Hong Wan

Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas

Gliomas arising in the brainstem and thalamus are devastating tumors that are difficult to surgically resect. To determine the genetic and epigenetic landscape of these tumors, we performed exomic sequencing of 14 brainstem gliomas (BSGs) and 12 thalamic gliomas. We also performed targeted mutational analysis of an additional 24 such tumors and genome-wide methylation profiling of 45 gliomas. This study led to the discovery of tumor-specific mutations in PPM1D, encoding wild-type p53–induced protein phosphatase 1D (WIP1), in 37.5% of the BSGs that harbored hallmark H3F3A mutations encoding p.Lys27Met substitutions. PPM1D mutations were mutually exclusive with TP53 mutations in BSG and attenuated p53 activation in vitro. PPM1D mutations were truncating alterations in exon 6 that enhanced the ability of PPM1D to suppress the activation of the DNA damage response checkpoint protein CHK2. These results define PPM1D as a frequent target of somatic mutation and as a potential therapeutic target in brainstem gliomas.

The H3.3 K27M mutation results in a poorer prognosis in brainstem gliomas than thalamic gliomas in adults

Brainstem and thalamic gliomas are rare, and they are poorly understood in adults. Genetic aberrations that occur in these tumors are still unknown. In this study, we investigated whether thalamic gliomas have different genetic aberrations and clinical outcomes compared with brainstem gliomas in adults. Forty-three glioma samples were selected, including 28 brainstem and 15 thalamic gliomas. The frequency of the K27M mutation in adult midline gliomas was 58.1%. High-grade gliomas in the thalamus were statistically significantly more numerous than brainstem gliomas. Patients with K27M mutant brainstem gliomas had a significantly shorter overall survival than patients with wild-type tumors (P = .020) by Cox regression after adjustment for other independent risk factors. However, there was no statistical tendency toward a poorer overall survival in thalamic gliomas containing the K27M mutation compared with wild-type tumors. The presence of the K27M mutation significantly corresponded with mutations in TP53 in thalamic gliomas. Interestingly, the K27M mutation was mutually exclusive with mutations in IDH1, which was detected only in brainstem gliomas. The microarray data identified 86 differentially expressed genes between brainstem and thalamic gliomas with the K27M mutation. The cyclin-dependent kinase 6 (CDK6) gene, which plays an important role in cancer pathways, was found to be differentially expressed between brainstem and thalamic gliomas with K27M mutations. Although the K27M mutation was frequently observed in adult brainstem and thalamic gliomas, this mutation tended to be associated with a poorer prognosis in brainstem gliomas but not in thalamic gliomas. Brainstem gliomas may present different genetic aberrations from thalamic gliomas. These differences may provide guidance for therapeutic decisions for the treatment of adult brainstem and thalamic gliomas, which may have different molecular targets.

Expression of β-catenin and E- and N-cadherin in human brainstem gliomas and clinicopathological correlations

Brainstem gliomas are usually associated with serious dysfunction and poor prognosis especially for diffuse intrinsic brainstem gliomas; however, the reasons are still unclear. Some clinical studies have suggested that the invasive ability may be different among brainstem gliomas, and the dysfunction of β-catenin and E- and N-cadherin appears to be connected with tumor invasion and progression. In this study, the expression of β-catenin and E- and N-cadherin was detected in 40 brainstem glioma samples using immunochemistry and was further analyzed in 18 samples using reverse transcription-polymerase chain reaction. The clinicopathological characteristics were also analyzed. The results show that there was no obvious staining for E-cadherin, but weak expression at the messenger RNA (mRNA) level could be seen in a few samples. The protein and mRNA expression levels of β-catenin and N-cadherin were significantly associated with the pathological grades of brainstem gliomas. No significant differences in the expression levels of β-catenin and N-cadherin were observed for age, sex, location or diffuse growing pattern. The overall survival of patients with low β-catenin expression was longer than that with high β-catenin expression, and there was a trend toward increased expression of N-cadherin with shorter survival; however, both of them had no statistical differences. These results demonstrate that expression of β-catenin and N-cadherin is associated with the malignant progression of brainstem gliomas but not correlated with the diffuse and invasive growing pattern. β-catenin and N-cadherin are potential therapeutic targets and prognostic markers for brainstem glioma, which need to be validated in a larger patient cohort.

Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy.

OBJECT Facial nerve injury results in facial palsy that has great impact on the psychosocial conditions of affected patients. Reconstruction of the facial nerve to restore facial symmetry and expression is still a significant surgical challenge. In this study, the authors assessed a hypoglossal-facial nerve anastomosis method combined with neurotrophic factor gene therapy to treat facial palsy in adult rats after facial nerve injury. METHODS Surgery consisted of the interposition of a predegenerated nerve graft (PNG) that was anastomosed with the hypoglossal and facial nerves at each of its extremities. The hypoglossal nerve was cut approximately 50% for this anastomosis to conserve partial hypoglossal function. Before their transplantation, the PNGs were genetically engineered using lentiviral vectors to induce overexpression of the neurotrophic factor neurotrophin-3 (NT-3) to improve axonal regrowth in the reconstructed nerve pathway. Reconstruction was performed after facial nerve injury, either immediately or after a delay of 9 weeks. The rats were followed up for 4 months postoperatively, and treatment outcomes were then assessed. RESULTS Compared with the functional innervation in control rats that underwent facial nerve injury without subsequent treatment, functional innervation of the paralyzed whisker pad by hypoglossal motoneurons in rats treated 4 months after nerve reconstruction was evidenced by the retrograde transport of neuronal tracers, the recording of muscle action potentials conducted by the PNG, and the recovery of facial symmetry. Although a better outcome was observed when reconstruction was performed immediately after facial nerve injury, reconstruction with NT3-treated PNGs significantly improved functional reinnervation of the paralyzed whisker pad even when implantation occurred 9 weeks posttrauma. CONCLUSIONS Results demonstrated that hypoglossal-facial nerve anastomosis facilitates innervation of paralyzed facial muscle via hypoglossal motoneurons without sacrificing ipsilateral hemitongue function. Neurotrophin-3 treatment through gene therapy could effectively improve such innervation, even after delayed reconstruction. These findings suggest that the combination of surgical reconstruction and NT-3 gene therapy is promising for its potential application in treating facial palsy in humans.

The Brachyury Gly177Asp SNP Is not Associated with a Risk of Skull Base Chordoma in the Chinese Population

A recent chordoma cancer genotyping study reveals that the rs2305089, a single nucleotide polymorphism (SNP) located in brachyury gene and a key gene in the development of notochord, is significantly associated with chordoma risk. The brachyury gene is believed to be one of the key genes involved in the pathogenesis of chordoma, a rare primary bone tumor originating along the spinal column or at the base of the skull. The association between the brachyury Gly177Asp single nucleotide polymorphism (SNP) and the risk of skull base chordoma in Chinese populations is currently unknown. We investigated the genotype distribution of this SNP in 65 skull-base chordoma cases and 120 healthy subjects. Comparisons of the genotype distributions and allele frequencies did not reveal any significant difference between the groups. Our data suggest that the brachyury Gly177Asp SNP is not involved in the risks of skull-base chordoma, at least in the Chinese population.

Brachyury: A sensitive marker, but not a prognostic factor, for skull base chordomas

Patients with skull base chordomas have a poor prognosis, and the role of the protein expression of brachyury in chordomas remains to be fully elucidated. The present study used immunohistochemistry to analyze 57 cases of skull base chordoma, and analyzed the clinical data of the patients. The results demonstrated that the protein expression of brachyury was negative in 8.8% (5/57) of the cases. The weak/positive, positive and strong/positive rates were 5.3% (3/5 7), 21.1% (12/57) and 64.9% (37/57), respectively. The association between the expression of brachyury and recurrence was not statistically significant. Kaplan-Meier analysis revealed that the degree of surgery, rather than the expression of brachyury, was associated with tumor recurrence (P=0.001). In conclusion, the results of the present study demonstrated that the expression of Brachyury offers a sensitive marker, but not a risk factor, for skull base chordomas, and radical surgery is recommended.

Hypoglossal–facial nerve ‘side’-to-side neurorrhaphy using a predegenerated nerve autograft for facial palsy after removal of acoustic tumours at the cerebellopontine angle

Trial design Hypoglossal–facial nerve (HN-FN) neurorrhaphy is a method commonly used to treat facial palsy when the proximal stump of the injured FN is unavailable. Since the classic HN-FN neurorrhaphy method that needs to section the injured FN is not suitable for incomplete facial palsy, we investigated a modified method that consists of HN-FN ‘side’-to-side neurorrhaphy, retaining the remaining or spontaneously regenerated FN axons while preserving hemihypoglossal function. Methods To improve axonal regeneration, we used for the first time a predegenerated sural autograft for performing HN–FN ‘side’-to-side neurorrhaphy followed by postoperative facial exercise. We treated 12 patients who had experienced FN injury for 1–18 months as a result of acoustic tumour removal. All patients experienced facial grade V–VI paralysis according to the House-Brackmann scale, but their FN was anatomically preserved. No spontaneous facial reinnervation was detected before repair. Results Although we did not perform fresh nerve grafts and HN–FN ‘side’-to-end neurorrhaphy as controls for ethical reasons, the reparative outcomes after nerve reconstruction were remarkable: functional improvements were detected as soon as 3 months after repair, House-Brackmann grade II or III FN functions were achieved in five and four patients, respectively, and there were no apparent signs of synkinesis. The three patients who experienced less satisfactory outcomes had exhibited facial palsy for more than 1 year accompanied by muscle atrophy, consistent with a need for rapid surgical intervention. Conclusions Based on fundamental concepts and our experimental results, this new surgical method represents a major advance in the rehabilitation of FN injury. Trial registration number JS2013-001-02.

Donor nerve axotomy and axonal regeneration after end-to-side neurorrhaphy in a rodent model

OBJECTIVEIn this study, the authors used a surgical model of end-to-side neurorrhaphy between a nerve graft and a donor tibial nerve in adult rats to investigate the optimal conditions for axonal regeneration induced by the donor nerve. They also assessed the importance of a more favorable pathway using a predegenerated nerve graft to attract regenerating axons to regrow into the graft and then directing and improving their growth toward the target in comparison with results obtained with a fresh nerve graft.METHODSEnd-to-side neurorrhaphy was performed between a nerve graft and a donor tibial nerve. The nerve graft was obtained from the left tibial nerve, which was either freshly removed or predegenerated 1 week prior to neurorrhaphy. The donor right tibial nerve was injured by epineurium removal alone, injured by epineurium removal with cross section of 20% or 50% of the total axons at the coaptation site, or left intact. The animals were followed postoperatively for a 6-week period, and outcomes were evaluated by optical microscopy and retrograde labeling to detect the regenerated primary sensory neurons located in the lumbar dorsal root ganglia and spinal motor neurons located in the lumbar spinal ventral horn.RESULTSAt the end of the follow-up period, no regenerating axons were observed in the nerve grafts when the donor nerve was left intact, and very few axons were detected when the donor nerve was injured by epineurium removal alone. However, numerous regenerating axons appeared in the grafts when the donor nerve was axotomized, and the greatest number was achieved with a 50% cross section axotomized nerve. In the rats with a 50% cross section of the donor nerve, better nerve-like morphology of the grafts was observed, without connective adhesions. When a predegenerated nerve graft was used, more regenerating axons were attracted and elongated with a more regular shape and improved myelination.CONCLUSIONSAxonal regrowth into a nerve graft depends on axotomy of the donor nerve after end-to-side neurorrhaphy. More efficient attraction and an improved structure of the regenerating axons were achieved when a predegenerated nerve graft was used. Furthermore, a nerve graft may require a certain number of regenerating axons to maintain a nerve-like morphology.

Time-course of Changes in Activation Among Facial Nerve Injury: A Functional Imaging Study.

AbstractPatients suffering different intervals of facial nerve injury were investigated by functional magnetic resonance imaging to study changes in activation within cortex.Forty-five patients were divided into 3 groups based on intervals of facial nerve injury. Another 16 age and sex-matched healthy participants were included as a control group. Patients and healthy participants underwent task functional magnetic resonance imaging (eye blinking and lip pursing) examination.Functional reorganization after facial nerve injury is dynamic and time-dependent. Correlation between activation in sensorimotor area and intervals of facial nerve injury was significant, with a Pearson correlation coefficient of −0.951 (P < 0.001) in the left sensorimotor area and a Pearson correlation coefficient of 0.333 (P = 0.025) in the right sensorimotor area.Increased activation in integration areas, such as supramarginal gyrus and precunes lobe, could be detected in the early-middle stage of facial dysfunction compared with normal individuals. Decreased activation in sensorimotor area contralateral to facial nerve injury could be found in late stage of facial dysfunction compared with normal individuals. Dysfunction in the facial nerve has devastating effects on the activity of sensorimotor areas, whereas enhanced intensity in the sensorimotor area ipsilateral to the facial nerve injury in middle stage of facial dysfunction suggests the possible involvement of interhemispheric reorganization. Behavioral or brain stimulation technique treatment in this stage could be applied to alter reorganization within sensorimotor area in the rehabilitation of facial function, monitoring of therapeutic efficacy, and improvement in therapeutic intervention along the course of recovery.

Differences between brainstem gliomas in juvenile and adult rats.

Clinical studies have shown that gliomas of the brainstem behave differently in children and adults. The aim of the present study was to compare and analyze the differences between these gliomas in juvenile and adult rats with regard to tumor growth, survival, pathology and magnetic resonance imaging (MRI). A total of 25 juvenile and 25 adult Wistar rats were divided into groups A (15 juvenile rats), B (10 juvenile rats), C (15 adult rats) and D (10 adult rats). The rats of groups A and C (experimental) were injected with glioma cells, while groups B and D (control) were injected with a physiological saline solution. Rat neurological signs, survival time, tumor size, hematoxylin and eosin (HE) staining and immunohistochemical staining for MMP-2, MMP-9 and β-catenin were compared. The survival time of group A was 19.47±2.232 days, whereas that of group C was 21.47±2.232 days (P<0.05). The tumor sizes were 4.55 and 4.62 mm (P>0.05) in groups A and C, respectively. HE and immunohistochemical staining revealed no differences between the groups. The results suggest that the growth patterns and invasiveness of brainstem gliomas may vary in children compared with adults due to the varied biological behaviors of the tumor cells.