Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma.

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.

External validation of the Chicago Chiari Outcome Scale in adults with Chiari malformation type I.

OBJECTIVE: The Chicago Chiari Outcome Scale (CCOS) serves as a standardized clinical outcome evaluation tool among patients with Chiari malformation type I (CM-I). While the reliability of this scale has been proven for pediatric patients, the literature lacks CCOS validation when used solely in adults. Therefore, this study aimed to determine the validity of the CCOS in an external cohort of adult patients. METHODS: The authors retrospectively analyzed the medical records of symptomatic patients with CM-I who underwent posterior fossa decompression between 2010 and 2018 in six neurosurgical departments. Each patient was clinically assessed at the latest available follow-up. Gestalt outcome was determined as improved, unchanged, or worsened compared with the preoperative clinical state. Additionally, the CCOS score was calculated for each patient based on the detailed clinical data. To verify the ability of the CCOS to determine clinical improvement, the area under the receiver operating characteristic (AUROC) curve was evaluated. A logistic regression analysis using all four components of the CCOS (pain symptoms, nonpain symptoms, functionality, and complications) was performed to establish predictors of the improved outcome. RESULTS: Seventy-five individuals with a mean age of 42 ± 15.32 years were included in the study. The mean follow-up duration was 52 ± 33.83 months. Considering gestalt outcome evaluation, 41 patients (54.7%) were classified as improved, 24 (32%) as unchanged, and 10 (13.3%) as worsened. All patients with a CCOS score of 14 or higher improved, while all those with a CCOS score of 8 or lower worsened. The AUROC was 0.986, suggesting almost perfect accuracy of the CCOS in delineating clinical improvement. A CCOS score of 13 showed high sensitivity (0.93) and specificity (0.97) for identifying patients with clinical improvement. Additionally, a meaningful correlation was found between higher CCOS scores in each component and better outcomes. Patient stratification by total CCOS score showed that those categorized as improved, unchanged, and worsened scored prevalently between 13 and 16 points, 10 and 12 points, and 4 and 9 points, respectively. CONCLUSIONS: In this adult cohort, the CCOS was found to be almost perfectly accurate in reflecting postoperative clinical improvement. Moreover, all four CCOS components (pain symptoms, nonpain symptoms, functionality, and complications) significantly correlated with patient clinical outcomes.

Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy.

BACKGROUND: Surgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients' outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response. METHODS: EGFR-specific affibody molecule (ZEGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used. RESULTS: In vitro findings confirmed the ability of ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours. CONCLUSIONS: Our data underline the potential of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.

Wall shear stress gradient is independently associated with middle cerebral artery aneurysm development: a case-control CFD patient-specific study based on 77 patients.

BACKGROUND: Previously published computational fluid dynamics (CFD) studies regarding intracranial aneurysm (IA) formation present conflicting results. Our study analysed the involvement of the combination of high wall shear stress (WSS) and a positive WSS gradient (WSSG) in IA formation. METHODS: We designed a case-control study with a selection of 38 patients with an unruptured middle cerebral artery (MCA) aneurysm and 39 non-aneurysmal controls to determine the involvement of WSS, oscillatory shear index (OSI), the WSSG and its absolute value (absWSSG) in aneurysm formation based on patient-specific CFD simulations using velocity profiles obtained from transcranial colour-coded sonography. RESULTS: Among the analysed parameters, only the WSSG had significantly higher values compared to the controls (11.05 vs - 14.76 [Pa/mm], P = 0.020). The WSS, absWSSG and OSI values were not significantly different between the analysed groups. Logistic regression analysis identified WSS and WSSG as significant co-predictors for MCA aneurysm formation, but only the WSSG turned out to be a significant independent prognosticator (OR: 1.009; 95% CI: 1.001-1.017; P = 0.025). Significantly more patients (23/38) in the case group had haemodynamic regions of high WSS combined with a positive WSSG near the bifurcation apex, while in the control group, high WSS was usually accompanied by a negative WSSG (14/39). From the analysis of the ROC curve for WSSG, the area under the curve (AUC) was 0.654, with the optimal cut-off value -0.37 Pa/mm. The largest AUC was recognised for combined WSS and WSSG (AUC = 0.671). Our data confirmed that aneurysms tend to form near the bifurcation apices in regions of high WSS values accompanied by positive WSSG. CONCLUSIONS: The development of IAs is determined by an independent effect of haemodynamic factors. High WSS impacts MCA aneurysm formation, while a positive WSSG mainly promotes this process.

Near-infrared photoimmunotherapy targeting EGFR-Shedding new light on glioblastoma treatment.

Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12-15 months following standard therapy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR-specific affibody (ZEGFR:03115 ) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near-infrared light produces a cytotoxic response. ZEGFR:03115 -IR700DX EGFR-specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter-Glo® assay, and in vivo using subcutaneous U87-MGvIII xenografts. In addition, mice were imaged pre- and post-PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor-dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the ZEGFR:03115 -IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof-of-concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.

Morphological, hemodynamic, and clinical independent risk factors for anterior communicating artery aneurysms.

BACKGROUND AND PURPOSE: The pathogenesis of cerebral aneurysms still raises some controversies. The aim of this study was to identify morphological, hemodynamic, and clinical independent risk factors for anterior communicating artery (ACoA) aneurysm development. METHODS: Computed tomography angiography and transcranial color-coded sonography were performed in 77 patients with a nonbleeding ACoA aneurysm and in 73 controls. Symmetry of A1 segments of the anterior cerebral arteries, angles between A1 and A2 segments, tortuosity, diameter, mean velocity (Vm), pulsatility index, and volume flow rate in both A1 segments were determined. Moreover, all study participants completed a survey on their medical history. Multivariate backward stepwise logistic regression analysis was performed to identify independent risk factors for ACoA aneurysm development. RESULTS: Smoking, hypertension, asymmetry of A1 segments, the angle between A1 and A2 segments, A1 segment diameter, Vm, pulsatility index, and volume flow rate turned out to be associated with the occurrence of ACoA aneurysms on univariate analysis. Multivariate analysis identified smoking (odds ratio, 2.036; 95% confidence interval, 1.277-3.245), asymmetry of A1 segments>40% (odds ratio, 2.524; 95% confidence interval, 1.275-4.996), pulsatility index (odds ratio, 0.004; 95% confidence interval, 0.000-0.124), and the angle between A1 and A2 segments≤100° (odds ratio, 4.665; 95% confidence interval, 2.247-9.687) as independent strong risk factors for ACoA aneurysm development. CONCLUSIONS: The risk of ACoA aneurysm formation is determined by several independent clinical, morphological, and hemodynamic factors. The strongest independent risk factors include smoking, asymmetry of A1 segments>40%, low blood flow pulsatility, and the angle between A1 and A2 segments≤100°.

Type I atlanto-occipital dislocation complicated by non-communicating hydrocephalus - A case report.

Hydrocephalus, an extremely rare complication of craniocervical junction injuries, is postulated to result from compression of the fourth ventricular cerebrospinal fluid (CSF) outlets by fractured and displaced bone fragments, a swollen upper spinal cord or adhesions formed after a traumatic subarachnoid haemorrhage. We present the case of a 21-year-old woman for whom an injury to the cervical spine complicated by a type I atlanto-occipital dislocation contributed to the development of non-communicating hydrocephalus. The hydrocephalus was probably a consequence of impaired CSF circulation at the fourth ventricular outlets (the foramina of Luschka and Magendie), caused by post-haemorrhagic adhesions formed after severe injury to the craniocervical junction.

Kinin Receptors and Kinin-Related Gene Expression in Astrocytic Brain Tumors.

Kinins are a set of peptides present in tissues that are involved in the inflammatory response and cancer progression. However, studies showing the expression of kinin receptors in human glioma samples are still incomplete and contradictory. The aim of the present study was to ascertain the expression of BDKRB1 and BDKRB2 genes, as well as the level of B1R and B2R proteins in human gliomas, depending on the degree of malignancy. Additionally, representative kinin-dependent genes with altered expression were indicated. The expression profile of kinin-dependent genes was determined using oligonucleotide microarray technique. In addition, RT-qPCR was used to assess the expression level of selected differentiating genes. The location of kinin receptors in brain gliomas was assessed using immunohistochemical methods. The oligonucleotide microarray method was used to identify 12 mRNA IDs of kinin-related genes whose expression was upregulated or downregulated in gliomas of different grades. In immunohistochemically stained samples, the concentrations of BR1 and BR2 proteins, measured by optical density, were statistically significantly higher in grade G3 vs. G2 and G4 vs. G3. Increased expression of kinin receptors BDKRB1 and BDKRB2 in brain gliomas, depending on the degree of malignancy, suggests the involvement of kinins and their receptors in the disease's pathogenesis. Quantitative assessment of mRNA BDKRB1, PRKAR1A, MAP2K, and EGFR in patients with brain tumors may hold diagnostic and therapeutic significance.

Reoperations of patients with low-grade gliomas in eloquent or near eloquent brain areas.

BACKGROUND AND PURPOSE: Reoperations of patients with recurrent low-grade gliomas (LGG) are not always recommended due to a higher risk of neurological deficits when compared to initial surgery. The purpose of the present study was to evaluate surgical outcomes of patients operated on for recurrent LGG. MATERIAL AND METHODS: Sixteen patients who had surgery for recurrent LGG out of 68 LGG patients who underwent surgery at the Department of Neurosurgery in Sosnowiec, Poland between 2005 and 2011 were enrolled in the study. RESULTS: A large tumour volume prior to the initial surgery was the most significant parameter influencing LGG progression (96.6 cm³ vs. 47.9 cm3, p = 0.01). Increased incidence of epileptic seizures and decreased mental ability according to Karnofsky score were the most common symptoms associated with tumour recurrence. In the group of patients with malignant transformation, the relative cerebral blood volume (rCBV) was considerably increased (1.21 vs. 2.41, p < 0.01). No statistically significant difference was found in terms of the extent of resection between initial surgery and reoperation. Similarly, no significant difference was found in the number of patients with a permanent neurological deficit after initial surgery and reoperation. CONCLUSIONS: Reoperations of the patients with recurrent LGG are not burdened with a higher risk of neurological sequelae when compared to initial surgery. The extent of resection during the surgery for LGG recurrence is comparable to initial surgery. The increase of rCBV seems to be a significant biomarker that indicates malignant transformation.

Polyspikes and Rhythmic Polyspikes During Volatile Induction of General Anesthesia With Sevoflurane Result in Bispectral Index Variations.

BACKGROUND: Although electroencephalography (EEG)-based indices may show artifactual values, raw EEG signal is seldom used to monitor the depth of volatile induction of general anesthesia (VIGA). The current analysis aimed to identify whether bispectral index (BIS) variations reliably reflect the actual depth of general anesthesia during presence of different types of epileptiform patterns (EPs) in EEGs during induction of general anesthesia. METHODS: Sixty patients receiving either VIGA with sevoflurane using increasing concentrations (group VIMA) or vital capacity (group VCRII) technique or intravenous single dose of propofol (group PROP) were included. Monitoring included facial electromyography (fEMG), fraction of inspired sevoflurane (FiAA), fraction of expired sevoflurane (FeAA), minimal alveolar concentration (MAC) of sevoflurane, BIS, standard EEG, and hemodynamic parameters. RESULTS: In the PROP group no EPs were observed. During different stages of VIGA with sevoflurane in the VIMA and VCRII groups, presence of polyspikes and rhythmic polyspikes in patients' EEGs resulted in artifactual BIS values indicating a false awareness/wakefulness from anesthesia, despite no concomitant change of FiAA, FeAA, and MAC of sevoflurane. Periodic epileptiform discharges did not result in aberrant BIS values. CONCLUSION: Our results suggest that raw EEG correlate it with values of BIS, FiAA, FeAA, and MAC of sevoflurane during VIGA. It seems that because artifactual BIS values indicating false awareness/wakefulness as a result of presence of polyspikes and rhythmic polyspikes in patients' EEGs may be misleading to an anesthesiologist, leading to unintentional administration of toxic concentration of sevoflurane in ventilation gas.

Immuno-PET Imaging of Tumour PD-L1 Expression in Glioblastoma.

There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (ZPD-L1) radiolabelled with F-18 (Al18F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. ZPD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that 18F-AlF-NOTA-ZPD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. 68Ga-NOTA-ZPD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.

[Partial transcondylar approach - analysis of the surgical technique in cadaver simulation]. / Dostep przezklykciowy czesciowy - analiza techniki operacyjnej na podstawie symulacji na zwlokach.

The aim of the study was to present consecutive stages of the partial transcondylar approach. Six simulations of the partial transcondylar approach were performed on non-fixed human cadavers without any known pathologies in the head and neck. The consecutive stages of the procedure were documented with photographs and diagrams. The starting point for the partial transcondylar approach is a posterior repositioning of the suboccipital segment of the vertebral artery. The approach is achieved by partial removal of the occipital condyle and lateral mass of the atlas as well as by suboccipital craniectomy. Elevation of the cerebellar hemisphere presents an important supplement of the approach. The partial transcondylar approach is a reproducible technique, which provides surgical penetration of the anterior part of the cranio-cervical junction and related regions. This approach is particularly useful in the treatment of intradural tumours localized ventrally to the medulla.

Meningiomas of the anterior portion of the craniovertebral junction: immediate and late outcome following surgical removal using a partial transcondylar approach.

BACKGROUND AND PURPOSE: The partial transcondylar approach (PTA) is an alternative to the suboccipital approach in the surgical treatment of meningiomas of the anterior portion of the craniovertebral junction (APCVJ). The purpose of this study is to present our results of treatment of these meningiomas using PTA. MATERIAL AND METHODS: Fourteen patients (11 women, 3 men) with meningioma of the APCVJ were included in the study. Neurological status of the patients was assessed before and after surgery as well as at the conclusion of the treatment. The approximate volume of the operated tumour, its relation to large blood vessels, cranial nerves and brainstem, along with its consistency and vascularisation were assessed. RESULTS: The symptom duration ranged from 1 to 36 months (median: 11 months). In 79% of patients, motor deficits of the extremities were predominant symptoms. Less frequent symptoms included headache, cervical pain and sensory deficits of cervical nerves C2 to C5. Approximate volume of the tumours ranged from 2.5 mL to 22.1 mL (mean: 11.7 mL). Gross total or subtotal resection was achieved in 86% of patients. The postoperative performance status improved in 57%, did not change in 36% and deteriorated in 7% of the patients. CONCLUSIONS: The PTA is a useful technique for removal of meningiomas expanding intradurally of the APCVJ without significant compression of the medulla. The results of treatment were good in most patients.

Biology, Physics and Genetics of Intracranial Aneurysm Formation: A Review.

Intracranial aneurysms (IAs) are persistent, localised dilatations of the arterial wall that are found in approximately 3% of the general population. The most severe complication of IAs is rupture, which results in devastating consequences such as subarachnoid haemorrhage and brain damage with serious neurological sequelae. Numerous studies have characterised the mechanisms underlying IA development and growth and identified a number of environmental modifiable (smoking, hypertension) and nonmodifiable risk factors (related to the histology of cerebral arteries and genetic factors) in its pathogenesis. Haemodynamic stress also likely plays a crucial role in the formation of IAs and is conditioned by the geometry and morphology of the vessel tree, but its role in the natural history of unruptured IAs remains poorly understood; it is believed that changes in blood flow might generate the haemodynamic forces that are responsible for damage to the vascular wall and vessel remodelling that lead to IA formation. This review summarises the most relevant data on the current theories on the formation of IAs, with particular emphasis on the roles of special conditions resulting from the microscopic anatomy of intracranial arteries, haemodynamic factors, bifurcation morphometry, inflammatory pathways, and the genetic factors involved in IA formation.

Glioblastoma: Pitfalls and Opportunities of Immunotherapeutic Combinations.

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system tumour in adults. It has extremely poor prognosis since the current standard of care, comprising of gross total resection and temozolomide (TMZ) chemoradiotherapy, prolongs survival, but does not provide a durable response. To a certain extent, this is due to GBM's heterogeneous, hostile and cold tumour microenvironment (TME) and the unique ability of GBM to overcome the host's immune responses. Therefore, there is an urgent need to develop more effective therapeutic approaches. This review provides critical insights from completed and ongoing clinical studies investigating novel immunotherapy strategies for GBM patients, ranging from the use of immune checkpoint inhibitors in different settings of GBM treatment to novel combinatorial therapies. In particular, we discuss how treatment regimens based on single antigen peptide vaccines evolved into fully personalised, polyvalent cell-based vaccines, CAR-T cell, and viral or gene therapies. Furthermore, the results of the most influential clinical trials and a selection of innovative preclinical studies aimed at activating the immunologically cold GBM microenvironment are reviewed.

Differences in the Expression Patterns of TGFß Isoforms and Associated Genes in Astrocytic Brain Tumors.

Genes associated with the TGFß isoforms are involved in a number of different cancers, and their effect on the progression of brain tumors is also being discussed. Using an oligonucleotide microarray method, we assessed differences in expression patterns of genes in astrocytic brain tumor sections from 43 patients at different stages of disease. Quantitative mRNA assessment of the three TGFß isoforms was also performed by real-time RT-qPCR. Oligonucleotide microarray data were analyzed using the PL-Grid Infrastructure. The microarray analysis showed a statistically significant (p < 0.05) increase in TGFß1 and TGFß2 expression in G3/G4 stage relative to G2, whereas real-time RT-qPCR validation confirmed this change only for the TGFß2 isoform (p < 0.05). The oligonucleotide microarray method allowed the identification of 16 differential genes associated with TGFß isoforms. Analysis of the STRING database showed that the proteins encoded by the analyzed genes form a strong interaction network (p < 0.001), and a significant number of proteins are involved in carcinogenesis. Differences in expression patterns of transcripts associated with TGFß isoforms confirm that they play a role in astrocytic brain tumor transformation. Quantitative assessment of TGFß2 mRNA may be a valuable method to complement the diagnostic process in the future.

Preservation of the Hypoxic Transcriptome in Glioblastoma Patient-Derived Cell Lines Maintained at Lowered Oxygen Tension.

Despite numerous efforts aiming to characterise glioblastoma pathology (GBM) and discover new therapeutic strategies, GBM remains one of the most challenging tumours to treat. Here we propose the optimisation of in vitro culturing of GBM patient-derived cells, namely the establishment of GBM-derived cultures and their maintenance at oxygen tension mimicking oxygenation conditions occurring within the tumour. To globally analyse cell states, we performed the transcriptome analysis of GBM patient-derived cells kept as spheroids in serum-free conditions at the reduced oxygen tension (5% O2), cells cultured at atmospheric oxygen (20% O2), and parental tumour. Immune cells present in the tumour were depleted, resulting in the decreased expression of the immune system and inflammation-related genes. The expression of genes promoting cell proliferation and DNA repair was higher in GBM cell cultures when compared to the relevant tumour sample. However, lowering oxygen tension to 5% did not affect the proliferation rate and expression of cell cycle and DNA repair genes in GBM cell cultures. Culturing GBM cells at 5% oxygen was sufficient to increase the expression of specific stemness markers, particularly the PROM1 gene, without affecting neural cell differentiation markers. GBM spheroids cultured at 5% oxygen expressed higher levels of hypoxia-inducible genes, including those encoding glycolytic enzymes and pro-angiogenic factors. The genes up-regulated in cells cultured at 5% oxygen had higher expression in parental GBMs compared to that observed in 20% cell cultures, suggesting the preservation of the hypoxic component of GBM transcriptome at 5% oxygen and its loss in standard culture conditions. Evaluation of expression of those genes in The Cancer Genome Atlas dataset comprising samples of normal brain tissue, lower-grade gliomas and GBMs indicated the expression pattern of the indicated genes was specific for GBM. Moreover, GBM cells cultured at 5% oxygen were more resistant to temozolomide, the chemotherapeutic used in GBM therapy. The presented comparison of GBM cultures maintained at high and low oxygen tension together with analysis of tumour transcriptome indicates that lowering oxygen tension during cell culture may more allegedly reproduce tumour cell behaviour within GBM than standard culture conditions (e.g., atmospheric oxygen tension). Low oxygen culture conditions should be considered as a more appropriate model for further studies on glioblastoma pathology and therapy.

Improvements in Quality Control and Library Preparation for Targeted Sequencing Allowed Detection of Potentially Pathogenic Alterations in Circulating Cell-Free DNA Derived from Plasma of Brain Tumor Patients.

Malignant gliomas are the most frequent primary brain tumors in adults. They are genetically heterogenous and invariably recur due to incomplete surgery and therapy resistance. Circulating tumor DNA (ctDNA) is a component of circulating cell-free DNA (ccfDNA) and represents genetic material that originates from the primary tumor or metastasis. Brain tumors are frequently located in the eloquent brain regions, which makes biopsy difficult or impossible due to severe postoperative complications. The analysis of ccfDNA from a patient's blood presents a plausible and noninvasive alternative. In this study, freshly frozen tumors and corresponding blood samples were collected from 84 brain tumor patients and analyzed by targeted next-generation sequencing (NGS). The cohort included 80 glioma patients, 2 metastatic cancer patients, and 2 primary CNS lymphoma (PCNSL) patients. We compared the pattern of genetic alterations in the tumor DNA (tDNA) with that of ccfDNA. The implemented technical improvements in quality control and library preparation allowed for the detection of ctDNA in 8 out of 84 patients, including 5 out of 80 glioma patients. In 32 out of 84 patients, we found potentially pathogenic genetic alterations in ccfDNA that were not detectable in tDNA. While sequencing ccfDNA from plasma has a low efficacy as a diagnostic tool for glioma patients, we concluded that further improvements in sample processing and library preparation can make liquid biopsy a valuable diagnostic tool for glioma patients.

Mucocoele and mucopyocoele of the frontal sinus penetrating to the cranial cavity and the orbit.

BACKGROUND AND PURPOSE: Mucocoele of the paranasal sinuses falls within the scope of interest for neurosurgery when erosion of the sinus wall and the osseous structures of the skull base develops and the lesion extends towards the cranial cavity, the orbit, the cavernous sinus or the sella turcica. The pa-per aims to present the method of treatment of extensive mucocoele which is used in our clinic. MATERIAL AND METHODS: We treated 7 patients (2 women and 5 men; age range: 27-68 years). Mucopyocoele was diagnosed in two cases, and mucocoele in the other five. In 5 cases, extension of the mucocoele to the cranial cavity and the orbit or to the ethmoid sinus and the orbit was observed. In the remaining 2 cases, mucopyocoele extended to the ethmoid sinus, the sphenoid and maxillary sinuses, cranial cavity and the orbit. The purpose of surgery was to remove the mucocoele or the mucopyocoele and to prevent recurrence. RESULTS: The postoperative course in all 7 patients was uneventful. All symptoms gradually receded. No relapse was observed in any patient during a follow-up period that varied from 10 months to 8 years; nor did incidents of inflammation of collateral sinuses occur. CONCLUSIONS: The treatment of mucocoele or mucopyocoele of the frontal sinus penetrating to the cranial cavity and the orbit consists of the following stages: cranialization of the frontal sinus, complete resection of the mucosa, tight closing of the frontal-nasal duct, and separating the air space of the opened collateral nasal sinuses from the cranial cavity with a large pedicled periosteal flap.