A neural tract-inspired conduit for facile, on-demand biopsy of glioblastoma.

Background: A major hurdle to effectively treating glioblastoma (GBM) patients is the lack of longitudinal information about tumor progression, evolution, and treatment response. Methods: In this study, we report the use of a neural tract-inspired conduit containing aligned polymeric nanofibers (i.e., an aligned nanofiber device) to enable on-demand access to GBM tumors in 2 rodent models. Depending on the experiment, a humanized U87MG xenograft and/or F98-GFP+ syngeneic rat tumor model was chosen to test the safety and functionality of the device in providing continuous sampling access to the tumor and its microenvironment. Results: The aligned nanofiber device was safe and provided a high quantity of quality genomic materials suitable for omics analyses and yielded a sufficient number of live cells for in vitro expansion and screening. Transcriptomic and genomic analyses demonstrated continuity between material extracted from the device and that of the primary, intracortical tumor (in the in vivo model). Conclusions: The results establish the potential of this neural tract-inspired, aligned nanofiber device as an on-demand, safe, and minimally invasive access point, thus enabling rapid, high-throughput, longitudinal assessment of tumor and its microenvironment, ultimately leading to more informed clinical treatment strategies.

Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma.

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1.  Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.

ABL1 and ABL2 promote medulloblastoma leptomeningeal dissemination.

Background: Medulloblastoma is the most common malignant pediatric brain tumor, and leptomeningeal dissemination (LMD) of medulloblastoma both portends a poorer prognosis at diagnosis and is incurable at recurrence. The biological mechanisms underlying LMD are unclear. The Abelson (ABL) tyrosine kinase family members, ABL1 and ABL2, have been implicated in cancer cell migration, invasion, adhesion, metastasis, and chemotherapy resistance, and are upstream mediators of the oncogene c-MYC in fibroblasts and lung cancer cells. However, their role in medulloblastoma has not yet been explored. The purpose of this work was to elucidate the role of ABL1/2 in medulloblastoma LMD. Methods: ABL1 and ABL2 mRNA expression of patient specimens was analyzed. shRNA knockdowns of ABL1/2 and pharmacologic inhibition of ABL1/2 were used for in vitro and in vivo analyses of medulloblastoma LMD. RNA sequencing of ABL1/2 genetic knockdown versus scrambled control medulloblastoma was completed. Results: ABL1/2 mRNA is highly expressed in human medulloblastoma and pharmacologic inhibition of ABL kinases resulted in cytotoxicity. Knockdown of ABL1/2 resulted in decreased adhesion of medulloblastoma cells to the extracellular matrix protein, vitronectin (P = .0013), and significantly decreased tumor burden in a mouse model of medulloblastoma LMD with improved overall survival (P = .0044). Furthermore, both pharmacologic inhibition of ABL1/2 and ABL1/2 knockdown resulted in decreased expression of c-MYC, identifying a putative signaling pathway, and genes/pathways related to oncogenesis and neurodevelopment were differentially expressed between ABL1/2 knockdown and control medulloblastoma cells. Conclusions: ABL1 and ABL2 have potential roles in medulloblastoma LMD upstream of c-MYC expression.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX , defining molecular alterations in IDH -mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX knockout glioma models in the presence and absence of the IDH1 R 132 H mutation. ATRX-deficient glioma cells were sensitive to dsRNA-based innate immune agonism and exhibited impaired lethality and increased T-cell infiltration in vivo . However, the presence of IDH1 R 132 H dampened baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1 R132H inhibition. IDH1 R132H co-expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1 R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytoma.

Polio virotherapy targets the malignant glioma myeloid infiltrate with diffuse microglia activation engulfing the CNS.

BACKGROUND: Malignant gliomas commandeer dense inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) promoting immune suppression, evasion, and tumor progression. Like all cells in the mononuclear phagocytic system, GAMM constitutively express the poliovirus receptor, CD155. Besides myeloid cells, CD155 is widely upregulated in the neoplastic compartment of malignant gliomas. Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, yielded long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). This scenario raises questions about the contributions of myeloid versus neoplastic cells to polio virotherapy of malignant gliomas. METHODS: We investigated PVSRIPO immunotherapy in immunocompetent mouse brain tumor models with blinded, board-certified neuropathologist review, a range of neuropathological, immunohistochemical, and immunofluorescence analyses, and RNAseq of the tumor region. RESULTS: PVSRIPO treatment caused intense engagement of the GAMM infiltrate associated with substantial, but transient tumor regression. This was accompanied by marked microglia activation and proliferation in normal brain surrounding the tumor, in the ipsilateral hemisphere and extending into the contralateral hemisphere. There was no evidence for lytic infection of malignant cells. PVSRIPO-instigated microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the Programmed Cell Death Ligand 1 (PD-L1) immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to durable remissions. CONCLUSIONS: Our work implicates GAMM as active drivers of PVSRIPO-induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the brain-resident myeloid compartment by PVSRIPO.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

CT Density Changes with Rapid Onset Acute, Severe, Focal Cerebral Ischemia in Monkeys.

Computerized tomography (CT) is the most often used imaging modality in the evaluation of acute clinical stroke. However, the rapidity with which CT density changes occur after acute, severe, focal ischemia cannot be determined clinically. Even if the time of symptom onset is known, clinical stroke severity is highly variable. We studied the time course of CT density change after severe, rapid onset, acute, focal ischemia as documented by stable xenon CT cerebral blood flow (CBF) in monkeys. Eight monkeys (Macaca mulatta) were subjected to transorbital occlusion of the left posterior cerebral, anterior, middle, and internal carotid arteries to induce focal ischemia. CT density Hounsfield units (HU), CBF by stable xenon CT, arterial blood pressure, and blood gases were measured before occlusion, immediately after occlusion, at 30 min, and hourly for up to 6 h. Occlusion of the cerebral arteries decreased CBF to 8 ± 5 ml/100 g/ min within 15 min postocclusion. At 6 h, CBF was unchanged at 9 ± 4 ml/100 g/ min. CT density within the ischemic core fell from 42 to 38 HU immediately after occlusion (P < 0.05), rose transiently, then fell at 2 h (P < 0.01) and plateaued at 36 ± 5 HU for a total decrease of 4-5 HU between 4 and 6 h poststroke. Changes in CT density lag severe focal ischemia by 2 h. Thus, when CT hypodensity is seen in acute stroke, it is likely 2 h old. It also provides an explanation for the phenomenon of clinical CT mismatch with clinical deficits and normal CT.

Pursuing cleanliness in a field surgical environment.

Perioperative military nurses often are faced with the challenge of providing a clean surgical milieu in environments that are decidedly hostile to cleanliness. The combination of temperature extremes, blowing sand, dirt, mud, infection vectors, and the nature of battlefield wounds thwart the best intentions of maintaining cleanliness. Mitigating these problems requires setting priorities, teamwork, a rigorous daily regimen of cleaning, and a willingness to make improvements as opportunities become available.

Isolated sagittal and coronal craniosynostosis associated with TWIST box mutations.

Craniosynostosis, the premature fusion of one or more cranial sutures, affects 1 in 2,500 live births. Isolated single-suture fusion is most prevalent, with sagittal synostosis occurring in 1/5,000 live births. The etiology of isolated (nonsyndromic) single-suture craniosynostosis is largely unknown. In syndromic craniosynostosis, there is a highly nonrandom pattern of causative autosomal dominant mutations involving TWIST1 and fibroblast growth factor receptors (FGFRs). Prior to our study, there were no published TWIST1 mutations in the anti-osteogenic C-terminus, recently coined the TWIST Box, which binds and inhibits RUNX2 transactivation. RUNX2 is the principal master switch for osteogenesis. We performed mutational analysis on 164 infants with isolated, single-suture craniosynostosis for mutations in TWIST1, the IgIIIa exon of FGFR1, the IgIIIa and IgIIIc exons of FGFR2, and the Pro250Arg site of FGFR3. We identified two patients with novel TWIST Box mutations: one with isolated sagittal synostosis and one with isolated coronal synostosis. Kress et al. [2006] reported a TWIST Box "nondisease-causing polymorphism" in a patient with isolated sagittal synostosis. However, compelling evidence suggests that their and our sequence alterations are pathogenic: (1) a mouse with a mutation of the same residue as our sagittal synostosis patient developed sagittal synostosis, (2) mutation of the same residue precluded TWIST1 interaction with RUNX2, (3) each mutation involved nonconservative amino acid substitutions in highly conserved residues across species, and (4) control chromosomes lacked TWIST Box sequence alterations. We suggest that genetic testing of patients with isolated sagittal or coronal synostosis should include TWIST1 mutational analysis.

Application of the spanning plate concept to frontal orbital advancement: techniques and clinical experience in 60 patients.

Frontal orbital advancement (FOA), is the procedure of choice in treatment of coronal and metopic synostosis. Resorbable plates and screws have been widely accepted for use in pediatric craniofacial surgery, including FOA. We have applied the concept of extended resorbable spanning plates to FOA for metopic, unilateral, and bilateral coronal synostosis in infants and children during a 5-year period. We report on 60 patients, ages 4 to 15 months (mean, 7 months); 28 girls, 32 boys. Follow-up ranged from 12 to 36 months (mean, 24 months). There were no structural failures, no infections, and no complications related to the use of extended spanning plates. Extended spanning plates decrease mobility between bone segments, confer greater stability to the construct, and reduce both the number of plates and of screws that are necessary and reduce the operative time. Application of these plates simplifies FOA surgery and represents a step in the evolution of plating technology.

Chiari Type II malformation: past, present, and future.

OBJECT: The Chiari Type II malformation (CM II) is a unique hindbrain herniation found only in patients with myelomeningocele and is the leading cause of death in these individuals younger than 2 years of age. Several theories exist as to its embryological evolution and recently new theories are emerging as to its treatment and possible prevention. A thorough understanding of the embryology, anatomy, symptomatology, and surgical treatment is necessary to care optimally for children with myelomeningocele and prevent significant morbidity and mortality. METHODS: A review of the literature was used to summarize the clinically pertinent features of the CM II, with particular attention to pitfalls in diagnosis and surgical treatment. CONCLUSIONS: Any child with CM II can present as a neurosurgical emergency. Expeditious and knowledgeable evaluation and prompt surgical decompression of the hindbrain can prevent serious morbidity and mortality in the patient with myelomeningocele, especially those younger than 2 years old. Symptomatic CM II in the older child often presents with more subtle findings but rarely in acute crisis. Understanding of CM II continues to change as innovative techniques are applied to this challenging patient population.

A large planum sphenoidale meningioma with sinonasal extension in a child. Case report and review of the literature.

Anterior cranial base meningiomas are rare tumors in children. Due to the extensive involvement of orbit, paranasal sinuses, midface, and anterior skull base, a multidisciplinary approach is warranted. We present a case of a child with a large planum sphenoidale meningioma extending into subfrontal region, ethmoid and maxillary sinuses inferiorly, and orbits laterally. The patient, a 4-year-old girl, presented with long-standing nasal stuffiness and swelling of the midface. An extended frontobasal approach through a bifrontal craniotomy was used to resect the intracranial portion of this mass. The tumor had eroded through nasal septum, medial orbital walls, and left maxilla, structures which were not readily accessible from a cranial approach alone. A modified Weber-Ferguson incision was used for a transfacial approach to resect the residual mass below the skull base. The advantages of combining the bifrontal craniotomy with a transfacial split provided the added exposure to maximize the extent of resection.

Delayed intracranial migration of cervical sublaminar and interspinous wires and subsequent cerebellar abscess. Case report.

Delayed complications associated with sublaminar and interspinous wiring in the pediatric cervical spine are rare. The authors present a case of delayed complication in which a cervical fusion wire migrated into the cerebellum, causing subsequent cerebellar abscess 2 years after posterior cervical arthrodesis. A craniotomy was required to remove the wire and drain the abscess. Despite their history of safety and successful fusion, procedures involving sublaminar and interspinous wiring carry a risk of neurological injury secondary to wire migration. A thorough neuroimaging evaluation is required in patients who have undergone fusion and who have neurological complaints to detect late instrumentation-related sequelae.