Nucleolar organizer region score and Ki-67 labelling index in high-grade gliomas and metastatic brain tumours

Summary Sixteen cases of malignant brain tumours comprising 6 anaplastic astrocytomas, 3 glioblastoma multiforme, 1 medulloblastoma and 6 metastatic brain tumours were investigated independently by a silver colloid method for nucleolar organizer regions (NORs) and an immunohistochemistry using a monoclonal antibody against a nuclear antigen, Ki-67, in proliferating cells. The correlation between the mean number of NORs and the percentage of Ki-67 labelled cells (Ki-67 labelling index) was examined. In addition, four normal brain tissue samples without neoplastic cells were stained for NOR.The mean number of NORs in these malignant brain tumours was significantly greater than that in normal astrocytes (p<0.001). Moreover, both the mean number of NORs and the Ki-67 labelling index in metastatic brain tumours were significantly greater than those in high-grade gliomas (p<0.001). The Ki-67 labelling index and the mean number of NORs in malignant brain tumours including metastatic brain tumours were found to be linearly related (r=0.86).These results suggest that the proliferative potential of malignant brain tumours could be evaluated by NOR score as well as Ki-67 labelling index and that such indices provide clear discrimination between high-grade gliomas and metastatic brain tumours.     

Correlation between nucleolar organizer region staining and Ki-67 immunostaining in human gliomas.

Fourteen cases of various grades of gliomas were investigated by an immunohistochemical method using a monoclonal antibody, Ki-67, which reacted with a nuclear antigen in proliferating cells, and the correlation between Ki-67 labeling index and the number of nucleolar organizer regions stained by an argyrophilic method. Four normal brain tissue samples without neoplastic cells, which were obtained at surgery, were also examined for nucleolar organizer region staining. Both the mean number of nucleolar organizer regions and the percentage of Ki-67 positive cells well reflected the histological grade of gliomas (astrocytoma grade 2: Ki-67, 0.5% and nucleolar organizer regions, 1.6; astrocytoma grade 3: Ki-67, 3% and nucleolar organizer regions, 2.5; astrocytoma grade 4: Ki-67, 5.2% and nucleolar organizer regions, 2.8). Also, the percentage of Ki-67 positive cells and the mean number of nucleolar organizer regions were found to be linearly related (r = 0.91). Both values of high-grade astrocytomas were significantly greater than those of low-grade astrocytomas (p less than 0.01 and p less than 0.001). Thus, the mean number of nucleolar organizer regions as well as Ki-67 labeling index reflect the proliferating potential of gliomas.     

Identification of proliferating cells in human gliomas using the monoclonal antibody Ki-67

Ki-67 is a monoclonal antibody directed against a nuclear antigen present only in proliferating cells in the G1, S, G2, and M phases of the cell cycle. Fifty-one frozen glioma specimens were stained with Ki-67 using the avidin-biotin immunoperoxidase system. For each tumor, six different randomly selected fields were examined. The percentage of Ki-67-positive cells in the total number of cells in the five fields counted with counterstaining has been calculated. The areas of necrosis and the vascular endothelial cells when they were distinguishable were not included in the calculation. The indices determined on this material ranged from 0% to 4.5% (mean, 1.0; SD, 1.5) for 16 low grade astrocytomas; from 0.7% to 7.4% (mean, 3.5; SD, 2.2) for 8 anaplastic astrocytomas; and from 1.7% to 32.2% (mean, 11.1; SD, 8.2) for 27 glioblastomas. The differences among the means of each group are statistically significant. Five patients with malignant gliomas with an index of less than 2.5 had survival times of more than 40 weeks. These results show that the Ki-67 index of proliferating cells in human gliomas correlates with the usual histological classification of these tumors. There is a potential interest in using this technique in routine histopathology because it is simple and more rapid than the classic methods of evaluation of proliferating cells.     

Cell proliferation in serial biopsies through human malignant brain tumours: measurement using Ki67 antibody labelling

Cell proliferation was assessed in brain tumours using the monoclonal antibody Ki67 which recognizes a nuclear antigen expressed by proliferating cells. Using a novel stereotactic biopsy procedure, serial 1 cm biopsies were taken along a trajectory through six malignant brain tumours. Specimens were also obtained from 10 other brain tumours during conventional surgery. The percentage of Ki67 positive cells was determined as a fraction of the total number of tumour cells present. The Ki67 index for anaplastic astrocytomas and glioblastomas was significantly higher (Ki67 index range 11-18%) than that for benign or low grade tumours. Significant variation in proliferation was measured along the biopsy track through individual tumours (e.g. 0-12.3%) which correlated well with histological appearance. The Ki67 indices of normal brain were very low. In general the Ki67 indices increased with increasing histological grade and also appear to be a useful indicator of the active tumour volume and margin. This method provides spatial information about tumour proliferation which may be used to decide between different treatments and relate to prognosis.     

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.     

The use of the monoclonal antibody Ki-67 in the identification of proliferating cells: application to surgical neuropathology

In order to test its potential application to surgical neuropathology, the monoclonal antibody Ki-67 was used to demonstrate immunohistochemically the proliferating cells in 40 neoplasms of the nervous system. The antibody, which reacts with a nuclear protein expressed in the G1, G2, S, and M phases of the cell cycle, was demonstrated in frozen sections of all lesions. The highest incidence of stained nuclei was found in a metastatic carcinoma (57%). The percentage of stained cells in gliomas was in general agreement with the histologic grade and known biologic behavior of the lesions, ranging from 0.6% in a pilocytic astrocytoma to 12.4% in a glioblastoma multiforme. In the fibrillary astrocytic neoplasms of low cellularity, there were good correlations between the percentages of stained cells and the degrees of nuclear pleomorphism and chromatin density. In meningiomas, schwannomas, and a cerebellar hemangioblastoma, the fractions of labeled nuclei were less than 1%. The percentage of stained cells in pituitary adenomas showed considerable variation among the four cases (0.2-1.5%), the biologic significance of which is unknown. In four of the above cases, Ki-67 staining was performed on air-dried squash preparations with excellent visualization of immunoreactive nuclei. In one case, a hemangioblastoma, no stained nuclei were seen. The results confirm that Ki-67 staining is technically suitable as a diagnostic method, with good correlations between frozen sections and smear preparations. Determination of the replicating cell fraction could become an important additional criterion to predict the biologic behavior of nervous system neoplasms.     

Cell kinetics of human meningiomas and neurinomas--Ki-67 PAP stain

Seventeen surgically resected meningiomas and six neurinomas including one neurofibroma were labelled with frozen sections using a monoclonal antibody Ki-67, which reacts with a nuclear antigen expressed by proliferating cells. In thirteen classic non-malignant meningiomas, the percentage of stained cells ranged from 0.2 to 3.2% (mean, 1.4%). Those of three recurrencies consisted of anaplastic one and another one with anaplastic component, and one hemangiopericytic type ranged from 2.9 to 8.7% (mean, 4.8%). Four classic non-malignant neurinomas and one neurofibroma had the range from 0.3 to 2.7% (mean, 1.3%). However, one anaplastic neurinoma showed the largest fraction (13.0%) of positive nuclei in all these tumors. These results show, in conclusion, that the determination of Ki-67 immunoreactivity in the cell nuclei of meningiomas and neurinomas can contribute to more objectiveness in histological grading and the higher positive rate could be one of various factors relating to recurrence.     

Vascular permeability and cell kinetics of ethylnitrosourea (ENU)-induced rat brain tumours

Summary Vascular permeability and proliferative activity of ethylnitrosourea (ENU)-induced rat brain tumours were studied by intravenous injection of Evans blue dye (EB) and by bromodeoxyuridine (BrdU) uptake examinations. Tumours induced by ENU showed various histologial types, and they were oligodendrogliomas, mixed oligo-astrocytomas, mixed oligo-ependymomas, astrocytomas, anaplastic astrocytomas, polymorphic gliomas, and ependymomas. The labelling indexes (LIs: the ratio of BrdU-labelled cells to total cells) of tumour and vascular component cells in the tumour were high in anaplastic astrocytomas, polymorphic gliomas and ependymomas, but low in oligodendrogliomas. EB stained anaplastic astrocytomas, polymorphic gliomas and ependymomas deeply, but did not penetrate oligodendrogliomas. In mixed gliomas, EB staining and the LIs of tumour cells were not uniform. After intracarotid infusion of hyperosmolar mannitol into tumour-bearing rats, tumour staining with EB and the LIs of tumour cells were not increased, whereas the penetration of EB into the normal brain was drastically increased. Therefore it is not likely that the delivery of chemotherapeutic drugs to the tumour could be increased by intracarotid infusion of hyperosmolar manitol. Our data suggest that the vascular permeability of tumour vessels is highly correlated with the high proliferative activity of tumour and its vascular cells.     

Use of positron emission tomography (PET) in stereotactic conditions for brain biopsy

Summary In order to take advantage of the metabolic information provided by positron emission tomography (PET) in cases of brain tumour, we have developed a technique to integrate PET images routinely in the planning of stereotactic brain biopsy. We used stereotactic PET with [¹⁸F]-labelled fluorodeoxyglucose (PET-FDG) in 38 patients undergoing brain biopsy. To evaluate the contribution of PET-FDG in guiding brain biopsy, we analyzed the diagnosis provided by the 78 Stereotactic trajectories obtained in these patients.We found that stereotactic PET-FDG seemed to provide more information in cases of anaplastic astrocytomas and glioblastomas than in low-grade gliomas. Our results also show that biopsy trajectories performed in areas where increased FDG uptake is found within the lesion boundaries always provide interpretable specimens; this was not the case for trajectories guided by CT only. Therefore, the routine integration PET-FDG in the planning of stereotactic brain biopsy may lead to a reduction in sampling. Recently, we also tested consecutive stereotactic PET with [¹¹C]-labelled methionine (PET-Met) and PET-FDG. This technique allowed us to compare accurately the tumoural glucose metabolism and protein synthesis.Our results suggest that stereotactic PET may increase the diagnostic yield of brain biopsy and may improve the understanding of PET in neuro-oncology.     

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.     

Ability of navigated 3D ultrasound to delineate gliomas and metastases – comparison of image interpretations with histopathology

Summary Background. The objective of the study was to test the ability of a 3D ultrasound (US) based intraoperative imaging and navigation system to delineate gliomas and metastases in a clinical setting. The 3D US data is displayed as reformatted 2D image slices. The quality of the displayed 3D data is affected both by the resolution of the acquired data and the reformatting process. In order to investigate whether or not 3D US could be used for reliable guidance in tumour surgery, a study was initiated to compare interpretations of imaged biopsy sites with histopathology. The system also enabled concomitant comparison of navigated preoperative MR with histopathology. Method. Eighty-five biopsies were sampled between 2–7 mm from the tumour border visible in the ultrasound images. Biopsies were collected from 28 operations (7 low-grade astrocytomas, 8 anaplastic astrocytomas, 7 glioblastomas and 6 metastases). Corresponding cross-sections of preoperative MR T1, MR T2 and intraoperative US were concomitantly displayed, steered by the biopsy forceps equipped with a positioning sensor. The surgeons’ interpretation of the images at the electronically indicated biopsy sites were compared with the histopathology of the samples. Findings. The ultrasound findings were in agreement with histopathology in 74% (n = 31) for low-grade astrocytomas, 83% (n = 18) for anaplastic astrocytomas, 77% (n = 26) for glioblastomas and 100% (n = 10) for metastases. Excluding irradiated patients, the results for glioblastomas improved to 80% concurrence (n = 20). As expected tumour cells were found in biopsies outside the US visible tumour border, especially in low-grade gliomas. Navigated 3D US have a significantly better agreement with histopathology than navigated MR T1 for low-grade astrocytomas. Conclusion. Reformatted images from 3D US volumes give a good delineation of metastases and the solid part of gliomas before starting the resection. Navigated 3D US is at least as reliable as navigated 3D MR to delineate gliomas and metastases.     

The correlation of Ki-67 staining indices with tumour doubling times in regrowing non-functioning pituitary adenomas

Summary In order to improve our ability to predict the regrowth of nonfunctioning pituitary adenomas, we tried to assess the correlation between growth fractions with Ki-67 and PCNA (proliferating cell nuclear antigen) and tumour doubling times in regrowing tumours, and also to find out any difference of growth fractions between the regrowing and the cured cases.In 33 patients with non-functioning pituitary adenomas, 14 cases including 11 with cavernous sinus invasion showed residual tumour on MRI after the operation (regrowing group) and 19 cases had no tumour regrowth on MRI within 5 years after the operation (cured group). Immunocytochemical studies were done with monoclonal antibodies (anti-PCNA, anti-Ki-67: MIB-1). The growth fraction of each tumour was estimated by calculating the ratio of the positive nuclei to the total number of tumour cells with the aid of an image analyser (Mac SCOPE). The tumour doubling times were estimated from serial CT or MRI with the aid of the image analyser (NIH image).Ki-67 staining indices ranged from 0.2% to 1.5% (n = 14, 0.86±0.10%; mean±SEM) in the regrowing group, and from 0.1% to 0.5% (n = 19, 0.23±0.03%) in the cured group. PCNA staining indices of the regrowing group ranged from 0.6% to 24% (n = 14, 3.7±1.6%). In the regrowing group, the tumour doubling times ranged from 200 to 2550 days (930±180 days), and showed a significant inverse correlation with Ki-67 staining indices, but no correlation with PCNA staining indices. The regrowing group showed a significantly higher Ki-67 staining index (n = 14, 0.86±0.10%) than the cured group (n = 19, 0.23±0.03%) (p<0.01).These results indicate that immunocytochemical studies using MIB-1 may be better than those with PCNA for the prediction of regrowth in non-functioning pituitary adenomas. Immunocytochemical study with MIB-1 could lead to the accurate prediction of the rapid regrowing lesions in non-functioning adenomas.     

Role of thallium SPECT for evaluating the effect of maintenance chemotherapy against malignant gliomas

Thallium (Tl)-201 single-photon emission computed tomography (SPECT) is a useful tool for detecting brain tumors. In this study, we evaluated the utility of Tl-201 SPECT for determining the effect of maintenance chemotherapy with ACNU (nimustine hydrochloride)/VCR (vincristine sulfate) against malignant gliomas. The cases were comprised of 16 glioma cases; 6 astrocytomas, 2 anaplastic astrocytomas, and 8 glioblastomas. We first analyzed the correlation between Tl-201 uptake ratio and proliferative activity of the tumor, using Ki-67 immunohistochemistry in 13 cases of glioma. The uptake ratio of Tl-201 correlated with the Ki-67 staining indices (SI), and a closer correlation was obtained using Tl-201 delayed images than with the early images. We also analyzed the chronological changes of Tl-201 uptake ratio and volume of abnormal area evaluated by MRI T2-weighted imaging (MRI T2WI), in 10 cases of malignant glioma during maintenance chemotherapy. The Tl-201 uptake ratio gradually decreased with the effect of maintenance chemotherapy, and the sensitivity was superior to MRI findings. Together with MRI, Tl-201 SPECT is considered to be a useful indicator for evaluating the effect of maintenance chemotherapy against malignant gliomas.     

Cellular targets of exogenous tumour necrosis factor-alpha (TNFα) in human gliomas

Summary To identify the cellular targets of TNF in human gliomas, a total of 30 surgical specimens (12 glioblastomas, 4 anaplastic astrocytomas, 3 astrocytomas, 7 brains adjacent to tumour (BAT), 4 histologically normal-appearing brains) were examined by in vitro binding technique using biotinylated TNFa. The TNF-binding sites (TNF-BS) were recognized in the tumour cells in 8 of the 12 glioblastomas. 3 of the 4 anaplastic astrocytomas and in all the 3 astrocytomas. The TNF-BS were also recognized in the vascular endothelial cells in all these cases. The presence of TNF-BS in blood vessels ranged from 7.7 to 74.4% of the background vessels. This wide range of variation in the presence of TNF-BS within the tumour cells and tumour blood vessels may be relevant to the variable response of individual tumours to TNF therapy. Since the tissue of normal brain, which lacks TNF-BS, might hardly be affected by this cytokine, administration of TNFa may be considered as an adjuvant therapy in selected groups of patients.     

Management of deep-seated gliomas

The management of thalamic and brain stem astrocytomas remains controversial. Treatment options are: (a) clinical observation, (b) radiotherapy without biopsy, (c) stereotactic biopsy followed by radio and/or chemotherapy, and (d) surgical removal with or without adjuvant therapy. Stereotactic surgical techniques have improved the morbidity and mortality rates of biopsies and surgical resection of deep-seated gliomas. The biologic behavior of these lesions is not well known and proliferation cell index tests may help in the choice of therapy. In this review, seven recent papers on the management of deep-seated gliomas are presented. Radical removal of thalamic pilocytic astrocytoma may cure the patient. In cases of low-grade astrocytomas, stereotactic guided surgical removal has low morbidity. Adjuvant radiotherapy should be used only in selected cases. Sterotactic biopsy followed by radio- and/or chemotherapy is the best option for thalamic or brain stem anaplastic astrocytomas and glioblastomas.     

Evaluation of radiation therapy in pediatric brain stem glioma by computed tomography: CT findings and tumor response to radiotherapy

The effects of radiation therapy on 29 brain stem gliomas in childhood were evaluated by computed tomography (CT). The patients received radiation of 2 Gy/day as a single fraction, 5 day a week with a total dose of 40 to 60 Gy. Initial CT findings of brain stem gliomas were divided into two types: diffuse and localized. Of 29 children, 5 had localized and 24 had diffuse tumor. Histological diagnoses were available for 18 patients, 4 with localized and 14 with diffuse tumor. All of the localized tumors were astrocytomas and diffuse tumors included 13 anaplastic gliomas (glioblastomas), 3 anaplastic astrocytomas, and one astrocytoma. Complete response or partial response to radiation therapy was observed on CT in 100% (5/5) of the localized tumors and 46% (11/13) of the diffuse tumors at the first evaluation. Contrary to expectation, low-grade gliomas responded much better to radiation therapy than high-grade gliomas. The response rates were 80% (4/5) in astrocytoma, 67% (2/3) in anaplastic astrocytoma, and 38% (5/13) in anaplastic glioma. In the follow-up CT after radiation therapy, a delayed effect was observed in only one of the 24 diffuse tumors. Nine of 10 children who had a re-irradiation following the recurrence experienced very little benefit. None of the patients with localized tumors have shown evidence of tumor progression or recurrence, and the quality of their life has been excellent. On the other hand, all of the patients with diffuse tumor died within 20 months after initial treatment. The results of this study suggest that radiation therapy is beneficial for localized tumors but not for diffuse tumors, and new treatments need to be developed for diffuse tumors.     

Immunohistochemical demonstration of DNA polymerase alpha in human brain-tumor cells

The proliferative capacity of brain-tumor cells was analyzed in vitro and in situ using monoclonal antibody (MAb) against deoxyribonucleic acid (DNA) polymerase alpha. For the in vitro studies, two cultured human glioma cell lines were investigated using MAb against DNA polymerase alpha, the MAb Ki-67, a serum against proliferating cell nuclear antigen (PCNA/cyclin), bromodeoxyuridine (BUdR), and an anti-BUdR MAb. During exponential growth of the cells, the percentage of polymerase alpha-positive cells (the "polymerase alpha score") ranged from 72.0% to 77.1%, the Ki-67-positive cells (the "Ki-67 score") ranged from 43.4% to 59.4%, the PCNA/cyclin-positive cells from 30.9% to 41.4%, and the BUdR labeling index from 28.6% to 39.3%. For the in situ studies, tissue from 60 human brain tumors and from two normal human brains was investigated and the polymerase alpha scores and Ki-67 scores were compared. In normal brain tissue, no immunostaining was found by either method. In brain tumors, both the polymerase alpha scores and the Ki-67 scores correlated with the histological grade of malignancy. Polymerase alpha scores were generally higher than Ki-67 scores in the same specimen, especially in malignant brain tumors. These findings suggest that immunostaining of DNA polymerase alpha is a convenient and important new method by which to estimate the cellular proliferation rate of brain tumors. Polymerase alpha scores may be closer to the growth fraction of the individual tumor than the MAb Ki-67 or other scores.     

Stereotactic biopsy guidance in adults with supratentorial nonenhancing gliomas: role of perfusion-weighted magnetic resonance imaging

OBJECT: The. diagnosis of low-grade glioma (LGG) cannot be based exclusively on conventional magnetic resonance (MR) imaging studies, and target selection for stereotactic biopsy is a crucial issue given the high risk of sampling errors. The authors hypothesized that perfusion-weighted imaging could provide information on the microcirculation in presumed supratentorial LGGs. METHODS: All adult patients with suspected (nonenhancing) supratentorial LGGs on conventional MR imaging between February 2001 and February 2004 were included in this study. Preoperative MR imaging was performed using a dynamic first-pass gadopentate dimeglumine-enhanced spin echo-echo planar perfusion-weighted sequence, and the tumors' relative cerebral blood volume (rCBV) measurements were expressed in relation to the values observed in contralateral white matter. In patients with heterogeneous tumors a stereotactic biopsy was performed in the higher perfusion areas before resection. Among 21 patients (16 men and five women with a mean age of 36 years, range 23-60 years), 10 had diffuse astrocytomas (World Health Organization Grade II) and 11 had other LGGs and anaplastic gliomas. On perfusion-weighted images demonstrating heterogeneous tumors, areas of higher rCBV focus were found to be oligodendrogliomas or anaplastic astrocytomas on stereotactic biopsy; during tumor resection, however, specimens were characterized predominantly as astrocytomas. Diffuse astrocytomas were associated with significantly lower mean rCBV values compared with those in the other two lesion groups (p < 0.01). The rCBV ratio cutoff value that permitted better discrimination between diffuse astrocytomas and the other lesion groups was 1.2 (80% sensitivity and 100% specificity). CONCLUSIONS: Perfusion-weighted imaging is a feasible method of reducing the sampling error in the histopathological diagnosis of a presumed LGG, particularly by improving the selection of targets for stereotactic biopsy.