Summary p53 and the murine double minute 2 (MDM2) oncoprotein expression was evaluated in paraffin-embedded tissue from 61 patients with central nervous system gliomas (53 astrocytomas and eight oligodendrogliomas) and related to proliferation-associated markers [i.e. proliferating cell nuclear antigen (PCNA), Ki-67 and nuclear organizer regions (NORs)] and epidermal growth factor receptor (EGFR). We used the monoclonal antibodies PC-1 0, MIB-1, DO-1, 1 Bi 0 and EGFR 113 and the colloid silver nitrate (AgNOR) technique. MDM2 and p53were co-expressed in 28% of cases. A p53-positive/MDM2-negative phenotype was observed in 15% and a p53-negative/MDM2-positive phenotype in 20% of cases. There was a positive correlation of p53 and MDM2 expression with grade and proliferation indices. Univariate analysis in the group of diffuse astrocytomas showed that older age, high histological grade, high PCNA labelling index (LI) and high AgNOR score were associated with reduced overall survival (P < 0.05). p53 LI, Ki-67 LI, AgNOR score, tumour location and grade influenced diseasefree survival (P < 0.05), whereas the only parameters affecting post-relapse survival were histological grade and Ki-67 LI (P < 0.1). Multivariate analysis revealed that age, radiotherapy, PCNA LI and p53 LI were the independent predictors of overall survival. p53 LI, Ki-67 LI, MDM2 LI, EGFR LI, grade and type of therapy were independent predictors of disease-free survival, and grade was the only independent predictor of post-relapse survival. Our results indicate that p53 LI and MDM2 LI, EGFR expression as well as proliferation markers (PCNA and Ki-67) are useful indicators of overall and disease-free survival in diffuse astrocytoma patients.Keywords: proliferating cell nuclear antigen; Ki-67; MIB-1; AgNORs; p53; MDM2; epidermal growth factor receptor; gliomas Central nervous system (CNS) gliomas range in clinical behaviour and histological appearance from indolent well-differentiated lesions to highly anaplastic, rapidly growing neoplasms. A cardinal property of almost all types of gliomas is a propensity to recur and undergo anaplastic change (Russel and Rubinstein, 1989). A major stimulus to the study of cell proliferation in gliomas has been the widely held belief that quantification of this fundamental process will be of value in the objective categorization of these tumours. However, while cell kinetic information is an important aspect of the biology of gliomas, it has become clear that neoplastic evolution towards glioblastoma is a multistep process that involves deregulation of several genes related to both cellular proliferation and differentiation. The molecular determinants of glioma progression are still under investigation, with considerable attention directed towards the tumour-suppressor gene p53. On the basis of findings from molecular genetic analysis, Bigner and Vogelstein (1990)

Using immunohistochemistry we evaluated the expression of two negative regulators of the cell cycle, the retinoblastoma gene product (pRb) and the WAF1/Cip1 gene product (p21), in consecutive paraffin sections from 54 gliomas (49 astrocytomas and 5 oligodendrogliomas) and related it to clinicopathological parameters, proliferative fraction, p53 expression and survival. Survival analysis was restricted to the group of diffuse astrocytomas (48 patients). pRb expression did not correlate with histological type, grade or p53 expression, while a moderately strong correlation existed between pRb expression and the percentages of proliferating cell nuclear antigen (PCNA) and MIB-1-positive cells. In 30% of cases we observed diminished pRb expression (i.e., a low pRb/Ki-67 ratio), irrespective of grade or histological type. p21 protein was elevated in 50% of cases, especially within the higher grades. The percentage of p21-positive cells was not related to histological type or grade but correlated loosely with PCNA and pRb expression. A p53-negative/p21-negative phenotype was characteristic of oligodendrogliomas and low-grade astrocytomas, whereas the p53-positive/p21-positive, p53-positive/p21-negative and p53-negative/p21-positive phenotypes were almost equally distributed among high-grade tumors. In survival analysis (either univariate or multivariate) diminished pRb expression was not a statistically significant prognostic indicator. In contrast, p21 expression emerged as an important indicator of shortened disease-free survival, in both univariate and multivariate analyses. Moreover, the double-positive p53/p21 phenotype tended to be associated with a shorter overall survival. Our results suggest that Rb gene deregulation does not significantly affect prognosis but p21 expression may play an important role in disease-free survival of astrocytoma patients.

The relationship between proliferating cell nuclear antigen (PCNA) expression and various clinicopathological indices (age, sex, tumour location, histological type and grade and treatment) and post-operative survival were studied in patients with central nervous system gliomas using univariate and multivariate analysis. The expression of PCNA (PC10 score) was examined immunohistochemically using the monoclonal antibody PC10 on paraffin sections from 45 cases. Univariate analysis showed that a high PC10 score as well as older age, high histological grade and the histological type (astrocytoma) were associated with reduced survival. However, multivariate analysis revealed that only PC10 score and histological type had independent prognostic significance. The most important feature influencing PC10 score was the tumour grade. Regarding the patients who relapsed, the survival from the time of original diagnosis was related to the relapse-free period, while the PC10 score of the primary tumour emerged as the only independent predictor of survival following the first recurrence. These results indicate that PCNA expression is an independent prognostic indicator in CNS gliomas.

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The expression of two novel proliferation-associated markers, mitosin and topoisomerase II␣ (Topo II␣), was evaluated immunohistochemically in consecutive paraffin sections from 60 diffuse astrocytomas (grades 2 to 4) in relation to clinicopathologic parameters, proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1) expression and survival. The percentage of mitosin and Topo II␣-positive cells (LI) increased with grade and Ki-67 LI, but could not discriminate between grade 3 on the one hand and grades 2 or 4 on the other hand. In 51% of cases, Ki-67 LI exceeded Topo II␣ LI, especially within grade 4. Topo II␣ and mitosin expression was adversely related to overall and disease-free survival in the entire cohort and in grades 2/3. However, only Topo II␣ LI affected disease-free survival in grade 4 tumors. Multivariate analysis selected only mitosin LI along with the age of the patient, as the independent parameters predicting overall survival, whereas Topo II␣ emerged as the single independent predictor of disease-free survival. It is concluded that the proliferative potential of astrocytomas, as measured by mitosin and Topo II␣ immunostaining, conveys useful prognostic information, in addition to that obtained by standard clinicopathologic parameters.