 molecule, which is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 has multiple isoforms generated through alternative splicing, with CD44V6 being one of these variants. This specific isoform includes an additional variable exon (exon v6) in its extracellular domain. CD44v6-directed CAR-T cells effectively controlled tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. We developed CD44V6-directed CAR-T cells and investigated the capacity of Anti-CD44v6 CAR-T cells to execute cytotoxicity against ATRT tumor cells. We see significant cell killing by Anti-CD44v6 CAR-T cells in vitro and a significant increase in the survival of ATRT tumor-bearing mice.
Abstract BACKGROUND Immunotherapy is a developing but challenging field of research for the treatment of central nervous system malignancies. Chimeric antigen receptor (CAR)-T cell therapy has been ...effective in treating hematologic malignancies, but that success has not been clinically translated to solid tumors in children. B7-H3 has been identified as a promising immunotherapy target as it is highly expressed in pediatric glioblastoma and correlates with tumor progression and poor prognosis. We aim to evaluate a nanobody-based CAR-T cell therapy that binds B7-H3 with high affinity and hypothesize that anti-B7-H3 nanobody CAR-T cells can effectively kill B7-H3-expressing tumor cells. METHODS B7-H3 nanobodies were isolated from camel phage libraries and cloned into lentiviral CAR-T cell constructs. Human T cells were transduced by lentivirus to express CAR on the surface. Nanobody CAR-T cell functionality was evaluated by an impedance-based cytotoxicity assay and cytokine release was measured by ELISA. CAR-T avidity was quantified by the Lumicks z-Movi assay. In vivo studies are evaluating the efficacy of B7-H3 CAR-T cells in an orthotopic glioblastoma model in immunodeficient mice. Mice were treated with B7-H3 CAR-T cells by intracerebroventricular or intravenous delivery. RESULTS We confirmed high expression of B7-H3 on the surface of three pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. We confirmed effective killing of B7-H3-expressing tumor cells and the release of high levels of cytokines. B7-H3 nanobody CAR-T cells were observed to have high avidity compared to control CAR-T cells. In vivo studies are currently ongoing and treatment response will be evaluated by luminescence imaging and endpoint histologic analysis. CONCLUSION We have shown that B7-H3 nanobody CAR-T cells demonstrate significant preclinical efficacy. It is critical to study CAR-T cell therapy in immunocompetent in vivo models for further development and clinical application.
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