Object. Bone morphogenetic proteins (BMPs) have been shown to have significant osteoinductive activity in numerous in vitro and in vivo assay systems, and BMP-2 and BMP-7 are currently being evaluated in human clinical studies. In the spinal region, BMPs have been shown to promote spinal arthrodesis at a higher rate than autologous bone alone. The delivery of BMPs via direct or ex vivo gene therapy techniques is also currently being evaluated and has shown promise in several mammalian models. The present study was designed to evaluate the efficacy of the use of direct, percutaneous BMP-9 adenoviral gene therapy to promote spinal fusion in the rodent. Methods. Each animal was injected with 7.5 × 108 pfu of a BMP-9 adenoviral vector in the lumbar paraspinal musculature and allowed to survive 16 weeks. Computerized tomography studies and histological analysis demonstrated massive bone induction at the injection sites, clearly leading to solid spinal arthrodesis, without evidence of pseudarthroses, nerve root compression, or systemic side effects. Conclusions. The results of this study strongly support the advancement of BMP gene therapy techniques toward clinical use.

The present study was performed to determine the histological, ultrastructural, and radiographic changes that occur over time at intramuscular BMP-9 gene therapy treatment sites. Several members of the bone morphogenetic protein (BMP) family have the potential to induce osteochondrogenesis when the protein is delivered to rodents, canines, rabbits, and nonhuman primates. Previous studies have also demonstrated that BMP gene therapy utilizing adenoviral vectors can also stimulate orthotopic and heterotopic bone formation in rodents and rabbits. Athymic nude and Sprague-Dawley rats were injected with Ad-BMP-9 or Ad-beta-Gal (3.75 x 10(9) particles) in their thigh musculature and light microscopic, electron microscopic, and computerized tomography analysis was performed 3, 6, 9, 12, 15, 18, 21, and 100 days later. To assess early mesenchymal cell proliferation, a bromodeoxyuridine (BrdU) immunohistochemical analysis was also performed 48, 60, and 72 hr postinjection in athymic nude rats. All animals demonstrated extensive endochondral bone formation at the Ad-BMP-9 treatment sites within 3 weeks. The Sprague-Dawley rats also exhibited a massive, acute inflammatory infiltrate during the first week. Proliferating mesenchymal stem cells were clearly evident as early as 2 days after treatment, which differentiated into small or hypertrophied chondrocytes during the next week. During the third week, the cartilaginous matrix mineralized and formed woven bone, which converted to lamellar bone by 3 months. No evidence of bone formation was demonstrated at the Ad-beta-Gal injection sites in the athymic nude or Sprague-Dawley rats. In addition, no cellular proliferation was seen at the Ad-beta-Gal treatment sites in the athymic nude animals as assessed by light microscopy and BrdU immunohistochemistry. The extensive bone formation induced by Ad-BMP-9 suggests that BMP gene therapy may have potential utility in the treatment of degenerative, rheumatic, or traumatic bone pathology.

Background and PurposeIntraoperative neurophysiological monitoring (IONM) using upper and lower somatosensory evoked potentials (SSEPs) is an established technique used to predict and prevent neurologic injury during intracranial tumor resections. Endoscopic port surgery (EPS) is a minimally-invasive approach to deep intraparenchymal and intraventricular brain tumors. The authors intended to evaluate the predictive value of SSEP monitoring during resection of intracranial brain tumors using a parallel endoscopic technique.MethodsA retrospective review was conducted of patients operated on from 2007-2010 utilizing IONM in whom endoscopic ports were used to remove either intraparenchymal or intraventricular tumors. Cases were eligible for review if an endoscopic port was used to resect an intracranial tumor and the electronic chart included all intraoperative monitoring data as well as pre- and post-operative neurologic exams.Results139 EPS cases met criteria for inclusion. Eighty five patients (61%) had intraparenchymal and fifty four (39%) had intraventricular tumors or colloid cysts. SSEP changes were seen in eleven cases (7.9%), being irreversible in three (2.2%) and reversible in eight cases (5.8%). Seven patients (5.0%) with intraparenchymal tumors had SSEP changes which met our criterea for significant changes while there were four (2.9%) with intraventricular (p-value=0.25). Five patients suffered post operative deficits, two reversible and two irreversible SSEP changes. Only one case exhibited post operative hemiparesis with no SSEP changes. The positive predictive value of SSEP was 45.4% and the negative predictive value was 99.2%.ConclusionsBased on the high negative and low positive predictive values, the utility of SSEP monitoring for cylindrical port resections may be limited. However, the use of SSEP monitoring can be helpful in reducing the impact of endoscopic port manipulation when the tumor is closer to the somatosensory pathway.

NEURO-ONCOLOGY • MAY 2017 stem malignant gliomas have one-year progression-free survival rates below 25% and median overall survival of 9 to 10 months with current treatment options and hence represent a significant unmet medical need. Genomewide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene (H3.3.K27M mutation). Tumor-specific missense mutations are not subjected to self-tolerance and can be suitable targets (i.e. neoantigen) for cancer immunotherapy. We evaluated whether the H3.3.K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2 + human T cells. In vitro stimulation of HLA-A2+ donor-derived CD8 + T-cells with a synthetic peptide encompassing the H3.3.K27M mutation (H3.3.K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3.K27M epitope peptide but also lysed HLA-A2 + DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2 + H3.3.K27M-negative DIPG cell lines or H3.3K27M-positive but HLA-A2-negative cells. The H3.3.K27M epitope peptide, but not the non-mutant counterpart, indicated an excellent affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer, cDNAs for T cell receptor (TCR) α-and β-chains were cloned into a retroviral vector. Human HLA-A2 + T-cells transduced with the TCR demonstrated antigen-specific reactivity as well as anti-glioma responses in vitro. Peptide titration assay suggested that the H3.3.K27M-specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino-acid sequence motif in the epitope for the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T-cells transduced with the H3.3.K27M-specific TCR significantly inhibited the growth of intracranial HLA-A2 + H3.3.K27M-positive glioma xenografts in immune-deficient mice. These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the H3.3.K27M-specific TCR.

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