Tord D. Alden

Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.

Cellular injury and neuroinflammation in children with chronic intractable epilepsy

ObjectiveTo elucidate the presence and potential involvement of brain inflammation and cell death in neurological morbidity and intractable seizures in childhood epilepsy, we quantified cell death, astrocyte proliferation, microglial activation and cytokine release in brain tissue from patients who underwent epilepsy surgery.MethodsCortical tissue was collected from thirteen patients with intractable epilepsy due to focal cortical dysplasia (6), encephalomalacia (5), Rasmussens encephalitis (1) or mesial temporal lobe epilepsy (1). Sections were processed for immunohistochemistry using markers for neuron, astrocyte, microglia or cellular injury. Cytokine assay was performed on frozen cortices. Controls were autopsy brains from eight patients without history of neurological diseases.ResultsMarked activation of microglia and astrocytes and diffuse cell death were observed in epileptogenic tissue. Numerous fibrillary astrocytes and their processes covered the entire cortex and converged on to blood vessels, neurons and microglia. An overwhelming number of neurons and astrocytes showed DNA fragmentation and its magnitude significantly correlated with seizure frequency. Majority of our patients with abundant cell death in the cortex have mental retardation. IL-1beta, IL-8, IL-12p70 and MIP-1beta were significantly increased in the epileptogenic cortex; IL-6 and MCP-1 were significantly higher in patients with family history of epilepsy.ConclusionsOur results suggest that active neuroinflammation and marked cellular injury occur in pediatric epilepsy and may play a common pathogenic role or consequences in childhood epilepsy of diverse etiologies. Our findings support the concept that immunomodulation targeting activated microglia and astrocytes may be a novel therapeutic strategy to reduce neurological morbidity and prevent intractable epilepsy.

The use of bone morphogenetic protein gene therapy in craniofacial bone repair.

&NA; Bone morphogenetic proteins (BMPs) are capable of inducing endochondral bone formation when applied on biologic carriers in numerous mammalian in vivo assay systems. Bone morphogenetic protein gene therapy is also currently being developed to promote osteogenesis for clinical indications such as spinal fusions, craniofacial bone loss, and osteoporosis. In this study, critical‐sized mandibular defects were treated with a control adenoviral vector (Ad‐&bgr;‐gal), a BMP‐2 adenoviral vector (Ad‐BMP‐2), or a BMP‐9 adenoviral vector (Ad‐BMP‐9). Gross tissue examination, radiographic analysis, and histologic analysis demonstrated significant bony healing in the BMP treated groups compared to controls. Osteogenesis was limited to the bony defect, without extension into the surrounding soft tissues. The study suggests that with further development, BMP gene therapy may be potentially useful for repair of bony defects in the craniofacial region.

Fluoroscopic Frameless Stereotaxy for Transsphenoidal Surgery

OBJECTIVETo assess the value of frameless fluoroscopy-guided stereotactic transsphenoidal surgery using the FluoroNav Virtual Fluoroscopy System (Medtronic Sofamor Danek, Inc., Memphis, TN). METHODSTwenty consecutive patients undergoing transsphenoidal surgery for sellar lesions were assigned to transsphenoidal surgery with or without computer-assisted fluoroscopic image guidance using the FluoroNav system. Prospective data regarding patient age, sex, lesion characteristics, operative time, and treatment cost were obtained. RESULTSAlthough patients in the FluoroNav group were, on average, 17 years younger than the patients in the control group, more patients with recurrent adenomas were treated in the image guidance group. No other significant differences between the groups were found. FluoroNav provided accurate, continuous information regarding the anatomic midline trajectory to the sella turcica as well as anatomic structures (e.g., sella, sphenoid sinus) in the lateral view. No patient required reversion to intraoperative videofluoroscopy. No statistically significant differences were found with regard to preincision setup time, operative time, or cost. FluoroNav allowed procedures to be performed with significantly fewer x-rays being taken. CONCLUSIONFluoroscopic computer-assisted frameless stereotaxy furnishes accurate real-time information with regard to midline structures and operative trajectory. Although it is useful in first-time transseptal transsphenoidal surgery, its primary benefit is realized in recurrent surgery.

Morphologic analysis of BMP-9 gene therapy-induced osteogenesis

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.

Intrathecal baclofen withdrawal: a case report and review of the literature.

Abstract Introduction. Spasticity is an endpoint of a variety of neurologic disorders with upper motor neuron damage. There have been several studies demonstrating improvement in spasticity through administration of intrathecal baclofen. Withdrawal from oral baclofen has been well described. Intrathecal baclofen withdrawal has been less frequently reported. We present a case of withdrawal after intrathecal baclofen pump catheter failure. Patient. A 14-year-old boy presented with fevers, which were thought to be related to recent spine surgery and possible pneumonia. Eventual workup revealed evidence of intrathecal baclofen withdrawal owing to pump catheter failure. His fevers, with temperatures of up to 40°C, and painful muscle spasms resolved and his clinical condition improved after pump exploration and resumption of intrathecal delivery. Conclusions. Intrathecal baclofen withdrawal can be life threatening. Prompt recognition and restoration of an adequate intrathecal baclofen dose is essential for recovery.

Bone Morphogenetic Proteins and Bone Morphogenetic Protein Gene Therapy in Neurological Surgery: A Review

OBJECTIVE To review the uses of bone morphogenetic proteins (BMPs) and BMP gene therapy for the treatment of neurosurgical disorders. METHODS Literature review. RESULTS BMPs are members of the transforming growth factor beta superfamily, and they play an important role in the growth and development of numerous tissues, including bone, brain, and spinal cord. Although the majority of previous studies have focused on the regulatory functions of BMPs in the normal growth and differentiation of the skeletal system, BMPs also seem to be exquisitely involved in the regulation of cellular proliferation, survival, differentiation, apoptosis, and lineage commitment in the central nervous system. When specific BMPs are delivered on biological matrices, they have the capacity to induce bone, cartilage, ligament, and tendon at both heterotopic and orthotopic sites, suggesting that they may play a major role in the future treatment of spinal and craniofacial pathology. For example, recent studies have clearly demonstrated the usefulness of BMPs and BMP gene therapy for the induction of spinal arthrodesis in several animal models. In addition, several BMPs have been shown to have a neuroprotective effect in animal models of head injury, cerebral ischemia, and Parkinsons disease and may therefore have direct clinical applications for the treatment of central nervous system disorders. CONCLUSION As the physiological activity of BMPs in the development and pathology of the central nervous system and spine are more fully elucidated, BMP therapeutics and gene therapy will probably have numerous applications in neurological surgery.

The transsphenoidal resection of pediatric craniopharyngiomas: a case series

OBJECT The majority of pediatric craniopharyngiomas are treated using a transcranial approach. Although there is an increasing acceptance of transsphenoidal resection in adults, there are few reports describing this approach in the pediatric population. The purpose of this study is to report the outcomes after transsphenoidal surgery in a consecutive series of pediatric patients with craniopharyngiomas treated at a single institution with the goal of gross-total resection (GTR). METHODS Twenty-three patients with pathologically proven craniopharyngiomas were identified who were 18 years of age or less at the time of surgery. The medical records and imaging studies of the patients were retrospectively reviewed. One patient who was lost to follow-up after surgery was excluded. RESULTS Among the 22 patients included in the study, 11 underwent transsphenoidal surgery as the primary procedure and 11 underwent transsphenoidal surgery as a secondary procedure after a previous procedure. All patients had at least some sellar component to their tumor and all had either anterior or posterior pituitary dysfunction at presentation. In the entire cohort, a GTR was achieved in 15 (68%) of 22 patients, a radical subtotal resection in 4 (18%) of 22 patients, a subtotal resection in 1 patient, and a partial resection in 2 patients. The degree of resection was higher in the primary transsphenoidal group. After a mean follow-up of 82 months, 4 patients (18%) experienced recurrence. Recurrence occurred in 13% after GTR compared with 28.5% after all other degrees of resection. Tumor recurred in 9% of the primary transsphenoidal group and in 30% of patients who had undergone other therapies prior to the transsphenoidal operation. No patient who had panhypopituitarism experienced a gain of function postoperatively, 67% developed new panhypopituitarism, and 56% experienced new diabetes insipidus. Vision improved or normalized in 9 (64%) of 14 patients presenting with visual loss. Complications included 1 death 3 weeks postoperatively, 2 CSF leaks, and new obesity in 37%. CONCLUSIONS Transsphenoidal resection of pediatric craniopharyngiomas results in a high rate of both visual improvement and GTR with a low associated risk of recurrence. The transsphenoidal approach should be considered in selected pediatric patients with craniopharyngioma, especially those with infradiaphragmatic origin.

Bone Morphogenetic Protein Gene Therapy

Study Design. A retrospective analysis of previous BMP gene therapy and general gene therapy publications. Objective. To present the potential role of BMP gene therapy for the induction of osteogenesis and spinal fusion. Summary of Background Data. A variety of viral and non-viral techniques have been utilized to insert foreign transgenes into cells, both in vivo and in vitro. These techniques are now being used to transduce cells with a BMP gene to express significant amounts of BMP. This secreted BMP can subsequently stimulate osteogenesis in a variety of locations, including in the paraspinal regions. Methods. A retrospective analysis of the literature. Results. Direct and ex vivo BMP gene therapy has been shown to successfully promote bone healing and regeneration in a variety of animal models. Long-term and regulated transgene expression are clear advantages of BMP gene delivery, compared to direct BMP application. To date, BMP gene delivery with adenoviral vectors have been the most effective approach for stimulating bone induction in vivo. Conclusions. Although BMP gene therapy techniques have significant potential for the treatment of spine pathology, further preclinical and clinical research and development are required before this technology will have direct clinical applications.