Production of Urokinase-Type Plasminogen Activator (u-PA) and Plasminogen Activator Inhibitor-1 (PAI-1) in Human Brain Tumours

Summary We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-enviroment contributes (ascribes) to tumour cell invasion.     

Study of correlation between expression of bradykinin B2 receptor and pathological grade in human gliomas

In clinical practice there is a difference in response of the blood-tumour barrier (BTB) permeability induced by bradykinin in brain tumours with the same pathology. The variability in response of tumours to bradykinin is likely to be related to the expression level of bradykinin B(2) receptor. This study used fresh human glioma samples to determine the expression level of bradykinin B(2) receptor on gliomas with different pathological grades. The grade of tumour was classified using the WHO classification. To determine the bradykinin B(2) receptor expression level in gliomas, Immunohistochemistry and Western blot methods were used. In 24 cases of gliomas there were eight cases of WHO I glioma, eight cases of WHO II glioma and eight cases of WHO III glioma. Both Western blot and immunohistochemistry showed bradykinin B(2) receptors localized on tumour cells, whilst brain cells at the edge of the glioma hardly expressed B(2) receptor. There were significant differences of bradykinin B(2) receptor expression level among different pathological grades of glioma. The expression of B(2) receptor in the three grades of glioma was in the order of WHO I < WHO II < WHO III. Determination of bradykinin B(2) receptor expression level in human glioma may be useful in screening glioma patients to predict whether they will be suitable for opening of the blood - tumour barrier with bradykinin or its analogue.     

Expression of cALLa/NEP on gliomas: A possible marker of malignancy

Summary First described on pre-B leukemia cells, the common acute lymphoblastic leukemia antigen (cALLa) is also expressed on glioma cellsin vitro. Its identity to neutral endopeptidase (NEP) (E.C.3.24.11) was corroborated by our finding that cALLa positive glioma cells had NEP activity. To study cALLa/NEP distribution on glial tumours in vivo, we examined 76 brain tumour biopsies by immunostaining techniques on frozen tissue sections using anticALLa (FAH99) and anti-NEP (135 A 3) monoclonal antibodies. We found that 96% of grade 4 gliomas (25/26) expressed NEP. Whereas only 45% (4/9) of grade 3 or anaplastic astrocytomas did. In low grade gliomas, we found 2 positive tumours out of 21 tested (10%). Double immunostaining procedures revealed that NEP was co-expressed with GFAP. However no NEP could be detected on non-glial brain tumours nor on reactive astrocytes. These results suggest that cALLa/NEP expression could be linked to malignant progression of gliomas.     

The potential for strategies using micronutrients and heterocyclic drugs to treat invasive gliomas

Summary ¶Local invasion of neoplastic cells into the surrounding brain is perhaps the most important aspect of the biology of gliomas that precludes successful therapy. Despite significant advances in neuro-imaging, neurosurgery and radiotherapy, the median survival for patients with a malignant glioma is still less than one year. With the increasing knowledge of the biology of brain tumours, derived from cellular and molecular studies, new methods of treatment are being developed with some success. Approaches studied already include anti-invasive, pro-apoptotic and anti-angiogenesis strategies and clinical trials are imminent.In this article we review two new approaches to the management of gliomas: nutraceutical intervention and heterocyclic drugs. The first approach uses a combination of naturally occurring agents, including citrus flavonoids, chokeberry extract, red grape seed extract, lycopene, selenium and red clover extract. These agents can either trigger apoptosis or affect the pathways underlying diffuse invasion. The second approach involves the use of a heterocyclic drug, clomipramine, which selectively triggers apoptosis in neoplastic cells but not in normal glia. The article refers to the results of recent studies performed in our laboratory which suggest that these new approaches can be translated into benefit to patients.Published online July 31, 2003     

Prognostic value of perfusion-weighted imaging in brain glioma: a prospective study

Summary Object. Biopsy targeting based on MR imaging alone may fail to identify malignant areas in brain gliomas. Considering the differences in relative Cerebral Blood Volume (rCBV) ratios reported among tumour grades, we evaluated whether perfusion-weighted MR imaging (PWI) could usefully implement the routine preoperative imaging by detecting those areas bearing a higher yield for malignancy to guide the stereotactic biopsy or the surgical removal. Clinical material and methods. We studied a series of 55 consecutive patients with newly diagnosed brain glioma using both conventional MR imaging and PWI in the preoperative assessment. The pathological diagnosis was established by stereotactic biopsy in 29 cases and by craniotomy in 24 cases. We evaluated the patient survival to detect undergrading. Discussion. Independent from contrast-enhancement, perfusion-weighted MR imaging improved the target selection in stereotactic biopsy guidance and the removal of malignant areas in tumours amenable to surgery. Particularly sensitive to the perfused part of the tumour as to small regional changes, rCBV maps allowed a better detection of malignant areas. The rCBV ratios correlated significantly to the tumour grade and the final outcome (p < 0.01). Conclusions. We found PWI valuable in the preoperative assessment of brain gliomas, discriminating high from low-grade gliomas. PWI can easily be performed on widely available MR imaging systems as part of the routine imaging of gliomas.     

111Indium (DTPA-octreotide) scintigraphy in patients with cerebral gliomas

Summary Somatostatin receptors (SR) have been identified in vitro in normal brain tissue, in neuro-endocrine tumours and in cerebral gliomas WHO grade 1 or 2 by autoradiography or using somatostatin-gold conjugates. In vivo, SR detection has become possible by scintigraphy applying the somatostatin analogue octreotide, radio-labelled with¹¹¹Indium. It was supposed that expression of SR in cerebral gliomas corresponds to low grade tumour malignancy and that, in vivo, somatostatin receptor scintigraphy (SRS) could refine and improve the WHO grading system for cerebral gliomas.Nineteen patients with cerebral gliomas (grade 2: n=8, grade 3: n=3, grade4: n=8) were examined with¹¹¹In (DTPA-octreotide) to evaluate, whether SRS could improve the pre-operative estimation of tumour biology and the postoperative management. The results of SRS were related with the histological findings and with the in vitro demonstration of somatostatin-binding sites on cultured tumour cells incubated with a somatostatin-gold conjugate.In vivo, none of the patients with glioma grade 2 showed enhanced tracer uptake in the SRS, whereas in vitro SR were detected in cultured tumour tissue in 5 out of 5 cases. Every patient with glioma grade 3 or 4 demonstrated a high focal uptake of¹¹¹In (DTPA-octreotide), as shown by SRS. Three patients with glioma grade 4, additionally examined with 99mTc-DTPA, showed an increased tracer uptake within the tumour area when compared with results of SRS. In vitro, SR were detected on tumour cell surface in 5 out of 6 tissue samples from patients with gliomas grade 3 or 4. One patient harbouring a cerebral abscess presented with a high focal tracer uptake in the SRS but with absence of somatostatinbinding sites in vitro.We concluded, that in glioma patients enhanced tracer uptake in receptor scintigraphy with¹¹¹In (DTPA-octreotide) does not depend on the presence of SR in tumour tissue but on the dysfunction of the blood-brain barrier. Thus, SRS does not improve the preoperative glioma grading or postoperative management in patients with cerebral tumours of glial origin.The article is dedicated to Prof. H. Leonhardt on the occasion of his 75th birthday.     

Local blood flow changes in malignant brain tumours under induced hypertension

Summary Changes in tumour blood flow under an induced hypertensive state were examined in malignant brain tumours to know if the precondition for the effectiveness of induced hypertensive chemotherapy — relative increase in tumour blood flow — are fulfilled. Tumour blood flow was measured under both a resting and an induced hypertensive state in 12 patients with various malignant brain tumours (6 gliomas, 6 metastatic brain tumours) using xenon-enhanced computed tomography. The blood pressure was elevated 40% above the systemic blood pressure of the resting state by the infusion of angiotensin II. Tumour blood flow increased 30% on average above the normal brain tissue blood flow after the induction of an induced hypertensive state (p < 0.05). The tumour blood flow increased in 11 cases of malignant tumours, but decreased in one case with massive brain oedema after induced hypertension. The increase in blood flow was higher in hypervascular tumours and less in hypovascular tumours. Therefore, induced hypertensive chemotherapy probably will be more effective in hypervascular malignant brain tumours with small mass effects.     

Multicentric malignant glioma.

Multicentre gliomas are a well recognized entity but the occurrence of such tumours both above and below the tentorium remains uncommon. We report the case of an 11-year-old boy who underwent stereotactic biopsy of a brain stem ring enhancing tumour with histology of an anaplastic astrocytoma (Grade 3). Nine months following his radiotherapy a large left frontal mass was biopsied and found to be a malignant glioma (Grade 4). Advances in neuroradiological imaging will readily show multiple cerebral lesions and multicentre glioma should be considered in the differential diagnosis of such lesions and biopsy is indicated.     

Cellular immunity and complement levels in hosts with brain tumours

Summary As a major defence mechanism against cancer, host immunological surveillance is composed of a cellular immunity as well as humoral immunity including antibody and a complement system. In the course of the progress of brain tumours alone, serum complement level (CH 50) as a humoral immunological factor and the tuberculin skin reactivity as an index of cellular immune activity, were serially measured in brain tumour patients. One hundred and fifty-seven cases of brain tumours, including 75 cases of glioma, 25 benign tumours, and 42 metastatic tumours, were examined. Most cases of benign tumour belong to stage I oder II, in which both tuberculin reaction and complement are active. Many cases of glioblastoma and metastatic tumour belong to stage III; that is, they show negative tuberculin reaction and increased complement activity. The relation of immunological response to the tumour size and the clinical severity in patients with gliomas is revealed by the fact that complement titres rise in accordance with the degree of progress of the tumour and a negative tendency in the tuberculin reaction runs parallel to this. All cases of glioma, even in the terminal stages, remain in stage III. On the other hand, cases of metastatic tumour progress to stage IV and V, in which the tuberculin reaction is negative and complement titres decrease. The combined results of elevated complement level and depressed status of tuberculin reaction in patients with gliomas may be explained by the concept that complement activity rises to compensate for depressed cell-mediated immunity, in order to preserve the activity of the biophylaxis mechanism against cancer.     

Proliferation,migration, and invasion of human glioma cells exposed to fractionated radiotherapy in vitro

Radiotherapy is a well established treatment for malignant gliomas. This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines (GaMg and U-87 Mg) grown as multicellular tumour spheroids after radiotherapy. Migration and proliferation studies were performed using conventional and accelerated fractionation up to 60 Gy and 59.4 Gy, respectively. A dose-dependent growth and migratory response to irradiation independent of the type of fractionation was observed. A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. Marked invasion of the glioma spheroids into the brain aggregates occurred with or without radiotherapy. For the GaMg cells, flow cytometric DNA histograms after treatment with 10 Gy and 40 Gy showed an accumulation of cells in the G2/M phase of the cell cycle. Radiotherapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.     

Nitric oxide and glioma: a target for novel therapy?

The biological activities of nitric oxide (NO) include vasodilatation, inhibition of platelet aggregation, neurotransmission, neural plasticity, and modulation of inflammatory and immunological functions. NO synthase (NOS), which is the enzyme that produces NO, has been detected in resected human glioma specimens, and both human and rodent glioma cell lines. NO production in gliomas can alter several important pathophysiological processes, such as local host immune response, tumour cell apoptosis, tumour invasion/metastasis, free radical injury to tumour cells and adjacent normal brain tissues, tonic vasodilatation of tumour vessels, vascular permeability and neovascularization. Recently, some therapeutic strategies for gliomas using NO manipulation have been proposed, and evaluated both experimentally and indirectly in preliminary clinical trials. These include NO manipulation designed to modify tumour cell oncogenesis, tumour blood flow and disposition of anti-cancer drugs in tumour tissue. This review will discuss the biological role of NO in the central nervous system and gliomas and its current and future possibilities in neuro-oncology.     

Cranial neuronavigation with direct integration of (11)C methionine positron emission tomography (PET) data -- results of a pilot study in 32 surgical cases

BACKGROUND: MRI detects small intracranial lesions, but has difficulties in differentiating between tumour, gliosis and edema. (11)C methionine-PET may help to overcome this problem. For its appropriate intra-operative use, it must be integrated into neuronavigation. We present the results of our pilot study with this method. METHOD: 32 patients with 34 intracranial lesions detected by MRI underwent additional (11)C methionine-PET, because the pathophysiological behaviour or the tumour delineation was unclear. All lesions were treated surgically. In 25 patients PET data could be integrated directly into cranial neuronavigation. FINDINGS: (11)C methionine uptake was observed in 27/34 lesions, 26 of them were tumours: 14 malignant and 7 benign gliomas, 3 gliomas without further histological typing, one Ewing sarcoma and one non-Hodgkin lymphoma. Only one (11)C methionine positive lesion was non-tumourous: it was staged as post-irradiation necrosis in a patient operated on for a malignant glioma. 3/7 (11)C-methionine negative lesions were classified as gliosis (n=2) and M. Whipple (n=1), but 4/7 were tumours: 2 astrocytomas WHO(degrees)II, 1 DNT and one astrocytoma WHO(degrees)III. The sensitivity of (11)C methionine-PET was 87%, the specificity 75%, the positive predictive value 96% and the negative predictive value 43%. In all tumourous cases with positive tracer uptake the borderline area of the tumour was better defined by (11)C methionine-PET than by MRI. INTERPRETATION: A positive (11)C methionine-PET is highly suspicious of a tumour, a negative one does not exclude it. (11)C methionine-PET seems to be more sensitive than MRI for differentiating between tumour and edema or gliosis. Simultaneous integration MRI and (11)C methionine-PET into cranial neuronavigation can facilitate cross total tumour removal in glioma surgery.     

Tumours of the central nervous system

Surgery plays a key role in the management of brain tumours. The prognosis for a brain tumour diagnosis remains poor, but without the input of the surgical team, the outcomes would be bleaker. Innovations in understanding of the molecular biology of brain tumours may identify new targets for future therapy, but for now we should maximize the opportunities offered by surgery. In this article, we will examine recent updates in understanding of brain tumour biology and the implications for management. We will look at the latest research on brain tumour diagnosis, and then focus on management of the three most common brain tumours – glioma, meningiomas and cerebral metastases. We examine the surgical approach and adjuncts that should be employed to optimize patient outcomes.     

Tumours of the central nervous system

Surgery plays a key role in the management of brain tumours. The prognosis for a brain tumour diagnosis remains poor, but without the input of the surgical team, the outcomes would be bleaker. Innovations in understanding of the molecular biology of brain tumours may identify new targets for future therapy, but for now we should maximize the opportunities offered by surgery. In this article, we will examine recent updates in understanding of brain tumour biology and the implications for management. We will look at the latest research on brain tumour diagnosis, and then focus on management of the three most common brain tumours – glioma, meningiomas and cerebral metastases. We examine the surgical approach and adjuncts that should be employed to optimize patient outcomes.     

Proliferation,migration, and invasion of human glioma cells exposed to fractionated radiotherapy in vitro

Radiotherapy is a well established treatment for malignant gliomas. This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines (GaMg and U-87 Mg) grown as multicellular tumour spheroids after radiotherapy. Migration and proliferation studies were performed using conventional and accelerated fractionation up to 60 Gy and 59.4 Gy, respectively. A dose-dependent growth and migratory response to irradiation independent of the type of fractionation was observed. A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. Marked invasion of the glioma spheroids into the brain aggregates occurred with or without radiotherapy. For the GaMg cells, flow cytometric DNA histograms after treatment with 10 Gy and 40 Gy showed an accumulation of cells in the G2/M phase of the cell cycle. Radiotherapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.     

Heme oxygenase (HO) isoforms in experimental C6 glioma: an immunocytochemical study.

Abstract Because of their potential to regulate tumoural blood flow and interactions with nitric oxide the expression of the type 1 and 2 isoforms of heme oxygenase (HO-1 and HO-2) were evaluated in implanted C6 striatal gliomas. Immunocytochemistry using antibodies specific for HO-1 and HO-2 were used in 20 C6 glioma tumours. The bulk of the tumour parenchyma and endothelium was negative for both HO isoforms. Isolated, but weak staining for HO-1 was seen in most tumours with focally increased expression in perinecrotic regions. Cells morphologically resembling macrophages stained with both HO-1 and HO-2, but were not numerous. These findings suggest that carbon monoxide, unlike nitric oxide, does not have a major role in regulating tumoural blood flow in this experimental glioma model. These findings once again demonstrate the differences between human malignant glioma and experimental implantation glioma models.     

Nitric oxide synthase is expressed in experimental malignant glioma and influences tumour blood flow

Summary The distribution and function of nitric oxide synthase (NOS) was studied in the rodent C6 implantation glioma model. Using a histochemical stain for NADPH diaphorase, which colocalises with NOS, morphological studies revealed non homogenous staining of the constituent tumour cells and the neoplastic endothelium. Immunocytochemical staining for macrophages (ED1, ED2) showed dense positivity at the tumour brain interface with more patchy positivity within the tumour mass. This finding suggests that both macrophages, which are known to produce large amounts of NO, and the C6 cells contribute to the NADPH diaphorase positivity. Administration of the NOS inhibitor N^g-nitro-L-argine methyl ester (L-NAME) significantly reduced both tumour (40%) and contralateral local cerebral blood flow (20%) compared to control animals. These findings demonstrate that (i) NOS is present in experimental malignant glioma; (ii) NO mediated mechanisms contribute to tumour blood vessel dilatation and blood flow regulation; and (iii) using this model there is a significant differential sensitivity of the tumour and brain parenchymal vascular beds to a NOS inhibitor. Further investigations are required to determine the potential therapeutic and biological relevance of these findings and the relative contributions of tumour cells, neoplastic endothelium and reactive macrophages to NO mechanisms in gliomas.Presented at the 2nd Meeting of the British Neurosurgical Research Group, Newcastle, March 1995     

激光间质热治疗对大鼠脑胶质瘤细胞凋亡的影响

目的：探讨激光间质内热疗（ILTT）是否诱导大鼠C6脑胶质瘤细胞凋亡。方法：SD大鼠70只,尾状核接种C6鼠脑胶质瘤细胞20d,随机分为：空白对照组,实验对照组和ILTT后2,6,12,24,48h实验组,每组10只。ILTT后不同时段按不同的检测要求保存标本。电镜观察,流式细胞仪分析和脱氧核苷酸转移酶（TdT)原位末端标记（TUNEL)法分析ILTT诱导C6胶质瘤细胞凋亡的作用。结果：电镜观察其呈现典型的凋亡形态学变化,可见凋亡小体形成;TdT原位末端标记显示凋亡发生在ILTT效应灶周边部,流式细胞仪检查ILTT后6h为凋亡发生的高峰时间（19．4％）;24h明显下降（2．2％）;48h与对照组差异无显著性（P＞0．05）。结论：ILTT确能诱导C6细胞凋亡。     

Randomized Phase III controlled trials of therapy in malignant glioma: where are we after 40 years?

The objective of this study was to review the results of randomized Phase III controlled trials (RCTs) that involve initial treatments of malignant glioma and determine changes in median survival times (MST) over the last 40 years. An electronic database search identified RCTs for patients undergoing initial treatment for supratentorial high-grade malignant glioma. MSTs were analysed with respect to the date that patient accrual to the trial started, to identify the time course of changes in MST. Linear regression was used for statistical analysis. The review included 44 clinical trials that recruited patients between 1966 and 2004. Overall, there was a steady significant improvement in MST for the novel treatment cohorts over this period (r(2) = 0.43, p < 0.001), with MST increasing from around 8 to 15 months. There was also consistent improvement in the MST of the control cohorts, from around 7 months to 14 months, that reached statistical significance (r(2) = 0.41, p < 0.001). However, analysis including a quadratic term revealed a trend towards the rate of improvement in MST decreasing in the last two decades in the control, but not novel treatment, groups. The differences, either positive or negative, in MSTs between the control and novel treatment cohorts, and number of trials performed have all decreased with time. Subgroup analysis of the three most recent clinical trials report statistically significant better outcomes in MST after either >90% or 'complete' tumour resection. Despite tremendous advances in both the understanding of the biology of malignant gliomas and treatments in neuro-oncology, the prognosis for patients with malignant gliomas, although improved, remains very poor. The limitations of this type of analysis, including how trial design can bias outcomes and militate against comparison of trials are discussed.     

Noisy intracranial tumours

Summary Transorbital sound recordings were obtained from 21 patients with intracranial tumours, 28 patients with intracranial aneurysms and 20 control patients. The group of patients with tumours consisted of 12 patients with gliomas, of whom 6 had low-grade gliomas and 6 had high-grade gliomas, and 9 patients with meningiomas. All patients with gliomas, including the subgroup of patients with low-grade gliomas, as well as patients with aneurysms, had significantly different sound recordings in comparison to control patients. Recordings from glioma patients did not differ significantly from recordings of aneurysm patients.Radiological evaluation of the tumours was performed in order to establish which tumour characteristics were associated with abnormal sound recordings. It was found that the type of tumour, i.e., histology or malignancy grade, was a significant associated factor, whereas other tumour characteristics such as size, mass effect and amount of oedema were not.In conclusion, patients with specific types of intracranial tumours produced abnormal sounds which could not be distinguished from abnormal sounds recorded in patients with aneurysms. These results may be important for the interpretation of sounds recorded for the detection of intracranial pathology, especially for aneurysm screening.