Chromosome breakpoints near CpG islands in double minutes.

Double minute chromosomes (DMs) are the principal genetic vehicles for amplifying oncogenes in human tumors and drug resistance genes in cultured mouse cells. Mouse EMT-6 cells resistant to methotrexate (MTX) generally contain circular DMs, approximately 1 megabase (Mb) in size, that amplify the dihydrofolate reductase (DHFR) gene. The 1 Mb DMs generally have CpG islands located 500 kb upstream of the DHFR gene. The purpose of this study was to determine the relationship between CpG islands and chromosomal breakpoints giving rise to the DM. We show that EMT-6 cells growing in very low levels of MTX that do not yet contain the 1 Mb DHFR-amplifying DM, develop a NotI/EagI site 500 kb upstream of the DHFR gene. This NotI site is close to, if not identical with, one of the chromosomal breakpoints giving rise to the DM. We show that 500 kb of DM DNA from upstream of the DHFR gene is derived from 500 kb of chromosomal DNA upstream of the chromosomal DHFR gene. The downstream breakpoint maps to a region approximately 200 kb downstream of the DHFR gene near a chromosomal SstII/EagI site. Therefore, approximately 700 kb of DM DNA was derived from the genomic region surrounding the DHFR gene. To confirm the organization of the DM DNA, we isolated DNA probes from the 1 Mb DM. Using pulsed field gel electrophoresis and Southern hybridization, we determined the approximate location of each probe with respect to the CpG island in both the DM and the chromosome. Approximately 300 kb of chimeric DNA from a region unrelated to the DHFR gene was incorporated during DM formation. Implications for the mechanism of DM formation are discussed.

An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells.

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

Paradoxical ventricular enlargement after successful shunting.

We present two cases of ventricular obstruction in which shunting of intracranial cysts resulted in the unexpected finding of ventricular enlargement. By imaging criteria this suggested shunt malfunction [corrected]; however, the clinical presentation dramatically improved. The probable mechanism for this diagnostic pitfall is discussed.

Cystic meningiomas: MR characteristics and surgical correlations.

PURPOSE: To describe the MR appearance of cystic meningiomas, and to correlate the MR appearance with the surgical and neuropathologic findings. METHODS: Eight patients with cysts associated with meningiomas were studied on a 1.5-T MR system. Unenhanced sagittal T1- and axial T2-weighted images were obtained in all patients. Axial and coronal gadopentetate dimeglumine-enhanced T1-weighted spin-echo images were obtained in seven patients. Additional sagittal T1-weighted spin-echo contrast-enhanced images were obtained in four patients. RESULTS: The cystic components were intratumoral and eccentric in two cases, intraparenchymal in one case, and extraparenchymal (trapped cerebrospinal fluid) in five cases. Cyst wall enhancement was present in two of seven cases performed with intravenous gadopentetate dimeglumine. There was no correlation between cyst signal intensity and cyst content. A preoperative diagnosis of cystic meningioma was possible in all eight cases. CONCLUSIONS: MR demonstrates the extradural location of the tumor and its cystic component, correlates well with the surgical presentation and the neuropathologic results, and allows the preoperative diagnosis of cystic meningioma based on the MR findings. Division into three types of cysts aids the neurosurgeon, who must decide whether total resection is feasible. To obtain total resection and reduce the risk of recurrence with an intratumoral cyst, the surgeon must ensure that the plane of resection is in fact between the thin enhancing membrane of the tumor cyst and the adjacent arachnoid. In cases in which the cyst is trapped cerebrospinal fluid or intraparenchymal in location, the cyst wall adjacent to or within the brain parenchyma is not included in the resection.

Gangliocytoma of the cervicothoracic spinal cord.

We present a case of an extensive cervicothoracic gangliocytoma in an asymptomatic 9-year-old boy with progressive scoliosis. MR findings were that of a moderately enhancing cervicothoracic intramedullary mass, which throughout most of its length could not be distinguished from the normal spinal cord.

Oscillopsia and primary cerebellar ectopia: case report and review of the literature.

Oscillopsia, the visual sensation of stationary objects swaying back and forth or vibrating, has been described in association with downbeat nystagmus in patients with primary cerebellar ectopia (Chiari I malformation). A patient with symptomatic oscillopsia without downbeat nystagmus, who was diagnosed by magnetic resonance imaging to have displaced cerebellar tonsils below the foramen magnum, is presented here. Suboccipital craniectomy and upper cervical laminectomy completely relieved the visual disturbance of the patient. The pathogenesis and surgical management of oscillopsia are discussed with respect to the current literature. Early recognition and surgical management of oscillopsia are lead to complete recovery in patients with minimal symptomatology.

Duration of intracranial pressure monitoring does not predict daily risk of infectious complications.

A group of 205 patients (115 children and 90 adults) with a total of 212 intracranial pressure (ICP) monitors were retrospectively studied with attention to daily cerebrospinal fluid cultures, duration of monitoring, associated cranial injuries, and hospital site of the ICP monitor (intensive care unit or operating room). Only closed ICP monitoring systems without irrigation or compliance testing were used, and all patients received antibiotics as prophylaxis throughout the monitoring period. There were no complications associated with monitor placement. Incidence histograms and regression analysis were used to determine the daily risk of subsequent infections, in addition to evaluating the cumulative risk of infection, as has been previously described in the literature. No relation between the duration of ICP monitoring and the rate of daily infection through the period of maximal monitoring (1-2 weeks) was found in this series. The overall incidence of infection was 7.1% with a median duration of monitoring of 7.2 days. The age of the patient (adult vs. child), site of ICP monitor placement, and nature of the underlying disease (trauma vs. nontrauma) had no significant effect on the development of monitor-related infections in our study. These data indicate that the decision to continue ICP monitoring can be based solely on the clinical necessity for further monitoring rather than on concerns for monitor removal to prevent infection.

Hydroxyurea accelerates the loss of epidermal growth factor receptor genes amplified as double-minute chromosomes in human glioblastoma multiforme.

OBJECTIVE: We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions. METHODS: Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes. RESULTS: Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion. CONCLUSION: These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

OBJECTIVE: We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. METHODS: Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. CONCLUSION: The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.

Early clinical experience with the Syracuse I-Plate: an anterior spinal fixation device.

Sixteen patients were treated with a new anterior internal fixation device after thoracolumbar or lumbar decompression, and fusion with bone grafting. Ten patients had acute burst fractures, four had metastatic tumors, and two had old, healed fractures with deformity. In the acute fracture group, eight patients had neurologic deficits and seven patients experienced improvement. Six patients had lesions of the conus medullaris, all of which improved. The four patients with metastatic tumors underwent surgery for back and leg pain and all gained significant relief. Two patients had correction of old fracture deformity with satisfactory outcome. Complications were minimal. The new anterior stabilization device provided early stability, allowed early patient mobilization, was easy to insert, and has a low profile. Late collapse, non-union, and kyphotic deformity have not been noted thusfar.

Traumatic internal carotid artery aneurysm with rupture into the sphenoid sinus.

A case of cavernous-internal carotid artery aneurysm with delayed rupture into the sphenoid sinus is presented, and the literature reviewed. We stress important diagnostic features such as epistaxis, which may require emergent use of epistaxis balloon catheters. Cervical carotid ligation and intracranial clipping of the internal carotid artery proximal to the ophthalmic artery controlled the bleeding and resulted in no neurologic sequelae.

Cerebral venous thrombosis. Demonstration with magnetic resonance angiography.

Three patients with cerebral venous thrombosis (CVT) were imaged with magnetic resonance angiography (MRA). Initial spin-echo magnetic resonance (MR) demonstrated acute or subacute thrombosis in all patients. The presence of thrombosis was confirmed with MRA. Repeat MRA in the three patients revealed partial recanalization in one and almost complete recanalization in two patients. The etiology of CVT is reviewed, and the advantages of MRA are compared with conventional MR imaging and computed tomography in the evaluation of CVT.

A rare case of cerebellar glioblastoma multiforme in childhood: MR imaging.

Glioblastoma multiforme is a highly malignant brain neoplasm that is very rarely discovered in childhood but accounts for approximately 17% of intracranial tumors in the adult. Only approximately 25 children with glioblastoma multiforme in the cerebellum have been described in the literature. We report on a 4 1/2-year-old girl with this tumor in the cerebellar hemisphere and discuss the magnetic resonance imaging findings.

A simple approach to the technical aspects of radiosurgery treatments.

An approach to radiosurgery treatment that can be readily adopted in most radiotherapy centers with linear accelerators is presented. In our institution, a Leksell-type of neurosurgical frame, a computed tomography scanner, locally fabricated cones, and 6 MV X-ray beams are used to perform radiosurgery treatments. Collimated arcs with dose distributions, that conform to the shape of the lesion in the transverse and the sagittal planes are used. It is argued that the uncertainties in the localization of the isocenter within a lesion and the specifications of the size of the target volume do not justify high precision mechanical devices for most radiosurgery treatments.