Neuropathological features of pediatric laryngomalacia.

OBJECTIVE: Laryngomalacia is the most common pediatric laryngeal anomaly. The pathophysiology of laryngomalacia is not well defined; the leading hypothesis suggests weak laryngeal tone and neuromuscular discoordination. Only a few studies explored the histopathology of the laryngeal submucosal nerves, with reported nerve hypertrophy. Our study aims to describe the histopathology of submucosal nerves in specimens obtained from children with severe laryngomalacia compared to pediatric cadaveric controls. STUDY DESIGN: Prospective study. SETTINGS: Tertiary care children's hospital. METHODS: Histologic and immunohistochemical sections of supraglottic tissue from 26 children with severe laryngomalacia and six pediatric autopsies were digitally scanned and assessed with image analysis software (QuPath), resulting in the identification and measurement of 4561 peripheral nerves and over 100,000 foci of neurofilaments. RESULTS: Chronic inflammation was noted in all patients. Eosinophils were rare. The mean nerve area and perimeter were significantly smaller for patients with laryngomalacia compared to the control group (1594.0 ± 593.2 µm^2 vs. 2612.1 ± 2824.0 µm^2, p < 0.0001, and 158.8 ± 30.3 µm vs. 217.6 ± 165.0 µm, p < 0.0001). Nerve-per-area unit was significantly greater for patients with laryngomalacia compared to controls (1.39E-05 vs. 6.19 E-06, p = 0.009). The mean area and the number of neurofilaments per total nerve area were similar. Immunohistochemistry for calretinin, a marker for intestinal ganglion cells in Hirschsprung disease, was absent from all specimens. CONCLUSIONS: This series includes a comparison of all identifiable nerve fibers obtained from children with severe laryngomalacia and shows that the mucosal nerves are smaller on average than controls. These findings fail to provide support for significant morphologic peripheral nerve pathology in laryngomalacia.

A case of myxopapillary ependymoma with predominant giant cell morphology: A rare entity with comprehensive genomic profiling and review of literature.

In the evolving landscape of ependymoma classification, which integrates histological, molecular, and anatomical context, we detail a rare case divergent from the usual histopathological spectrum. We present the case of a 37-year-old man with symptomatic spinal cord compression at the L3-L4 level. Neuroradiological evaluation revealed an intradural, encapsulated mass. Histologically, the tumor displayed atypical features: bizarre pleomorphic giant cells, intranuclear inclusions, mitotic activity, and a profusion of eosinophilic cytoplasm with hyalinized vessels, deviating from the characteristic perivascular pseudorosettes or myxopapillary patterns. Immunohistochemical staining bolstered this divergence, marking the tumor cells positive for glial fibrillary acidic protein and epithelial membrane antigen with a characteristic ring-like pattern, and CD99 but negative for Olig-2. These markers, alongside methylation profiling, facilitated its classification as a myxopapillary ependymoma (MPE), despite the atypical histologic features. This profile underscores the necessity of a multifaceted diagnostic process, especially when histological presentation is uncommon, confirming the critical role of immunohistochemistry and molecular diagnostics in classifying morphologically ambiguous ependymomas and exemplifying the histological diversity within MPEs.

DNA Methylation Profiles Are Stable in H3 K27M-Mutant Diffuse Midline Glioma Neurosphere Cell Lines.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but the degree to which such cultures retain the characteristics of the source tumors is unknown. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided a comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

Primary Diffuse Leptomeningeal Melanomatosis in a Child with Extracranial Metastasis: Case Report.

Primary meningeal melanomatosis is an extremely rare tumor with very few documented responses to treatment. A 3-year-old male with a complex past medical history, including prematurity and shunted hydrocephalus, was diagnosed with primary meningeal melanomatosis with peritoneal implants. Molecular testing revealed an NRAS Q61R mutation. The patient received proton craniospinal radiation followed by immunotherapy with nivolumab (1 mg/kg) and ipilimumab (3 mg/kg) IV every 3 weeks and, upon progression, he was switched to a higher dose of nivolumab (3 mg/kg IV every 2 weeks) and binimetinib (24 mg/m2/dose, twice a day). The patient had significant improvement of CNS disease with radiation therapy and initial immunotherapy but progression of extracranial metastatic peritoneal and abdominal disease. Radiation was not administered to the whole abdomen. After two cycles of nivolumab and treatment with the MEK inhibitor binimetinib, he had radiographic and clinical improvement in abdominal metastasis and ascitis. He ultimately died from RSV infection, Klebsiella sepsis, and subdural hemorrhage without evidence of tumor progression. This is the first report of a child with primary meningeal melanomatosis with extracranial metastatic disease with response to a combination of radiation, immunotherapy and MEK inhibitor therapy.

Indoximod-based chemo-immunotherapy for pediatric brain tumors: A first-in-children phase I trial.

BACKGROUND: Recurrent brain tumors are the leading cause of cancer death in children. Indoleamine 2,3-dioxygenase (IDO) is a targetable metabolic checkpoint that, in preclinical models, inhibits anti-tumor immunity following chemotherapy. METHODS: We conducted a phase I trial (NCT02502708) of the oral IDO-pathway inhibitor indoximod in children with recurrent brain tumors or newly diagnosed diffuse intrinsic pontine glioma (DIPG). Separate dose-finding arms were performed for indoximod in combination with oral temozolomide (200 mg/m2/day x 5 days in 28-day cycles), or with palliative conformal radiation. Blood samples were collected at baseline and monthly for single-cell RNA-sequencing with paired single-cell T cell receptor sequencing. RESULTS: Eighty-one patients were treated with indoximod-based combination therapy. Median follow-up was 52 months (range 39-77 months). Maximum tolerated dose was not reached, and the pediatric dose of indoximod was determined as 19.2 mg/kg/dose, twice daily. Median overall survival was 13.3 months (n = 68, range 0.2-62.7) for all patients with recurrent disease and 14.4 months (n = 13, range 4.7-29.7) for DIPG. The subset of n = 26 patients who showed evidence of objective response (even a partial or mixed response) had over 3-fold longer median OS (25.2 months, range 5.4-61.9, p = 0.006) compared to n = 37 nonresponders (7.3 months, range 0.2-62.7). Four patients remain free of active disease longer than 36 months. Single-cell sequencing confirmed emergence of new circulating CD8 T cell clonotypes with late effector phenotype. CONCLUSIONS: Indoximod was well tolerated and could be safely combined with chemotherapy and radiation. Encouraging preliminary evidence of efficacy supports advancing to Phase II/III trials for pediatric brain tumors.

The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-[3-(3-methyl-1,2,4-triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.

Case report: ATIC-ALK fusion in infant-type hemispheric glioma and response to lorlatinib.

Introduction: Infant type hemispheric gliomas are a rare tumor with unique molecular characteristics. In many cases these harbor mutations in receptor tyrosine kinase pathways and respond to targeted therapy. Here we describe the case of an infant with this type of tumor with a novel ATIC-ALK fusion that has responded dramatically to the ALK inhibitor lorlatinib, despite being refractory to standard chemotherapy. Case description: The infant was initially treated with standard chemotherapy and found to have an ATIC-ALK fusion. When surveillance imaging revealed progressive disease, the patient was switched to the ALK-inhibitor lorlatinib at 47 mg/m2/day. The patient demonstrated a significant clinical and radiographic response to the ALK inhibitor lorlatinib after just 3 months of treatment and a near complete response by 6 months of therapy. Conclusion: The ALK inhibitor lorlatinib is an effective targeted therapy in infant type hemispheric glioma patients harboring ATIC-ALK fusion.

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth.

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.

Absence of lenadogene nolparvovec DNA in a brain tumor biopsy from a patient in the REVERSE clinical study, a case report.

BACKGROUND: Leber Hereditary Optic Neuropathy (LHON) is a rare, maternally-inherited mitochondrial disease that primarily affects retinal ganglion cells (RGCs) and their axons in the optic nerve, leading to irreversible, bilateral severe vision loss. Lenadogene nolparvovec gene therapy was developed as a treatment for patients with vision loss from LHON caused by the most prevalent m.11778G > A mitochondrial DNA point mutation in the MT-ND4 gene. Lenadogene nolparvovec is a replication-defective recombinant adeno-associated virus vector 2 serotype 2 (AAV2/2), encoding the human wild-type MT-ND4 protein. Lenadogene nolparvovec was administered by intravitreal injection (IVT) in LHON patients harboring the m.11778G > A ND4 mutation in a clinical development program including one phase 1/2 study (REVEAL), three phase 3 pivotal studies (REVERSE, RESCUE, REFLECT), and one long-term follow-up study (RESTORE, the follow-up of REVERSE and RESCUE patients). CASE PRESENTATION: A 67-year-old woman with MT-ND4 LHON, included in the REVERSE clinical study, received a unilateral IVT of lenadogene nolparvovec in the right eye and a sham injection in the left eye in May 2016, 11.4 months and 8.8 months after vision loss in her right and left eyes, respectively. The patient had a normal brain magnetic resonance imaging with contrast at the time of diagnosis of LHON. Two years after treatment administration, BCVA had improved in both eyes. The product was well tolerated with mild and resolutive anterior chamber inflammation in the treated eye. In May 2019, the patient was diagnosed with a right temporal lobe glioblastoma, IDH-wildtype, World Health Organization grade 4, based on histological analysis of a tumor excision. The brain tumor was assessed for the presence of vector DNA by using a sensitive validated qPCR assay targeting the ND4 sequence of the vector. CONCLUSION: ND4 DNA was not detected (below 15.625 copies/µg of genomic DNA) in DNA extracted from the brain tumor, while a housekeeping gene DNA was detected at high levels. Taken together, this data shows the absence of detection of lenadogene nolparvovec in a brain tumor (glioblastoma) of a treated patient in the REVERSE clinical trial 3 years after gene therapy administration, supporting the long-term favorable safety of lenadogene nolparvovec.