Cellular and tumoural heterogeneity of EGFR gene amplification in human malignant gliomas

It has been demonstrated that the epidermal growth factor receptor (EGFR) gene, the normal human counterpart of the viral erb-B oncogene is amplified and overexpressed in over 50% of human malignant gliomas (HMGs). In the present study, analysis of the immunohistological staining characteristics of 57 HMGs using an anti-EGFR monoclonal antibody (mab) showed positive staining in 65% of the tumours with large cellular and regional differences in staining pattern and intensity. Screening a smaller number of HMGs with molecular hybridization techniques revealed 10/21 glioblastomas (48%) amplified for the gene; of 11 glioblastomas studied by Northern blot hybridization, 7 tumours with gene amplification showed RNA overexpression, the remaining 4 without amplification did not. Regional differences in DNA levels were observed by Southern blot in 2 tumours: in one particular case, amplification and overexpression were found to be localized to one half of a single HMG, the other half showing neither EFGR gene amplification nor overexpression.     

A commentary on the biology and growth kinetics of low-grade and high-grade gliomas

Cell kinetics studies of patients with various gliomas published in the past decade have shown that the average labeling index (LI) obtained from a pulse of 3H-thymidine is very high in medulloblastomas (12.0% +/- 1.3%, standard error of the mean) and glioblastoma multiforme (9.3% +/- 1.0%), low in well differentiated gliomas (less than 1%), and intermediate in anaplastic astrocytomas (4.0% +/- 0.8%). The higher the LI, the faster the tumor grows, probably reflecting a larger growth fraction. In tumor tissues obtained at autopsy, two glioblastomas diluted out the labeling compound in the 2- to 4-month interval after labeling, whereas three glioblastomas and two anaplastic astrocytomas retained labeled neoplastic cells for 3 weeks to 5 months. Most patients whose tumors contained foci of labeled cells at autopsy survived longer. Well differentiated gliomas harbored labeled cells for 2 1/2 to 7 years. These findings indicate that the kinetics of proliferation in well differentiated gliomas are different from those in glioblastomas or anaplastic astrocytomas.     

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.     

The pyruvate kinase isoenzyme shift in human gliomas: a potential marker in the treatment of gliomas

In primary human brain tumours a shift occurs in the synthesis of isoenzymes of pyruvate kinase from the M towards the K-type. In astrocytomas, oligodendrogliomas and glioblastomas, which were localised in the cerebral hemispheres of adult patients over 20 years of age, the shift correlated well with histological grading and growth rate as observed in postoperative survival. Gliomas of adults, localised in midline structures, as well as childrens gliomas were characterised too by a strong shift from M towards the K type. However, in these tumours, less correlation with histological grading and growth rate was found. The isoenzyme shift can be rapidly demonstrated with an alanine inhibition test. The application of this assay may have a diagnostic value during operation for gliomas in grading of malignancy in adults as well as demarcation of the resection of gliomas in all age groups. The test can be performed within 10-15 min and can thus fit easily into a surgical procedure. A case report is presented for illustration.     

The reliability of computer tomography for the diagnosis and differential diagnosis of meningiomas,gliomas, and brain metastases

Summary The CT characteristics were studied in a series of 90 tumours, and the diagnostic criteria were determined for meningiomas glioblastomas, other gliomas and metastases. These criteria were then employed in another group of 46 tumours in which the histological diagnosis was not known at the time of examination. The over-all diagnostic accuracy was 85%, and it was largely the same for the various sub-groups.Careful combination of the CT characteristics, perfect techniques, and increased experience may further improve the diagnostic accuracy.     

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.     

Regrowth patterns of supratentorial gliomas: estimation from computed tomographic scans

To clarify the regrowth patterns of benign and malignant gliomas, we chose 27 intervals (between two operations or between an operation and autopsy) from 21 patients with pathologically verified recurrent supratentorial gliomas. Serial computed tomographic (CT) scans of these cases were analyzed to determine the doubling time (Td) calculated from the change in volume of enhanced and low density areas, the enhancement effect graded from 0 to 4 according to the Hounsfield number, and the presence of dissemination and contralateral extension. We studied 5 benign gliomas (including 1 case of radiation necrosis), 8 malignant astrocytomas, and 8 glioblastomas. The Td's of enhanced areas on CT scans of benign gliomas, malignant astrocytomas, and glioblastomas were 937 +/- 66.5 days, 65.1 +/- 29.4 days, and 48.1 +/- 20.9 days, respectively. The Td's of low density areas were 895 +/- 130.6 days, 70.8 +/- 22.2 days, and 50.5 +/- 14.7 days. There was a significant correlation between the Td's of the enhanced and low density areas (0.97). The enhancement effect increased at recurrence in 55% of the cases, with an average increase of 1.1 grades. The increase in enhancement effect at recurrence showed a tendency to become smaller as the tumor's degree of anaplasia increased. Radiotherapy was effective in significantly retarding the growth rate of malignant gliomas, whose Td's were doubled. Although the Td's of both enhanced and low density areas of benign gliomas were significantly longer than those of malignant gliomas, there was no significant difference in the Td's of enhanced areas between malignant astrocytomas and glioblastomas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experiences of surgical treatment of 400 consecutive ruptured cerebral arterial aneurysms

Summary Brain samples were taken in macroscopically oedematous zones during 21 operations for malignant glioma, and studied with the electron microscope. Oedema was found in 17 cases, mostly as serous extracellular fluid. Vascular changes in the oedema region are mostly secondary to oedema: the origin of oedema fluid ought to be found in the tumour proper. Cellular reaction is slight; neuronal lesions may be ascribed mostly to a perturbation of axonal flow. Oedema round malignant gliomas and oedema round other malignant or benign tumours are morphologically distinct.Presented at the Symposium on Glioblastoma, Graz, October 25, 1975.     

Clinical pathology of primitive gliomas in the cerebrum

Summary To clarify the biological features of primitive gliomas in the cerebrum and clearly distinguish them from malignant or anaplastic gliomas and glioblastomas, we studied eight cases clinically and pathologically. Our evaluations included immunohistochemical and electron microscopic observations. We divided the patients into two groups, children and young adults. Most tumours appeared as ring-like, enhanced masses on computed tomography and avascular or ring-like, vascular masses on angiography. Macroscopically, the tumours were well demarcated and contained cysts. Ocassionally we found tumour dissemination. Microscopically, the tumours were composed of small, round cells without remarkable structural features. Ependymal, astroglial, and oligodendroglial differentiation was evident, in varying proportions; tumours in which the differentiated areas constituted more than half of the mass were classified as poorly differentiated gliomas. By these criteria, this series comprised four undifferentiated and four poorly differentiated gliomas. Cell anaplasia and polymorphism were rare in both undifferentiated and differentiated areas of the tumours. Immunohistochemical and electron microscopic examinations also revealed glial differentiation. These primitive gliomas appear to be biologically similar, but not identical, to cerebellar medulloblastomas. In this series, five patients died because of recurrence or dissemination. Whole brain and spinal irradiation should be considered after total or subtotal surgical removal.     

Stereotactic guided laser-induced interstitial thermotherapy (SLITT) in gliomas with intraoperative morphologic monitoring in an open MR: clinical expierence.

Stereotactic guided laser-induced interstitial thermotherapy (SLITT) is a minimal invasive method to produce thermonecrosis in cerebral tumour tissue. Clinical data are sparse due to its limited application until now and the value of this approach for tumour control and survival time remain to be defined. Twenty-four patients (7 low-grade gliomas, 11 anaplastic gliomas, 6 glioblastomas) with brain tumours, most recurrences, were treated with SLITT, in total 30 laser procedures were performed. Under local anaesthesia a 600 micro m laser-fiber was inserted by the stereotactic-guided technique. In open low-field MR the denaturation of the tumour by a Nd-YAG-laser (1064 nm) was monitored using T 1 -weighted 3-D turbo FLASH sequences. The ablation procedure had to be stopped twice because of neurological deficit, one major infection occurred. In two cases neurological improvement was observed. Mean survival times for low grade astrocytomas, anaplastic gliomas and glioblastomas were 144 months, 39 months, 17 months, respectively. Mean survival times after SLITT were 34 months, 30 months and 9 months, respectively. Mean times to progression after SLITT for the 3 histological subgroups were 16 months, 10 months and 4 months, respectively. Five patients with low grade astrocytoma and a KI greater or equal 70 maintained a high quality functional status for 11, 20, 21, 33 and 43 months. In anaplastic tumours patients maintained a KI of 70 for a median time of 15 months and for those with glioblastoma the respective high quality duration was 7.5 months after SLITT. SLITT for selected patients with glioma could have a clinical value in a multimodality treatment schedule maintaining quality of live. Due to the minimal invasive technique, the method is a therapy of choice and may be favoured to reoperation. Major indications of this treatment are small tumours, in eloquent regions and deep seated, as well as in older patients or patients in poor functional status.     

Milieu-specific vascular changes in malignant brain tumours

Summary Changes in blood-vessels, in configuration similar to arteriosclerosis, are found in malignant gliomas, especially in glioblastomas, as well as in metastases of carcinomas. Since analogous changes in vessel walls were not seen in primary tumours pertaining to cerebral metastases and in metastases of the body, this can obviously be considered as a phenomenon milieu-specific in nature. It is reasoned that this phenomenon can essentially be attributed to decomposition of medullary substance and influx of resulting disintegration products (lipids and cholesterol) into the vessel walls.     

Tenascin distribution in human brain tumours

Summary Using a monoclonal antibody specific for human tenascin (TN), 180 intracranial growths were immunohistochemically studied.In 69 cases of meningioma, neoplastic cells were negative, with some positivity being observed only in the perivascular and the supporting stroma, especially in anaplastic meningiomas. In 57 cases of glioma different degrees of reactivity occurred in both the cellular conglomerates and the stromal components of the tumours. A higher variability in reactivity was observed in anaplastic astrocytomas and glioblastomas.The most constant finding of the study was the staining of the stroma, which was observed in all types of growths, including metastasis, abscess and tuberculoma.The results are consistent with the hypothesis that tenascin is a stromal marker rather than a true marker of malignant tumours.The heterogeneous distribution of TN in anaplastic gliomas may be a factor in the variable response to treatment with radiolabelled anti-TN monoclonal antibodies.     

The relationship between the capillary structure and hemorrhage in gliomas

A 48-year-old man was admitted with the sudden onset of symptoms of stroke caused by hemorrhage in an oligodendroglioma. Despite surgery and antiedema treatment, the patient died. Histological evaluation revealed an oligodendroglioma with calcified capillaries of the retiform type. To further investigate this phenomenon, a total of 160 gliomas were reviewed: 90 glioblastomas multiforme, 30 oligodendrogliomas, and 40 astrocytomas. Sufficient data were available for clinical evaluation in 100 cases. Of these, 5% (two oligodendrogliomas and three glioblastomas multiforme) were related to clinically significant hemorrhages. Of the remaining cases, microhemorrhages were found in 53.0% of the glioblastomas, in 56.7% of the oligodendrogliomas, and in 10.0% of the astrocytomas. In each case reviewed, the capillaries were assigned to one of three groups: axial, retiform, or glomeruloid. Statistical analysis revealed a significant association between hemorrhages and retiform capillaries in all three types of tumors, except that in oligodendrogliomas the statistical significance held true when calcification of the capillaries was also present. Glomeruloid-type capillaries were only weakly associated with hemorrhages, and no association was found for axial capillaries. A large-scale prospective study is necessary to more precisely assess the role of each of the three types of capillaries in hemorrhages of gliomas. Based on data available so far, patients with glial tumors with retiform capillaries, confirmed on biopsy, should be carefully monitored to exclude possible intratumoral hemorrhage.     

Amplification of the epidermal growth factor receptor gene in biopsy specimens from human intracranial tumours

Amplification and overexpression of proto-oncogenes are associated with the malignant nature of some human tumours. In this study we have determined the prevalence of amplification of the proto-oncogenes c-erb B1 (= epidermal growth factor receptor gene), c-erb B2 and c-myc in 44 human intracranial tumours (27 gliomas, six metastases to the brain and 11 meningiomas). None of the tumours had an amplified c-erb B2 gene and only two tumours had an amplified c-myc gene. Nineteen per cent (five out of 27) of the gliomas, 50% (three out of six) of the brain metastases and 0% (0 out of 11) meningiomas had an amplified EGF-receptor gene. Amplification of the EGF-receptor gene appeared to give a growth advantage when single-cell suspensions of the tumours were grown in agarose.     

Angiogenesis and Brain Oedema in Intracranial Meningiomas: Influence of Vascular Endothelial Growth Factor

Summary The correlation between angiographic neovascularization, peritumoural brain oedema (PTBOe) and the expression of vascular endothelial growth factor (VEGF) , was analysed in 30 patients with intracranial meningiomas. Pre-operative angiograms were examined for the existence of either an exclusively dural tumour blush or an additionally pial tumour supply from cerebral arteries. Furthermore the presence of macroscopic tumour-neovascularization and dysplastic changes of tumour-draining cerebral veins was evaluated. VEGF expression was investigated on histological tissue samples, using immunohistochemical techniques. VEGF immunohistochemistry and neuroradiological evaluations were performed in double blind fashion. Tumour volume and the amount of oedema were calculated by computerized tomography (CT) or magnetic resonance imaging (MRI). The oedema-tumour volume ratio was defined as oedema index (OeI). Compared to VEGF-negative meningiomas, tumours with striking VEGF staining revealed a significant higher mean oedema index (OeI=4,2 vs. OeI=1,5; p<0.018), and a higher oedema incidence (91,7% vs. 44,4%; p<0.046). Equally, meningiomas with additionally tumour supply from cerebral arteries were associated with a significant higher mean OeI (OeI=4.1 vs. OeI=1.2; p<0.01) and oedema incidence (94,7% vs. 20,0%; p<0,0023) than meningiomas with exclusively tumour supply from dural arteries. All meningiomas with striking VEGF-expression were associated with vascular tumour supply from cerebral arteries, but VEGF-negative tumours only in 50% (p<0.029). These data suggest a link between VEGF-expression, arterial tumour supply and peritumoural brain oedema. The development of tumour supply from cerebral arteries may be important for formation of meningioma-related oedema. Therefore, VEGF may represent a potent mediator in the evolution of this type of vascularization in meningiomas.     

Intratumoral arteriovenous shunting in malignant gliomas

OBJECTIVE: Intratumoral arteriovenous shunting in glioblastomas has been suspected but neither proven nor quantified. METHODS: Using a previously described technique of selective intra-arterial intratumoral injection of 99mTc-labeled microparticles (macroaggregated albumin), we measured the amount of radioactivity, by cerebral and pulmonary scintigraphy, in seven patients with malignant gliomas (six with glioblastomas and one with an anaplastic oligodendroglioma). The pulmonary shunt index was calculated as a percentage from the pulmonary/pulmonary plus cerebral radioactivity ratio. RESULTS: The results revealed a mean pulmonary shunt index of 67% (range, 47-89%), indicating that most of the microparticles injected into the tumor via the arterial route bypassed the tumor and reached the lungs. The measured arteriovenous shunting was greater when the injection was performed in an artery exclusively perfusing the tumor. CONCLUSION: Important intratumoral arteriovenous shunting exists in glioblastomas. The potential consequences of this finding for intra-arterial treatment strategies are discussed.     

Clinicopathological study of diffuse type brainstem gliomas: analysis of 40 autopsy cases

Diffuse type brainstem glioma is one of the most malignant types of brain tumors and the prognosis is extremely poor. The proliferative potential of these tumors is presumed to be very high, but there is little information about the cell kinetics of brainstem glioma because surgical resection is rarely performed. The histological grade, tumor spread, growth potential, and prognosis were evaluated in 40 autopsy cases of diffuse type brainstem glioma. To quantify the growth potentials of individual tumors, the proliferating cell indices of Ki-67 (MIB-1) and proliferating cell nuclear antigen (PCNA) monoclonal antibodies were measured. Mean MIB-1 and PCNA proliferating cell indices were 20.4% (24 cases) and 37.0% (28 cases), respectively, in 34 glioblastomas. The median survival time was 40 weeks in 22 treated patients. The mean PCNA proliferating cell index was 10.8% in four of five anaplastic astrocytomas and the median survival time in four treated patients was 91 weeks. The MIB-1 and PCNA proliferating cell indices of one astrocytoma were 2.9% and 20.3%, respectively, and the survival time was 56 weeks. The overall median survival time was 32 weeks. There was a significant difference in PCNA proliferating cell indices between glioblastomas and anaplastic astrocytomas (p < 0.05) and there was a significant difference in survival time between glioblastomas (40 weeks) and anaplastic astrocytomas plus astrocytoma (74 weeks) among the treated patients (p < 0.05). Supratentorial extension was more frequent in glioblastomas than in anaplastic astrocytomas (p < 0.05). Our results suggest that the majority of diffuse type brainstem gliomas are glioblastoma and the proliferative potential is probably as high as that of adult supratentorial glioblastoma. Supratentorial extension and dissemination are relatively frequent in the advanced stage. Anaplastic astrocytoma or astrocytoma is rarer and less infiltrative and proliferative, and carries a slightly better prognosis than glioblastoma.     

Oedema formation in experimental photo-irradiation therapy of brain tumours using 5-ALA

Summary Background. Five-aminolevulinic acid (5-ALA) induces the specific accumulation of photosensitising porphyrins in malignant gliomas and has been explored for photo-irradiation therapy of these tumours. However, information is unavailable on whether and to what extent this treatment modality may induce the formation of brain oedema, and how potential oedema might be treated.Methods. Rats were implanted with C6 gliomas. Eight days later magnetic resonance images (MRI) were obtained. On day 9 rats received 100mg 5-ALA/kg b.w. and were craniotomized for photo-irradiation of tumours 6 hours later (100J/cm², 635nm argon-dye laser). Part of the animals was treated with daily dexamethasone injections (0.3mg/kg), beginning 6 hours before phototherapy. 72 hours later, brains were removed and dissected according to tumour dimensions on pre-therapy MRI into tumour, brain around tumour (BAT), residual cortex and basal ganglia, for measurements of water contents. Measurements were also performed in untreated animals with tumours, with or without steroid treatment and in control animals. An additional group of animals lacking tumours, with or without steroid treatment, underwent 5-ALA-phototherapy to determine effects on normal brain.Results. C6 gliomas induced brain oedema, which responded to steroid treatment. 5-ALA-phototherapy resulted in additional oedema, which responded partly to steroids. 5-ALA-phototherapy of normal brain increased water content moderately in irradiated cortex. This oedema was also partly counteracted by steroids.Conclusions. Photo-irradiation therapy with 5-ALA induces oedema which is partly counteracted by steroid therapy. The possibility of steroid resistant oedema formation should be considered when planning human trials with this treatment modality.     

Cerebral glioblastoma with cerebrospinal fluid dissemination: a clinicopathological study of 14 cases examined by complete autopsy

During the last 17 years, complete autopsies were performed on 51 patients who died of cerebral glioblastoma, and 14 were found to have dissemination by cerebrospinal fluid (CSF). In these 14 cases of glioma, the extent of intraparenchymal invasion by the primary tumor and the degree of seeding were studied in connection with histological findings and immunohistochemical staining for glial fibrillary acidic protein (GFAP) as the most reliable marker of astrocytic differentiation. From the findings obtained, the cases were divided into two groups. In one group, consisting of 7 gliomas, autopsy revealed intense seeding, despite only slight invasion by the primary tumor. Among these 7 extensively disseminated gliomas, 4 expressed almost no GFAP, 2 contained only a few GFAP-positive cells, and only 1 displayed an immunohistochemically high degree of astrocytic differentiation. Clinically, 6 of the 7 affected patients developed symptoms attributable to CSF seeding. In the other group consisting of the remaining 7 gliomas, only slight dissemination was seen, despite extensive infiltration of the primary tumor. Each of these 7 gliomas contained many GFAP-positive cells. None of the affected patients developed symptomatic seeding. This study shows the existence of two clinicopathologically distinct groups of disseminated cerebral glioblastomas and suggests that, regardless of morphological features, glioblastomas showing immunohistochemically poor astrocytic differentiation tend to shed tumor cells more vigorously but are less invasive at the primary site than those with many GFAP-positive cells. It is also suggested that, as a consequence, the former glioma type produces symptomatic seeding more frequently than the latter type.