Giuseppe Cinalli

MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

Background Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (Pu200a=u200a0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Conclusions/Significance Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

Chiari malformation in craniosynostosis

IntroductionChiari malformation (CM) is a frequent finding in multisutural and syndromic craniosynostosis, occurring in 70% of patients with Crouzon’s syndrome, 75% with oxycephaly, 50% with Pfeiffer’s syndrome and 100% with the Kleeblattschädel deformity. The pathogenesis of this condition and rationale for treatment are still controversial.DiscussionSince its first description in 1972, several factors have been cited to play a role in inducing CM. In the light of recent publications, the roles of premature fusion of cranial vault and cranial base sutures, of congenital anomalies of the cerebellum and brain stem, of raised intracranial pressure, of venous hypertension and of hydrocephalus are reviewed. Evaluation and management of CM are also discussed.ConclusionChiari malformation appears to be an acquired and progressive condition that develops in the first months of life, because of a disproportion between hindbrain growth and an abnormally small posterior fossa, a consequence of the premature fusion of lambdoid and cranial base sutures. Venous hypertension caused by stenosis of the jugular foramen can also be present in these patients, resulting in intracranial hypertension and/or hydrocephalus. Careful MRI evaluation is recommended for the forms of craniosynostosis at a high risk of developing hindbrain herniation. The selection of posterior cranial vault expansion as the first surgical procedure is advocated. In selected cases, treatment of the posterior cranial deformity by occipital vault remodelling and treatment of the Chiari-like deformity by suboccipital decompression can be carried out using the same surgical procedure.

Application of neuroendoscopy to intraventricular lesions.

We present an overview of the history, development, technological advancements, current application, and future trends of cranial endoscopy. Neuroendoscopy provides a safe and effective management modality for the treatment of a variety of intracranial disorders, either tumoral or non-tumoral, congenital, developmental, and degenerative, and its knowledge, indications, and limits are fundamental for the armamentarium of the modern neurosurgeon.

MiR-34a Targeting of Notch Ligand Delta-Like 1 Impairs CD15+/CD133+ Tumor-Propagating Cells and Supports Neural Differentiation in Medulloblastoma

Background Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133+/CD15+ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1+/- p53-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. Conclusions/Significance Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSEnMedulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication.nnnPATIENTS AND METHODSnMolecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models.nnnRESULTSnSubgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas.nnnCONCLUSIONnCombining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

Endoscopic third ventriculostomy in the treatment of hydrocephalus in posterior fossa tumors in children

ObjectThe purpose of the present study is to assess the effectiveness of endoscopic third ventriculostomy (ETV) in children with hydrocephalus related to posterior fossa tumors.MethodsBetween September 1999 and December 2002, 63 children with posterior fossa tumors were treated at Santobono Hospital in Naples, Italy. Twenty-six patients had severe hydrocephalus. In order to relieve intracranial hypertension before tumor removal, 20 were treated with ETV, and 6 with ventriculo-peritoneal (VP) shunts. Twenty patients with mild hydrocephalus were treated with diuretics, corticosteroid agents, and early posterior fossa surgery, and 17 patients who did not have hydrocephalus were treated by elective posterior fossa surgery. Another 4 ETV were performed in the management of postoperative hydrocephalus.ResultsPreoperative ETV procedures were technically successful. One was complicated by intraventricular bleeding. The successful 19 preoperative ETV resolved intracranial hypertension before posterior fossa surgery in all cases. Three of these 19 patients developed postoperative hydrocephalus and were treated by VP shunt insertion after posterior fossa surgery. Out of the 4 ETV performed after posterior fossa surgery, only 2 were successful, both when the shunt malfunctioned.ConclusionsEndoscopic third ventriculostomy should be considered as an alternative procedure to ventriculo-peritoneal shunting and external ventricular draining for the emergency control of severe hydrocephalus caused by posterior fossa tumors, since it can quickly eliminate symptoms, and hence, can delay surgery scheduling if required. Even though ETV does not prevent postoperative hydrocephalus in all cases, it does protect against acute postoperative hydrocephalus due to cerebellar swelling. In addition, it eliminates the risks of cerebrospinal fluid (CSF) infection related to external drainage and minimizes the risk of overdrainage because it provides more physiological CSF drainage than the other procedures. Since postoperative hydrocephalus is very often physically obstructive, ETV should always be considered a possible treatment procedure.

Complications following endoscopic intracranial procedures in children.

BackgroundThe significant technological improvement of endoscopic instrumentation has allowed, in the last 10xa0years, a widespread diffusion of neuroendoscopic procedures. Nevertheless, severe, sometimes life-threatening, complications may occur during neuroendoscopic surgery, and the incidence and age specificity of complications in children have been underdescribed so far.Materials and methodsComplications recorded in a prospectively collected database of pediatric patients undergoing neuroendoscopic procedures were analysed; the medical histories of the patients and the surgical procedures were reviewed.ResultsComplications occurred in 32 out of 231 (13.8%) procedures performed for the management of obstructive hydrocephalus (137), multiloculated hydrocephalus (53), arachnoid cysts (29) and intraventricular tumors (12). Subdural hygroma occurred in 11 cases, seven requiring subdural shunting. In one of these cases, infection of the subdural space occurred and required a craniotomy. Cerebrospinal fluid (CSF) infection occurred in 11 cases. In one case, a frontal abscess developed and was managed with craniotomy. CSF leak occurred in nine cases, intraventricular haemorrhages in two, technical failures in seven, subcutaneous CSF collection (managed with lumbo-peritoneal shunt) in one, thalamic contusion and post-operative transient akinetic mutism in one. This patient suddenly died 6xa0months later, probably as a consequence of closure of the stoma. Two patients developed secondary compartmentalisation of the ventricles after complicated endoscopic third ventriculostomy. In nine cases, these complications were associated. Overall, no patient died after the procedure (operative mortality 0), one patient died 6xa0months after the procedure for unexplained events (sudden death rate 0.4%), and three patients presented permanent disability as a consequence of surgical complication (permanent morbidity 1.3%).ConclusionsComplication rate of neuro-endoscopic procedures is not negligible even in experienced hands. The majority are minor complications which do not affect the final outcome, but sporadically major events may occur, leading to significant problems in surgical management and, occasionally, to permanent disabilities. Careful selection of patients on pre-operative imaging studies and intensive training of surgeons are mandatory to improve results.

Current state and future development of intracranial neuroendoscopic surgery

Since the introduction of the modern, smaller endoscopes in the 1960s, neuroendoscopy has become an expanding field of neurosurgery. Neuroendoscopy reflects the tendency of modern neurosurgery to aim towards minimalism; that is, access and visualization through the narrowest practical corridor and maximum effective action at the target point with minimal disruption of normal tissue. Transventricular neuroendoscopy allows the treatment of several pathologies inside the ventricular system, such as obstructive hydrocephalus and intra-/paraventricular tumors or cysts, often avoiding the implantation of extracranial shunts or more invasive craniotomic approaches. Endoscopic endonasal transphenoidal surgery allows the treatment of pathologies of the sellar and parasellar region, with the advantage of a wider vision of the surgical field, less traumatism of the nasal structures, greater facility in the treatment of possible recurrences and reduced complications. However, an endoscope may be used to assist microsurgery in virtually any kind of neurosurgical procedures (endoscope-assisted microsurgery), particularly in aneurysm and tumor surgery. Basic principles of optical imaging and the physics of optic fibers are discussed, focusing on the neuroendoscope. The three main chapters of neuroendoscopy (transventricular, endonasal transphenoidal and endoscope-assisted microsurgery) are reviewed, concerning operative instruments, surgical procedures, main indications and results.

The role of Ommaya reservoir and endoscopic third ventriculostomy in the management of post-hemorrhagic hydrocephalus of prematurity

ObjectiveThe aim of this study is to retrospectively evaluate a series of consecutive patients affected by post-hemorrhagic hydrocephalus in prematurity, treated with an implant of an Ommaya reservoir followed by ventriculo-peritoneal (VP) shunt and/or endoscopic third ventriculostomy (ETV) to evaluate the safety and efficacy of these treatment options in the management of the condition.MethodsBetween 2002 and 2005, 18 consecutive premature patients affected by intra-ventricular haemorrhage (IVH) grades II to IV, presenting with progressive ventricular dilatation, were operated for implant of an intra-ventricular catheter connected to a sub-cutaneous Ommaya reservoir. Cerebrospinal fluid was intermittently aspirated percutaneously by the reservoir according with the clinical requirements and the echographic follow-up. The patients who presented a progression of the ventricular dilatation were finally operated for VP shunt implant or ETV according with the MRI findings.ResultsOne patient had grade II, 5 had grade III, and 12 had grade IV IVH. The mean age at IVH diagnosis was 5.2xa0days; the mean age at reservoir implant was 17.3xa0days. The Ommaya reservoir was punctured on an average basis of 11.4 times per patient (range 2–25), and the mean interval between aspirations was 2.7xa0days. The mean CSF volume per tap was 20xa0ml. One patient died for pulmonary complications during the study period. Out of the 17 survivors, 3 did not develop progressive ventricular dilatation, and their reservoir was removed; 14 developed progressive hydrocephalus, 5 of whom were implanted with a VP shunt and 9 received an ETV. Amongst the five shunted patients, two were re-admitted for shunt malfunction and had their shunt removed after ETV after 6.1 and 20.5xa0months, respectively. Amongst the nine patients who received an ETV, five had to be re-operated for VP shunt implant at an average interval of 2.17xa0months (range 9–172xa0days) because of increasing ventricular dilatation. Two of them had a redo third ventriculostomy with shunt removal at 11 and 25.1xa0months, respectively, after insertion. The first was reimplanted with a VP shunt 4xa0days later; the second remains shunt free. Therefore, at the end of the follow-up period, 10 out of 17 children affected by post-hemorrhagic hydrocephalus in prematurity were shunt free (59%).ConclusionsThe combination of Ommaya reservoir, VP shunt, and the aggressive use of ETV as a primary treatment or as an alternative to shunt revision allowed for a significant reduction of shunt dependency in a traditionally shunt-dependent population. Further studies are warranted to optimise the algorithm of treatment in these patients.

Intracranial pressure monitoring and lumbar puncture after endoscopic third ventriculostomy in children.

OBJECTIVE:The aim of this study is to analyze changes in intracranial pressure (ICP) after endoscopic third ventriculostomy (ETV) performed in children affected by noncommunicating hydrocephalus. METHODS:ICP was continuously recorded for an average of 7 days in 64 children who underwent 68 ETVs for obstructive triventricular hydrocephalus of various etiology. In the first group (44 children), ETV was performed as the primary treatment; in the second group (20 children), the patients presented with shunt malfunction and underwent ETV and shunt removal. Three of the patients in the second group were reoperated for obstruction of the stoma: two were reoperated once and one was reoperated twice. RESULTS:ICP changes after ETV were not homogeneous and varied according to etiology: the highest values were observed in patients affected by posterior fossa tumors and the lowest values were seen in patients operated on during shunt malfunction and who had their shunt removed. After 31 procedures (45.6%), ICP remained normal (<20 mmHg) for the entire duration of the monitoring. After 37 procedures (54.5%), ICP was persistently high on Day 1 (mean, 29.7) and decreased very slowly in the subsequent days, remaining high for 2–9 days (mean, 4.5). After 20 of the 37 procedures with high postoperative ICP, patients presented symptoms of intracranial hypertension that resolved, in most of the cases, with one or two lumbar punctures. Lumbar puncture was noted to be effective in bringing about fast normalization of the ICP and resolution of the symptoms. In 13 patients (19.1%), ETV failed and a ventriculoperitoneal shunt was implanted. After four procedures, the stoma obstructed and the patients were treated, reopening the stoma. Postoperative ICP was not statistically significant higher in the patients in whom ETV failed. CONCLUSION:The high ICP observed in a group of patients in the early postoperative days is probably related to the slow permeation of the subarachnoid spaces by the cerebrospinal fluid flowing out of the third ventriculostomy. Management of intracranial hypertension after ETV remains a matter of controversy. The role of the lumbar puncture in the faster normalization of the ICP is examined in this article. By increasing the compliance and the buffering capacities of the spinal subarachnoid spaces, it probably decreases the cerebrospinal fluid outflow resistance from the ventricular system, facilitating the decrease of the ventricular volume and allowing faster permeation of the intracranial subarachnoid spaces. High postoperative ICP can account for persistent symptoms of intracranial hypertension and ventricular dilatation on computed tomographic scans after third ventriculostomy. A cycle of one to three lumbar punctures should always be performed in patients who remain symptomatic and who show increasing ventricular dilatation after ETV, before ETV is assumed to have failed and an extracranial cerebrospinal fluid shunt is implanted.