Gamma Knife Radiosurgery of the Glomus Jugulare Tumour – Early Multicentre Experience

Leksell Gamma Knife was used to treat 66 patients with glomus jugulare tumour at 6 European sites between 1992-1998. The age of the patients ranged between 18-80 years (median 54 years). Gamma Knife radiosurgery was a primary treatment in 30 patients (45. 5%). Open surgery preceded radiosurgery in 24 patients (36.4%), embolisation in 14 patients (21.2%) and fractionated radiotherapy in 5 patients (7.6%). The volume of the tumour ranged 0.5-27 cm(3) (median 5,7 cm(3)). The minimal dose to the tumour margin ranged between 10-30 Gy (median 16.5 Gy). After radiosurgery 52 patients were followed, the follow up period was 3-70 months (median 24 months). Neurological deficit improved in 15 patients (29%) and deteriorated in 3 patients (5,8%), one transient and two persistant. Neuroradiological follow up using MRI or CT was performed in 47 patients 4-70 months (median 24 months) after radiosurgery. Tumour size decreased in 19 patients (40%) while in the remaining 28 patients (60%) no change in the tumour volume was observed. None of the tumours increased in volume during the observation period. Control angiography was performed in 6 patients. Pathological vascularisation completely disappeared in one patient, reduced in two and there was no change in the remaining three. Radiosurgery proves to be a safe treatment for glomus jugulare tumour with no mortality and no acute morbidity. Because of its naturally slow growth rate, up to 10 years of follow up will be necessary to establish a cure rate after radiosurgery for these lesions.     

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

QT dispersion and myocardial viability in patients after acute myocardial infarction

BACKGROUND: In recent years QT dispersion (QTd) in post-infarct patients was estimated in many studies, but still little is known about its association with the presence of dysfunctional but viable myocardium. AIM: We investigated the relation between dispersion of QT interval and myocardial viability in patients after acute myocardial infarction (AMI). MATERIAL AND METHODS: In 52 patients (mean age 67.2+/-11.7) treated thrombolytically because of AMI 12-lead ECG and low dose-high dose dobutamine echocardiography was performed on 14th day after treatment. QTd and regional myocardial contractility were estimated three times: at baseline, low dose dobutamine (LDD) (10-15 microg/kg per min) and high dose dobutamine (HDD) infusion (up to 40 microg/kg per min). RESULTS: Patients with viable myocardium had lower baseline QTd than patients with only necrosis in infarct zone. Significant increase in QTd was shown during LDD and HDD both in patients with and without myocardial viability. During infusion of HDD QTd was significantly higher in patients with myocardial ischemia. The greatest percentage increase of QTd at HDD was shown in patients with biphasic response to dobutamine infusion i.e. with myocardial viability evidenced at LDD and myocardial ischemia at HDD. CONCLUSION: Patients with preserved myocardial viability had lower QTd values compared to those with similar left ventricular dysfunction but caused only by post-infarction necrosis. Ischemia evoked on 14th day after AMI was accompanied by greater increase in QTd in patients with myocardial viability in infarct region than in patients without. It may be one of the reasons of greater risk of serious ventricular arrhythmias in such patients during myocardial ischemia.     

Air in the insufflation tube may cause fatal embolizations in laparoscopic surgery: an animal study

Background

The aim of this study was to evaluate the risk of an air embolization with the volume of the insufflation tube during induction of laparoscopy. A further objective was to determine the LD₅₀ of air in young piglets.

Methods

End-tidal carbon dioxide pressure ( $ P_{{{\text{CO}}_{2} ,{\text{et}}}} $ ), pulmonary arterial pressure (P _pa), heart rate (f _c), and mean arterial pressure (P _{a carot}) were measured in 17 piglets divided into three groups: group 1 (n = 6), bolus application (CO₂ embolization, followed by air embolization, 2 mL/kg each), group 2 (n = 7), continuous air embolization (30 min, 0.2 mL/kg/min), and group 3 (n = 4), continuous CO₂ embolization (30 min, 0.4 mL/kg/min).

Results

All animals survived CO₂ embolism. Air embolization as a bolus (2 mL/kg) or with an accumulated volume of 3.1 mL/kg led to death. Decreases in $ P_{{{\text{CO}}_{2} ,{\text{et}}}} $ indicated air or massive CO₂ embolization only. There was a good correlation between $ P_{{{\text{CO}}_{2} ,{\text{et}}}} $ and P _pa in case of air embolization (r = ?0.80, p < 0.0001). In contrast, no dependency was recognized during CO₂ embolism (r = ?0.17, p = 0.2).

Conclusions

In order to minimize the lethal risk of gas embolization, the insufflation system has to be completely filled with CO₂ before connecting to the patient.     

Functional outcome after gamma knife treatment in vestibular schwannoma

Radiosurgery (RS) is a noninvasive, ambulatory special neurosurgical procedure for the treatment of vestibular schwannoma (VS). We treated 123 patients with unilateral schwannomas between 1994 and 2000 at the gamma knife (GK) center in Munich using a primary stereotactic procedure. These patients were followed up until June 2004 in respect to audiological, neurological, neurootological and radiological features before and after radiosurgical intervention. The actual tumor control rate of 8.2 years (mean) after GK surgery for all patients and a single treatment was calculated to be 96.7%. The impairment of hearing was on average 18% after GK, ranking from 0% gain of hearing loss up to 90%. Facial nerve function, graded according to the House–Brackmann scale, deteriorated in none of the patients; 5.8% reported a trigeminal neuralgia. Tinnitus developed in 4.1% of the patients after RS; 13.3% had vertigo for the first time after the treatment, age apparently being a predisposing factor. Radiosurgical treatment for VS is an alternative to microsurgery (MS). It is associated with a lower rate of facial and trigeminal neuropathy, postoperative complications and hospital stay. The hearing preservation rate is equivalent to MS.     

Quality assurance in stereotactic radiosurgery/radiotherapy according to DIN 6875-1

The new DIN ('Deutsche Industrie-Norm') 6875-1, which is currently being finalised, deals with quality assurance (QA) criteria and tests methods for linear accelerator and Gamma Knife stereotactic radiosurgery/radiotherapy including treatment planning, stereotactic frame and stereotactic imaging and a system test to check the whole chain of uncertainties. Our existing QA program, based on dedicated phantoms and test procedures, has been refined to fulfill the demands of this new DIN. The radiological and mechanical isocentre corresponded within 0.2 mm and the measured 50% isodose lines were in agreement with the calculated ones within less than 0.5 mm. The measured absorbed dose was within 3%. The resultant output factors measured for the 14-, 8- and 4-mm collimator helmet were 0.9870 +/- 0.0086, 0.9578 +/- 0.0057 and 0.8741 +/- 0.0202, respectively. For 170 consecutive tests, the mean geometrical accuracy was 0.48 +/- 0.23 mm. Besides QA phantoms and analysis software developed in-house, the use of commercially available tools facilitated the QA according to the DIN 6875-1 with which our results complied.     

A technical triade for proteomic identification and characterization of cancer biomarkers

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.     

Stereotactic radiosurgery using the Gamma Knife for large uveal melanomas

BACKGROUND: We report the results over 3 years with stereotactic radiosurgery using the Gamma Knife for large and unsuitably located uveal melanomas. PATIENTS AND METHODS: A total of 100 patients (51 male, 49 female) have been treated since 1997 following a standardised treatment protocol (outpatient single-shot treatment, maximum dose 50 Gy, tumour margin dose min.25 Gy, retrobulbar anaesthesia alone for globe fixation). The localisation and/or dimension of the tumours did not allow radiation brachytherapy with Ru106 plaques. Of the tumours 18 were located in the ciliary body, 61 were located at the posterior pole, and 21 were located in the mid-periphery. All patients were followed and tested ophthalmologically and neuroradiologically at regular intervals. The 1-year follow-up data were available for 73 patients, 2-year follow-up data for 33 patients and 3-year follow-up-data for 17 patients. RESULTS: Before therapy the maximum apical tumour height (MAH) was median 7.8 mm (95% CI 2.9-12.5 mm): 1 year after treatment (73 patients) the MAH was median 5.7 mm (95% KI 2.4-10.2 mm),2 years after treatment (33 patients) the MAH was median 4.3 mm (95% KI 2.2-8.8 mm),and 3 years after treatment (17 patients) the MAH was median 4.6 mm (95% KI 2.4-8.5 mm). All differences to the MAH of the corresponding patients before treatment were statistically significant (paired t-test). Within the first year after treatment seven patients were enucleated due to a painful secondary glaucoma,within the second year after radiation two patients (one tumour recurrence, and one secondary glaucoma) and within the third year one more patient (tumour recurrence) was enucleated. CONCLUSIONS: Our 3-year results demonstrate that radiosurgery using the Gamma Knife is beneficial in achieving a local tumour control in 98% of eyes with large and unsuitably located uveal melanomas. The risk for a secondary enucleation is highest in the first year after treatment with a favourable overall rate of 10%. Due to the excellent local tumour control rate we decreased the maximum dose to 40 Gy (min.tumour margin dose 20 Gy) in the subsequently treated patients.     

Radiotherapy treatment planning for small complex-shaped lesions and its experimental verification

The Gamma Knife is used as a stereotactic tool for the conformal treatment of very small, complex-shape cranial lesions. The combination of planning software and treatment equipment enables a highly-precise conformal dose distribution and positioning. The purpose of the present study was to experimentally verify the precision actually achievable in case of extremely irregular, small target volumes. For this purpose, a complete treatment procedure was performed using a standard head phantom complemented with a specially developed insert that simulates an L-shaped lesion. The spatial precision of the irradiation was recorded by means of high-resolution film dosimetry using GafChromic films. The analysis of the films showed for the film in the center plane an excellent conformity of the 75% isodose line used to circumscribe the lesion. A very good agreement between planning and measurement resulted also for isodose lines residing outside of the target volume.