The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration.

Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.

What is inappropriate hospital use for elderly people near the end of life? A systematic review.

BACKGROUND: Older people with advance chronic illness use hospital services repeatedly near the end of life. Some of these hospitalizations are considered inappropriate. AIM: To investigate extent and causes of inappropriate hospital admission among older patients near the end of life. METHODS: English language publications in Medline, EMBASE, PubMed, Cochrane library, and the grey literature (January 1995-December 2016) covering community and nursing home residents aged ≥60years admitted to hospital. OUTCOMES: measurements of inappropriateness. A 17-item quality score was estimated independently by two authors. RESULTS: The definition of 'Inappropriate admissions' near the end of life incorporated system factors, social and family factors. The prevalence of inappropriate admissions ranged widely depending largely on non-clinical reasons: poor availability of alternative sites of care or failure of preventive actions by other healthcare providers (1.7-67.0%); family requests (up to 10.5%); or too late an admission to be of benefit (1.7-35.0%). The widespread use of subjective parameters not routinely collected in practice, and the inclusion of non-clinical factors precluded the true estimation of clinical inappropriateness. CONCLUSIONS: Clinical inappropriateness and system factors that preclude alternative community care must be measured separately. They are two very different justifications for hospital admissions, requiring different solutions. Society has a duty to ensure availability of community alternatives for the management of ambulatory-sensitive conditions and facilitate skilling of staff to manage the terminally ill in non-acute settings. Only then would the evaluation of local variations in clinically inappropriate admissions and inappropriate length of stay be possible to undertake.

OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma.

Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes.Significance: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 288-301. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Rapid Intraoperative Molecular Characterization of Glioma.

IMPORTANCE: Conclusive intraoperative pathologic confirmation of diffuse infiltrative glioma guides the decision to pursue definitive neurosurgical resection. Establishing the intraoperative diagnosis by histologic analysis can be difficult in low-cellularity infiltrative gliomas. Therefore, we developed a rapid and sensitive genotyping assay to detect somatic single-nucleotide variants in the telomerase reverse transcriptase (TERT) promoter and isocitrate dehydrogenase 1 (IDH1). OBSERVATIONS: This assay was applied to tissue samples from 190 patients with diffuse gliomas, including archived fixed and frozen specimens and tissue obtained intraoperatively. Results demonstrated 96% sensitivity (95% CI, 90%-99%) and 100% specificity (95% CI, 95%-100%) for World Health Organization grades II and III gliomas. In a series of live cases, glioma-defining mutations could be identified within 60 minutes, which could facilitate the diagnosis in an intraoperative timeframe. CONCLUSIONS AND RELEVANCE: The genotyping method described herein can establish the diagnosis of low-cellularity tumors like glioma and could be adapted to the point-of-care diagnosis of other lesions that are similarly defined by highly recurrent somatic mutations.

Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas.

PURPOSE: Isocitrate dehydrogenase (IDH) gene mutations occur in low-grade and high-grade gliomas. We sought to identify the genetic basis of malignant phenotype heterogeneity in IDH-mutant gliomas. METHODS: We prospectively implanted tumor specimens from 20 consecutive IDH1-mutant glioma resections into mouse brains and genotyped all resection specimens using a CLIA-certified molecular panel. Gliomas with cancer driver mutations were tested for sensitivity to targeted inhibitors in vitro. Associations between genomic alterations and outcomes were analyzed in patients. RESULTS: By 10 months, 8 of 20 IDH1-mutant gliomas developed intracerebral xenografts. All xenografts maintained mutant IDH1 and high levels of 2-hydroxyglutarate on serial transplantation. All xenograft-producing gliomas harbored "lineage-defining" mutations in CIC (oligodendroglioma) or TP53 (astrocytoma), and 6 of 8 additionally had activating mutations in PIK3CA or amplification of PDGFRA, MET, or N-MYC. Only IDH1 and CIC/TP53 mutations were detected in non-xenograft-forming gliomas (P = 0.0007). Targeted inhibition of the additional alterations decreased proliferation in vitro. Moreover, we detected alterations in known cancer driver genes in 13.4% of IDH-mutant glioma patients, including PIK3CA, KRAS, AKT, or PTEN mutation or PDGFRA, MET, or N-MYC amplification. IDH/CIC mutant tumors were associated with PIK3CA/KRAS mutations whereas IDH/TP53 tumors correlated with PDGFRA/MET amplification. Presence of driver alterations at progression was associated with shorter subsequent progression-free survival (median 9.0 vs. 36.1 months; P = 0.0011). CONCLUSION: A subset of IDH-mutant gliomas with mutations in driver oncogenes has a more malignant phenotype in patients. Identification of these alterations may provide an opportunity for use of targeted therapies in these patients. Clin Cancer Res; 20(11); 2898-909. ©2014 AACR.

Turner syndrome and meningioma: support for a possible increased risk of neoplasia in Turner syndrome.

Neoplasia is uncommon in Turner syndrome, although there is some evidence that brain tumors are more common in Turner syndrome patients than in the general population. We describe a woman with Turner syndrome (45,X) with a meningioma, in whom a second neoplasia, basal cell carcinomas of the scalp and nose, developed five years later in the absence of therapeutic radiation. Together with 7 cases of Turner syndrome with meningioma from a population-based survey in the United Kingdom, and 3 other isolated cases in the literature, we review this small number of patients for evidence of risk factors related to Turner syndrome, such as associated structural anomalies or prior treatment. We performed histological and fluorescent in situ hybridization (FISH) of 22q (NF2 locus) analyses of the meningeal tumor to search for possible molecular determinants. We are not able to prove causation between these two entities, but suggest that neoplasia may be a rare associated medical problem in Turner syndrome. Additional case reports and extension of population-based studies are needed.

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation.

Hemangioblastomas consist of 10-20% neoplastic "stromal" cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.

Wilms tumor is a pediatric kidney cancer associated with inactivation of the WT1 tumor-suppressor gene in 5 to 10% of cases. Using a high-resolution screen for DNA copy-number alterations in Wilms tumor, we identified somatic deletions targeting a previously uncharacterized gene on the X chromosome. This gene, which we call WTX, is inactivated in approximately one-third of Wilms tumors (15 of 51 tumors). Tumors with mutations in WTX lack WT1 mutations, and both genes share a restricted temporal and spatial expression pattern in normal renal precursors. In contrast to biallelic inactivation of autosomal tumor-suppressor genes, WTX is inactivated by a monoallelic "single-hit" event targeting the single X chromosome in tumors from males and the active X chromosome in tumors from females.

A histologic demonstration of siliceous materials in simian lung mite infected lung tissues by microincineration.

Approximately 90% of freshly imported macaques and other Old World Monkeys are known to be infected with respiratory mites. The lung associated pigments are integral components of pulmonary acariasis in Old World Monkeys; at least three distinctive pigmental bodies are identified in association with lung mite infection. Two major components of pigments are recently identified as silica by using elemental analysis using a high voltage electron microscope and an energy-dispersive X-ray analysis technique. Since a limited number of infected monkey lung tissues and associated pigments can be examined by this tedious procedure, it was important for us to examine much greater number of specimens to verify our initial observation. Ten microincineration technique described provided a unique and practical way to identify the mineral elements in as many 27 histologic sections within a short span of time. Silica and silicates are heat resistant whereas majority of organic materials including lung mite parasites disintegrated under the extreme temperature. Mineral elements were exclusively located within the polarizable white ash. More than 90% of total pigmental bodies identified were found to be related to siliceous materials in 20 incinerated infected monkey lung tissues whereas five noninfected lungs similarly examined did not reveal any pigmental bodies. Other than a small of fine granular mucin substances which were PAS positive, the majority of lung mite associated pigments such as large granules of hemosiderin, needle-like crystals and other fine granules engulfed by macrophages were identified to be siliceous materials as they have persisted even after microincineration. Mite parasites and other organic materials were completely disintegrated. Similar pigmental bodies examined by microscope X-ray analysis were positive for silicate. This finding suggests that lung mite infection in Old Monkeys apparently predisposed silicosis. Therefore, until the link between lung mite infection and silicosis is clarified, experimental inhalation toxicologic findings in mite-infected Old World monkeys should be interpreted cautiously.