Phase 2 Study of Pomalidomide (CC-4047) Monotherapy for Children and Young Adults With Recurrent or Progressive Primary Brain Tumors

INTRODUCTION: Treatment of recurrent primary pediatric brain tumors remains a major challenge, with most children succumbing to their disease. We conducted a prospective phase 2 study investigating the safety and efficacy of pomalidomide (POM) in children and young adults with recurrent and progressive primary brain tumors. BACKGROUND: METHODS: Patients with recurrent and progressive high-grade glioma (HGG), diffuse intrinsic pontine glioma (DIPG), ependymoma, or medulloblastoma received POM 2.6 mg/m2/day (the recommended phase 2 dose [RP2D]) on days 1-21 of a 28-day cycle. A Simon’s Optimal 2-stage design was used to determine efficacy. Primary endpoints included objective response (OR) and long-term stable disease (LTSD) rates. Secondary endpoints included duration of response, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: 46 patients were evaluable for response (HGG, n = 19; DIPG, ependymoma, and medulloblastoma, n = 9 each). Two patients with HGG achieved OR or LTSD (10.5% [95% CI, 1.3%-33.1%]; 1 partial response and 1 LTSD) and 1 patient with ependymoma had LTSD (11.1% [95% CI, 0.3%-48.2%]). There were no ORs or LTSD in the DIPG or medulloblastoma cohorts. The median PFS for patients with HGG, DIPG, ependymoma, and medulloblastoma was 7.86, 11.29, 8.43, and 8.43 weeks, respectively. Median OS was 5.06, 3.78, 12.02, and 11.60 months, respectively. Neutropenia was the most common grade 3/4 adverse event. CONCLUSIONS: Treatment with POM monotherapy did not meet the primary measure of success in any cohort. Future studies are needed to evaluate if POM would show efficacy in tumors with specific molecular signatures or in combination with other anticancer agents. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT03257631; EudraCT, identifier 2016-002903-25

Optimising biomarkers for accurate ependymoma diagnosis, prognostication and stratification within International Clinical Trials: A BIOMECA study

BACKGROUND: Accurate identification of brain tumour molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study. METHODS: Across six European BIOMECA laboratories we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via FISH and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. RESULTS: DNA Methylation profiling classified 65.3% (n=96/147) of cases as EPN-PFA and 15% (n=22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99-100% across three centres). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP1- fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-centre concordance and low sensitivity and specificity whilst MIP, MLPA and DNA methylation-based approaches demonstrated greater accuracy. CONCLUSIONS: We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q and CDKN2A loss whilst FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches

Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation

<p>Abstract</p> <p>Background</p> <p>The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrP^C) to an alternatively folded isoform (PrP^Sc). The accumulation of PrP^Scwithin the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined.</p> <p>Results</p> <p>We report the novel observation that prion infection altered the membrane composition and significantly increased total cholesterol levels in two neuronal cell lines (ScGT1 and ScN2a cells). There was a significant correlation between the concentration of free cholesterol in ScGT1 cells and the amounts of PrP^Sc. This increase was entirely a result of increased amounts of free cholesterol, as prion infection reduced the amounts of cholesterol esters in cells. These effects were reproduced in primary cortical neurons by the addition of partially purified PrP^Sc, but not by PrP^C. Crucially, the effects of prion infection were not a result of increased cholesterol synthesis. Stimulating cholesterol synthesis via the addition of mevalonate, or adding exogenous cholesterol, had the opposite effect to prion infection on the cholesterol balance. It did not affect the amounts of free cholesterol within neurons; rather, it significantly increased the amounts of cholesterol esters. Immunoprecipitation studies have shown that cytoplasmic phospholipase A₂(cPLA₂) co-precipitated with PrP^Scin ScGT1 cells. Furthermore, prion infection greatly increased both the phosphorylation of cPLA₂and prostaglandin E₂production.</p> <p>Conclusion</p> <p>Prion infection, or the addition of PrP^Sc, increased the free cholesterol content of cells, a process that could not be replicated by the stimulation of cholesterol synthesis. The presence of PrP^Scincreased solubilisation of free cholesterol in cell membranes and affected their function. It increased activation of the PLA₂pathway, previously implicated in PrP^Scformation and in PrP^Sc-mediated neurotoxicity. These observations suggest that the neuropathogenesis of prion diseases results from PrP^Scaltering cholesterol-sensitive processes. Furthermore, they raise the possibility that disturbances in membrane cholesterol are major triggering events in neurodegenerative diseases.</p

Upregulated IL-1β in dysferlin-deficient muscle attenuates regeneration by blunting the response to pro-inflammatory macrophages.

BACKGROUND: Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study. METHODS: To test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts. RESULTS: Dysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1β and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1β, which inhibited it. Importantly, blockade of IL-1β signaling significantly improved differentiation of dysferlin-deficient cells. CONCLUSIONS: We propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1β signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle

Changes in habitat associations during range expansion: disentangling the effects of climate and residence time

The distributions of many species are not at equilibrium with their environment. This includes spreading non-native species and species undergoing range shifts in response to climate change. The habitat associations of these species may change during range expansion as less favourable climatic conditions at expanding range margins may constrain species to use only the most favourable habitats, violating the species distribution model assumption of stationarity. Alternatively, changes in habitat associations could result from density-dependent habitat selection; at range margins, population densities are initially low so species can exhibit density-independent selection of the most favourable habitats, while in the range core, where population densities are higher, species spread into less favourable habitat. We investigate if the habitat preferences of the non-native common waxbill Estrilda astrild changed as they spread in three directions (north, east and south-east) in the Iberian Peninsula. There are different degrees of climatic suitability and colonization speed across range expansion axes, allowing us to separate the effects of climate from residence time. In contrast to previous studies we find a stronger effect of residence time than climate in influencing the prevalence of common waxbills. As well as a strong additive effect of residence time, there were some changes in habitat associations, which were consistent with density-dependent habitat selection. The combination of broader habitat associations and higher prevalence in areas that have been colonised for longer means that species distribution models constructed early in the invasion process are likely to underestimate species’ potential distribution

Molecular, Pathological, Radiological, and Immune Profiling of Non-brainstem Pediatric High-Grade Glioma from the HERBY Phase II Randomized Trial

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term “HGG” in the pediatric population

GENE-TRANSFER INTO SATELLITE CELL FROM REGENERATING MUSCLE - BUPIVACAINE ALLOWS BETA-GAL TRANSFECTION AND EXPRESSION IN-VITRO AND IN-VIVO

In Vitro Cell Dev Biol Anim. 1994 Feb;30A(2):131-3. Gene transfer into satellite cell from regenerating muscle: bupivacaine allows beta-Gal transfection and expression in vitro and in vivo. Cantini M, Massimino ML, Catani C, Rizzuto R, Brini M, Carraro U. Source Department of Experimental Biomedical Sciences, University of Padua, Italy. Abstract A large bulk of experimental evidence (15) suggests that myogenic cell transfer can be regarded as a promising therapeutic approach in the cure of inherited pathologies. In particular, it has been shown that primary myoblasts obtained from embryonic or neonatal muscles allows the recovery of the normal phenotype in defective muscle tissues. The utilization of this approach in clinical settings still bears heavy limitations. Apart from the legal and ethical difficulties, the use of muscles obtained from aborted fetus is challenged by a large risk of rejection, due to the incompatibility between donor and recipient. In this context based on the genetic alteration and reimplanting of the patient's own satellite cells, appears an approach attractive. Myoblasts derived from satellite cells are the obligate candidates for experiments, but the production of sufficient cell numbers is a major problem. Local anesthetics [Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) and related molecules] had been used to induce myofiber damage (and thus satellite cells proliferation) and thereby may represent a tool for increasing the yield of myoblasts from adult muscles (1,9,17). We will show that satellite cells obtained from adult muscles after bupivacaine injection can be transfected in vitro and that the transfected gene is expressed in vitro and in vivo, after reimplantation of the modified myoblasts in recipient muscles. PMID: 8012655 [PubMed - indexed for MEDLINE

Extracellular copper ions regulate cellular prion protein (PrPC) expression and metabolism in neuronal cells.

[ARTICOLO

Macrophages regulate proliferation and differentiation of satellite cells

Biochem Biophys Res Commun. 1994 Aug 15;202(3):1688-96. Macrophages regulate proliferation and differentiation of satellite cells. Cantini M, Massimino ML, Bruson A, Catani C, Dalla Libera L, Carraro U. Source Department of Biomedical Sciences, University of Padova, Italy. Abstract We used an in vitro model to investigate whether macrophages stimulate satellite cells proliferation. Satellite cells were obtained by tryptic digestion of adult muscle. Macrophages were obtained from peritoneal cavity by wash after injection of thioglycolate broth. Macrophages and satellite cells cocultures showed an increased number of differentiated myotubes as compared to control cultures. Moreover, in conditions of myoblast colony growth, the addition of macrophage-conditioned medium resulted in a greater number of muscle cell colonies, which are richer in large and differentiated myotubes. The experiments with macrophage-conditioned media suggest that the increased muscle cell proliferation and differentiation is mediated by soluble factor(s) released by macrophages. These results demonstrate that besides their scavenger role macrophages play a pivotal role in myoblast proliferation during muscle regeneration. PMID: 8060358 [PubMed - indexed for MEDLINE

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage.

Neurosci Lett. 1998 Aug 14;252(2):123-6. Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage. Sandri M, Massimino ML, Cantini M, Giurisato E, Sandri C, Arslan P, Carraro U. Source C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy. [email protected] Abstract Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells. PMID: 9756337 [PubMed - indexed for MEDLINE