PI3K/AKT pathway alterations are associated with clinically aggressive and histologically anaplastic subsets of pilocytic astrocytoma.

Pilocytic astrocytomas (PA) are well-differentiated gliomas having a favorable prognosis when compared with other diffuse or infiltrative astrocytomas. Molecular genetic abnormalities and activation of signaling pathways associated with clinically aggressive PA and histologically anaplastic PA have not been adequately studied. We performed molecular genetic, gene expression, and immunohistochemical studies using three PA subsets, including conventional PA (n = 43), clinically aggressive/recurrent PA (n = 24), and histologically anaplastic PA (n = 25). A clinical diagnosis of NF1 was present in 28% of anaplastic PA. Molecular cytogenetic studies demonstrated heterozygous PTEN/10q and homozygous p16 deletions in 6/19 (32%) and 3/15 (20%) cases of anaplastic PA, respectively, but in neither of the two other groups. BRAF duplication was identified in 33% of sporadic anaplastic PA and 63% of cerebellar examples. BRAF (V600E) mutation was absent in four (of 4) sporadic cases lacking duplication. IDH1(R132H) immunohistochemistry was negative in 16 (of 16) cases. Neither PDGFRA nor EGFR amplifications were present. pERK staining levels were similar among the three PA subsets, but a stepwise increase in cytoplasmic pAKT and to a lesser extent pS6 immunoreactivity was noted by immunohistochemistry in aggressive PA groups. This was particularly true in histologically anaplastic PA when compared with conventional PA (p < 0.001 and p = 0.005, respectively). In addition, PTEN expression at the mRNA level was decreased in histologically anaplastic PA when compared to the other groups (p = 0.05). In summary, activation of the PI3K/AKT in addition to MAPK/ERK signaling pathways may underlie biological aggressiveness in PA. Specifically, it may mediate the increased proliferative activity observed in histologically anaplastic PA.

A carrier for non-covalent delivery of functional beta-galactosidase and antibodies against amyloid plaques and IgM to the brain.

BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).

Phenotypic variations in NF1-associated low grade astrocytomas: possible role for increased mTOR activation in a subset.

Low grade astrocytomas are the most common CNS tumors in neurofibromatosis type 1(NF1) patients. While most are classic pilocytic astrocytomas (PA), some are difficult to classify, and have been termed "low grade astrocytoma subtype indeterminate" (LGSI). Some of these tumors exhibit peculiar morphologies, including plump cytoplasmic processes and macronucleoli. In the current study we performed electron microscopy, followed by gene expression, immunohistochemicai and western blot analyses in an effort to identify biological differences underlying phenotypic variation in NF1-associated low grade astrocytoma. Electron microscopy demonstrated intermediate filaments and frequent Rosenthal fiber material in both PA and LGSI. Dense core granules and/or aligned microtubules were present in the LGSI group (2 of 3 cases) and in the PA group (1 of 10 cases). Analysis of global gene expression data obtained using Affymetrix HG-U133 Plus2.0 chips (5 PA, 1 LGSI), and western blot analysis for phospho-S6 (1 LGSI, 2 PA) demonstrated a gene expression profile reflecting "neuronal differentiation" and increased phospho-S6 immunoreactivity consistent with mTOR activation in the LGSI compared with PA. These findings were confirmed by immunohistochemistry for neuronal markers, as well as combined phospho-S6/ phospho-p70S6K immunoreactivity in 4 (of 4) LGSI vs. 5 (of 13) NF1-associated PA (p=0.02), and 13 (of 39) sporadic PA. Phospho-ERK immunoreactivity was uniformly present in PA and LGSI groups, while BRAF duplication was absent by FISH in 8 NF1-associated low grade astrocytomas. In summary, differential expression of neuronal-related genes and increased mTOR activation may underlie phenotypic variations in NF1-associated low grade astrocytomas.