Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor.

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Increased compliance with tumor treating fields therapy is prognostic for improved survival in the treatment of glioblastoma: a subgroup analysis of the EF-14 phase III trial.

BACKGROUND: Tumor treating fields (TTFields) is a non-invasive, antimitotic therapy. In the EF-14 phase 3 trial in newly diagnosed glioblastoma, TTFields plus temozolomide (TTFields/TMZ) improved progression free (PFS) and overall survival (OS) versus TMZ alone. Previous data indicate a ≥ 75% daily compliance improves outcomes. We analyzed compliance data from TTFields/TMZ patients in the EF-14 study to correlate TTFields compliance with PFS and OS and identify potential lower boundary for compliance with improved clinical outcomes. METHODS: Compliance was assessed by usage data from the NovoTTF-100A device and calculated as percentage per month of TTFields delivery. TTFields/TMZ patients were segregated into subgroups by percent monthly compliance. A Cox proportional hazard model controlled for sex, extent of resection, MGMT methylation status, age, region, and performance status was used to investigate the effect of compliance on PFS and OS. RESULTS: A threshold value of 50% compliance with TTFields/TMZ improved PFS (HR 0.70, 95% CI 0.47-1.05) and OS (HR 0.67, 95% CI 0.45-0.99) versus TMZ alone with improved outcome as compliance increased. At compliance > 90%, median survival was 24.9 months (28.7 months from diagnosis) and 5-year survival rate was 29.3%. Compliance was independent of gender, extent of resection, MGMT methylation status, age, region and performance status (HR 0.78; p = 0.031; OS at compliance ≥ 75% vs. < 75%). CONCLUSION: A compliance threshold of 50% with TTFields/TMZ correlated with significantly improved OS and PFS versus TMZ alone. Patients with compliance > 90% showed extended median and 5-year survival rates. Increased compliance with TTFields therapy is independently prognostic for improved survival in glioblastoma.

[Mineralogical composition of urinary stones, risk factors and metabolic disturbances in patients with calcium-oxalate urolithiasis].

AIM: To identify the most likely metabolic disturbances and risk factors for stone formation in a group of patients with calcium oxalate urolithiasis, and to establish the relationship between the mineralogical composition of calculi and impaired excretion of inhibitors and promoters of stone formation. MATERIALS AND METHODS: Fifty patients with calcium oxalate urolithiasis were tested using a complex of physicochemical methods. Patients assessment included evaluation of quantitative mineralogical composition of calculi, daily urine pH profile and daily urinary excretion of urates, calcium, magnesium, oxalate, phosphate and citrate ions. RESULTS: The main mineralogical phase of the stones in over 80% of patients was calcium oxalate monohydrate; none of the patients had pure dihydrate stones. The most frequent metabolic disorders were hypocitraturia, hypercalciuria and hyperuricosuria. Predominant risk factors were excessive body weight and insufficient fluid intake. Only one patient had an idiopathic stone formation. It was established for the first time that patients with calcium oxalate stones, containing 10 or more mass percent of apatites had statistically significantly lower daily urinary calcium and oxalate excretion and simultaneously increased phosphate excretion. CONCLUSIONS: The study findings showed that patients with calculi based on calcium oxalate dihydrate should undergo testing for daily urinary excretion of calcium and citrate while pa-tients with calcium oxalate stones containing 10 or more mass percent of apatites should also be tested for daily phosphate excretion and urine pH-profile. Daily urinary citrate excretion was reduced in all study patients, and urate excretion was significantly increased, apparently due to an imbalanced diet and excessive intake of animal protein. Menopausal and postmenopausal women are at a particular risk due to low urinary citrate excretion and high urinary calcium excretion regardless of stone composition.

Centre-level variation in treatment and outcomes and predictors of outcomes in 5-year-old children with non-syndromic unilateral cleft lip treated within a centralized service: the Cleft Care UK study. Part 6: summary and implications.

OBJECTIVES: To summarize and discuss centre-level variation across a range of treatment and outcome measures and examine individual and ecological determinants of outcome in children in Cleft Care UK (CCUK). SETTING AND SAMPLE POPULATION: Two hundred and sixty-eight 5-year-old British children with non-syndromic unilateral cleft lip and palate (UCLP) recruited to CCUK and treated within a centralized service. MATERIALS AND METHODS: Children had a range of treatment and outcome measures collected at a 5-year audit clinic. These outcomes included dento-alveolar arch relationships from study models, measures of facial appearance from cropped photographs, hearing loss from audiological assessment, speech from speech recordings, self-confidence and strengths and difficulties from parental self-report. Data were collected on educational attainment at age 7 using record linkage. Centre variation was examined using hierarchical regression and associations between variables were examined using logistic or poisson regression. RESULTS: There was centre-level variation for some treatments (early grommet placement, fitting of hearing aids, fluoride treatment, secondary speech surgery and treatment for cleft speech characteristics) and for some outcomes (intelligibility of speech). Hearing loss was associated with a higher risk of poor speech while speech therapy was associated with a lower risk of poor speech. Children had high levels of caries but levels of preventative treatment (fluoride varnish and tablets) were low. CONCLUSIONS: Further improvements to and monitoring of the current centralized model of care are required to ensure the best outcomes for all children with cleft lip and palate.

Centre-level variation of treatment and outcome in 5-year-old children with non-syndromic unilateral cleft lip and palate: The Cleft Care UK study. Part 1: Methodology and results for dento-facial outcomes.

OBJECTIVES: Outline methods used to describe centre-level variation in treatment and outcome in children in the Cleft Care UK (CCUK) study. Report centre-level variation in dento-facial outcomes. SETTING AND SAMPLE POPULATION: Two hundred and sixty-eight five-year-old British children with non-syndromic unilateral cleft lip and palate (UCLP). MATERIALS AND METHODS: Between January 2011 and December 2012, data were collected on a comprehensive range of outcomes. Child facial appearance and symmetry were assessed using photographic pictures. Dental arch relationships were assessed from standardized dental study models. Hierarchical statistical models were used to predict overall means and the variance partition coefficient (VPC)-a measure of amount of variation in treatment or outcome explained by the centre. RESULTS: Data on dento-alveolar arch relationships and facial appearance were available on 197 and 252 children, respectively. The median age of the children was 5.5 years, and 68% were boys. Variation was described across 13 centres. There was no evidence of centre-level variation in good or poor dento-alveolar arch relationships with a VPC of 4% and 3%, respectively. Similarly, there was no evidence of centre-level variation in good or poor facial appearance with a VPC of 2% and 5%, respectively. CONCLUSIONS: There was no evidence of centre-level variation for dento-facial outcomes although this study only had the power to detect large variation between sites.

[Quantitative mineralogical analysis and structure of urinary stones in patients living in Ivanovo region].

This paper focuses on developing and implementing a method of quantitative mineralogical analysis of urinary stones based on powder diffraction data analysis using 4 Topas (Bruker) software. Mineralogical composition of 100 urinary stones from urolithiasis patients living in Ivanovo region was examined. More than 70% of stones consisted of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD), and their mixtures with hydroxylapatite. Forty four percent of urinary stones consisted of one component (COM, uric acid (UA) or, less frequently, hydroxyapatite (HA); 56% of urinary stones comprised two, three or four components. The most common mineral was COM (more than 70% of cases), the rarest were calcium oxalate trihydrate (CT), brushite and newberrite. The most common combinations of minerals in mixed stones were COM+HA, COM+COD and COM+COD+HA. The texture, the surface composition and its changes in the course of chemolysis in different types of stones were examined using scanning electron microscopy (SEM) and X-ray microanalysis (XRM). Implications for using analytical chemical and physical techniques for the diagnosis and treatment of urolithiasis were discussed.

Pathobiology of brain metastases.

Brain metastasis is a major cause of systemic cancer morbidity and mortality. Many factors participate in the development and maintenance of brain metastases. The survival of the metastasis depends upon crucial interactions between tumour cells and the brain microenvironment during its development at the new site. This review focuses on the pathobiological mechanisms involved in the establishment and regulation of brain metastases. Developments in molecular biology have vastly expanded our knowledge about the mechanisms of invasion, proliferation, metastatic cell signalling, and angiogenesis in brain metastases. Advances in this understanding of the pathobiology of brain metastasis may lead to novel targeted treatment paradigms and a better prognosis for patients with brain metastatic disease.

Detection of proliferating S-phase brain tumor cells by in situ DNA replication.

OBJECT: Current methods used to describe the proliferative status of brain tumors rely on labor-intensive, potentially costly procedures. This article provides a description of a rapid, inexpensive, uncomplicated technique used to identify proliferating cells in tissue obtained at the time of resection. METHODS: Touch preparations of 16 fresh astrocytic tumors and four fresh healthy temporal neocortical tissue samples were obtained at the time of surgery. Slides were placed in hypotonic potassium chloride to permeabilize their membranes, incubated in nucleotide precursors, and labeled with bromodeoxyuridine; they were later examined with the aid of a fluorescence microscope. The percentage of tumor cells in the S phase increased in conjunction with the grade of tumor and corresponded with the findings of immunohistochemical staining for the cell-cycle marker MIB-1. These results were confirmed in cell culture by using normal human astrocytes and two glioma cell lines. Slides can be analyzed in as little as 30 minutes after removal of tissue during surgery. CONCLUSIONS: In this study the authors describe a simple method by which cells in the S phase of the cell cycle. which are contained in fresh tumor obtained at the time of surgery, can be labeled. This method may prove a useful adjunct to frozen-section analysis and may permit discrimination of neoplastic tissues from other tissues observed in small specimen samples.

Oligodendroglial ganglioglioma with anaplastic features arising from the thalamus.

Anaplastic gangliogliomas with an oligodendroglial component are exceedingly rare tumors of uncertain growth potential. We report a 17-year-old female with a massive ganglioglioma containing anaplastic oligodendroglioma apparently arising from the thalamus. Two weeks after partial resection, she was started on a regimen including escalated doses of topotecan in combination with a fixed-dosage intensification regimen of cisplatin, cyclophosphamide and vincristine with subsequent hyperfractionated external beam radiotherapy. She currently has stable disease.

In vivo brain tumor demarcation using optical spectroscopy.

The applicability of optical spectroscopy for intraoperative detection of brain tumors/tumor margins was investigated in a pilot clinical trial consisting of 26 brain tumor patients. The results of this clinical trial suggest that brain tumors and infiltrating tumor margins (ITM) can be effectively separated from normal brain tissues in vivo using combined autofluorescence and diffuse-reflectance spectroscopy. A two-step empirical discrimination algorithm based on autofluorescence and diffuse reflectance at 460 and 625 nm was developed. This algorithm yields a sensitivity and specificity of 100 and 76%, respectively, in differentiating ITM from normal brain tissues. Blood contamination was found to be a major obstacle that attenuates the accuracy of brain tumor demarcation using optical spectroscopy. Overall, this study indicates that optical spectroscopy has the potential to guide brain tumor resection intraoperatively with high sensitivity.

Fiducial point placement and the accuracy of point-based, rigid body registration.

OBJECTIVE: To demonstrate that the shape of the configuration of fiducial points is an important factor governing target registration error (TRE) in point-based, rigid registration. METHODS: We consider two clinical situations: cranial neurosurgery and pedicle screw placement. For cranial neurosurgery, we apply theoretical results concerning TRE prediction, which we have previously derived and validated, to three hypothetical fiducial marker configurations. We illustrate the profile of expected TRE for each configuration. For pedicle screw placement, we apply the same theory to a common anatomic landmark configuration (tips of spinous and transverse processes) used for pedicle screw placement, and we estimate the error rate expected in placement of the screw. RESULTS: In the cranial neurosurgery example, we demonstrate that relatively small values of TRE may be achieved by using widely spread fiducial markers and/or placing the centroid of the markers near the target. We also demonstrate that near-collinear marker configurations far from the target may result in large TRE values. In the pedicle screw placement example, we demonstrate that the screw must be approximately 4 mm narrower than the pedicle in which it is implanted to minimize the chance of pedicle violation during placement. CONCLUSION: The placement of fiducial points is an important factor in minimizing the error rate for point-based, rigid registration. By using as many points as possible, avoiding near-collinear configurations, and ensuring that the centroid of the fiducial points is as near as possible to the target, TREs can be minimized.

December 2000: 6 month old boy with 2 week history of progressive lethargy.

This 6-month-old Caucasian boy presented with a 10-day history of lethargy, obtundation, inability to hold his head up and mild torticollis. MRI and CT scans showed a large solid and cystic mass involving the right temporal, parietal and occipital lobes, pineal, superior pons, mesencephalon and posterior right thalamus. He underwent craniotomy initially for a partial tumor resection with an intraoperative diagnosis of desmoplastic astrocytoma. With immunohistochemistry and special stains the diagnosis of desmoplastic infantile ganglioglioma (DIG) was made. A near total resection was performed a week after initial resection.The patient then was treated with chemotherapy. Two months later an MRI showed tumor growth. Following additional aggressive chemotherapy, an MRI at 5 months post-resection indicated further tumor progression. This case illustrates that some DIGs may behave more aggressively than typical WHO grade I lesions.

Brain tumor demarcation using optical spectroscopy; an in vitro study.

Optical spectroscopy for brain tumor demarcation was investigated in this study. Fluorescence and diffuse reflectance spectra were measured from normal and tumorous human brain tissues in vitro. A fluorescence peak was consistently observed around 460 nm (+/- 10 nm) emission from both normal and tumorous brain tissues using 337 nm excitation. Intensity of this fluorescence peak (F460) from normal brain tissues was greater than that from primary brain tumorous tissues. In addition, diffuse reflectance (Rd) between 650 and 800 nm from white matter was significantly stronger than that from primary and secondary brain tumors. A good separation between gray matter and brain tumors was found using the ratio of F460 and Rd at 460 nm (Rd460). Two empirical discrimination algorithms based on F460, Rd625, and F460/Rd460 were developed. These algorithms yielded an average sensitivity and specificity of 96% and 93%, respectively.

Basic surgical techniques in the resection of malignant gliomas.

Although progress is being made in the imaging and medical therapy of malignant gliomas, surgery continues to play a central role in the management of these tumors. Surgery remains necessary for histological diagnosis and relief of mass effect upon adjacent neurological structures. Surgery also achieves cytoreduction of tumor mass, allowing more effective treatment of residual tumor volume. Recent technical advances have made the goal of gross total tumor resection safer and more readily achieved. Surgical techniques and adjuncts that aid the aggressive surgical resection of malignant gliomas will be reviewed.

Gamma-ray mutagen sensitivity and survival in patients with glioma.

Despite advances in treatment of brain tumors, cerebral malignant gliomas are rapidly debilitating with poor survival. Patient age and tumor histology are known to influence survival in glioma patients, but these factors do not account for all of the variability in survival time. To identify additional useful predictors, we tested an assay that measures intrinsic gamma-ray mutagen sensitivity. Our hypothesis was that increased sensitivity of peripheral blood lymphocytes to chromatid breaks is associated with tumor aggressiveness and decreased patient survival. The eligible 76 patients were those with histologically confirmed malignant gliomas, seen at the University of Texas M. D. Anderson Cancer Center between 1994 and 1997, for whom we had sufficient blood for the in vitro gamma-radiation assay. After gamma-irradiation of each subject's lymphocytes, the frank chromatid breaks in 50 metaphases were averaged to calculate breaks/cell. On the basis of our patient series, we established a gamma-ray mutagen sensitivity cutoff point of 0.55 breaks/cell that was confirmed by bootstrap resampling techniques. Patients with values >0.55 breaks/cell were considered sensitive. Kaplan-Meier and Cox proportional hazards modeling were used for the analysis. The gamma-ray mutagen-sensitive patients had worse survival than the nonsensitive patients, with an unadjusted hazard rate ratio of 1.6 (95% confidence interval, 0.9-2.8; P = 0.15). After adjustment for age, tumor histology, and extent of surgical resection, the hazard rate ratio was 2.4 (95% confidence interval, 1.3-4.6; P = 0.0081). Our data suggest that gamma-ray mutagen sensitivity is a prognostic indicator of survival in glioma patients. The significance of these findings needs to be verified in studies with larger samples of patients with histologically similar gliomas.

Antagonist effect of insulin-like growth factor I on protein kinase inhibitor-mediated apoptosis in human glioblastoma cells in association with bcl-2 and bcl-xL.

OBJECT: Tamoxifen (TAM) has been found to be effective in inhibiting proliferation of glioblastoma cells in vitro, but clinical studies have been disappointing. The purpose of this study was to determine whether insulin-like growth factor I (IGF-I), a potential autocrine/paracrine mitogen produced by glioblastomas, interferes with the antimitogenic actions of TAM. METHODS: Human glioblastoma cells were treated with or without TAM and/or IGF-I in vitro and evaluated for: viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide cleavage assay; apoptosis by histochemical analysis of nuclear morphology and 3'-OH DNA fragments; and expression of the IGF-I receptor, and the bcl-2, bcl-xL, and bax proteins by immunoblot analysis. In addition, p53 status was determined by DNA sequencing and by transient transfection with luciferase reporter plasmids containing wild-type or mutant p53. Results indicated that after 72 hours of exposure to 2 mg/ml TAM in vitro, 56.3% of WITG3 and 43.8% of U87-MG glioblastoma cells contained apoptotic nuclei (p < 0.01 compared with untreated cells). Apoptosis was independent of the presence of p53 because the WITG3 cells, in contrast to the U87-MG cells, expressed a mutant, nonfunctional p53. The WITG3 cells expressed IGF-I receptor proteins and demonstrated IGF-I binding. Exogenous IGF-I stimulated WITG3 cell proliferation and significantly (p < 0.05) antagonized the cytotoxic effects of TAM in a dose-dependent fashion; IGF-I, but not TAM, enhanced expression of bcl-2 and bcl-xL proteins; however, bax protein expression was unchanged by either treatment. CONCLUSIONS: Because many gliomas secrete large amounts of IGF-I in autocrine/paracrine growth pathways, these data may, in part, explain the failure of TAM to achieve clinical results as dramatic as those in vitro.

Thyroid hormone depletion inhibits astrocytoma proliferation via a p53-independent induction of p21 (WAF1/CIP1).

BACKGROUND: Clinical hypothyroidism has been associated with prolonged survival in several types of malignancies, but the mechanism of this effect is unknown. MATERIAL AND METHODS: In vitro studies of thyroid hormone depletion (via culture in medium containing 5% thyroid hormone-depleted fetal bovine serum (FBS)) were carried out using a human glioblastoma cell line (WITG3) which expresses a mutant, non-functional p53. RESULTS: Thyroid hormone depletion inhibited WITG3 proliferation compared to control medium containing 5% euthyroid FBS. There was no evidence of apoptosis and viability was not compromised. Cell cycle analysis by flow cytometry indicated that thyroid hormone depletion accumulated WITG3 cells in G1, with fewer cells progressing into S than in euthyroid medium. By immunoblotting, p21 (WAF1/CIP1) was only slightly detectable in lysates from WITG3 cells grown in control euthyroid medium; however, in thyroid hormone-depleted FBS, a marked induction of p2 1 occurred which could be reversed by exogenous thyroid hormone CONCLUSIONS: These data indicate that thyroid hormone depletion may cause a G, arrest in astrocytoma mediated by a p53-independent induction of p21 (WAF1/CIP1). Results suggest a mechanism which may explain the effect of hypothyroidism on suppression of tumor cell growth.

The transforming activities of MDM2 in cultured neonatal rat astrocytes.

Although the molecular events regulating the pathogenesis of malignant astrocytomas remains unclear, the inactivation of tumor suppressor genes may be a key factor. The inactivation of p53 by mutation or deletion, however, is not the only obligatory step in astrocytoma genesis. The MDM2 protein has been shown to bind to and downmodulate p53 function, and to have oncogenic capacity. The MDM2 gene is also amplified and overexpressed in a subset of malignant astrocytomas without p53 mutation. Here we show that overexpression of MDM2 promoted the DNA synthesis of cultured neonatal rat astrocytes (RNB cells), abrogated the transcriptional activity of wild-type p53, conferred invasive activity, and subsequently induced the transformation from astrocytes to high-grade astrocytomas. Intriguingly, MDM2 enhanced the expression of angiogenic mitogens; basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in RNB cells. These results indicate that MDM2 may play an important role in the progression of astrocytomas, by not only conferring invasive activity but also stimulating the expression of angiogenic growth factors.