EVALUATION OF TUMOUR ANGIOGENESIS AS A PROGNOSTIC MARKER IN MALIGNANT MESOTHELIOMA

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumours. Malignant mesothelioma (MM) of the pleura is a highly invasive tumour with a poor prognosis. In the present study, microvascular quantification was undertaken on 25 specimens of mesothelioma and 15 specimens of non-neoplastic mesothelium (NNM), by staining for the antigens CD34 and CD31. Areas of highest intratumoural microvascular density (IMD) were identified and counted either manually (mIMD) or on a computerized image analysis system (CIAS; iIMD). The two IMDs were significantly correlated with each other ( r =0·736; P <0·001). The average IMD for MM was significantly ( P <0·001) higher than in NNM. Moreover, each unit increment in iIMD for MM, when regarded as a continuous variable, was significantly ( P =0·001) associated with an increased hazard of about 4 per cent. When regarded as a categorical variable, the patients in the highest tertile (>58 vessels/field) had a significantly ( P <0·01; log-rank test) shorter survival than patients in the lowest tertile (<45 vessels/field). This association was independent of the age of the patient and of the histological type or grade of the MM. No association was noted with p53 immunoexpression. Although the mean vascular area of blood vessels measured on the CIAS did not correlate with survival, assessment of IMDs can be an important independent prognostic indicator in malignant mesothelioma. © 1997 John Wiley & Sons, Ltd.     

Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival

The formation of new vessels (angiogenesis) is essential for primary tumour growth and metastasis and is induced by several angiogenic factors, including vascular endothelial growth factor (VEGF). The microvascular density (MVD) in tumours was assessed and the expression of VEGF and its receptors VEGF-R1-Flt1 and VEGF-R2-KDR/Flk1 was investigated in the different cellular compartments in vivo, in order to establish their interrelationship and their prognostic influence. Immunohistochemical study of 69 stage I–II non-small cell lung carcinomas (NSCLCs) was performed on paraffin sections with CD34 antibody to estimate MVD, using a Chalkley eye-piece graticule and VEGF, VEGF-R1, and VEGF-R2 antibodies. There was strong expression of VEGF and its receptors in tumour cells, endothelial cells, and stromal fibroblasts. In tumour cells, the level of VEGF was correlated with that of VEGF-R1 ( p = 0·018) but not that of VEGF-R2. In fibroblasts, high expression of VEGF was correlated with that of VEGF-R1 ( p = 0·0001) and VEGF-R2 ( p = 0·0001). In endothelial cells, expression of VEGF was correlated with that of VEGF-R1 ( p < 0·0001) and VEGF-R2 ( p = 0·04). The level of VEGF in fibroblasts was correlated with that of VEGF-R1 ( p = 0·0028) and VEGF-R2 ( p = 0·01) in endothelial cells. There was no correlation between the level of MVD and that of VEGF or VEGF-R1 or VEGF-R2. Neither the level of MVD, nor the level of expression of VEGF and VEGF receptors in any compartment influenced the patient's survival. In conclusion, although angiogenesis is essential for tumour growth, this study failed to demonstrate that MVD, VEGF, VEGF-R1, and VEGF-R2 are prognostic markers for stage I–II NSCLC. VEGF, however, might act as a direct autocrine growth factor for tumour cells via VEGF-R1 and angiogenesis could be promoted in a paracrine loop, where VEGF is produced by fibroblasts and tumour cells and then binds to endothelial cells via induced VEGF receptors. VEGF and its receptors thus appear as relevant therapeutic targets in NSCLC. Copyright © 1999 John Wiley & Sons, Ltd.     

Syndecan-1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome

Syndecan-1 binds basic fibroblast growth factor (bFGF), modulates neovascularization, plays a role in epithelial differentiation and is up-regulated by WT1. Malignant mesothelioma of the pleura is one of the most aggressive tumours known and expresses high levels of angiogenic growth factors. This study has analysed syndecan-1 expression in mesothelioma tumours and cell lines by immunohistochemistry and immunoblotting, using anti-syndecan-1 antibody directed against the core protein, and has examined its relation to morphology, bFGF, WT1, and intra-tumoural microvascular density (IMD). Shedding of syndecan-1 in the conditioned medium of mesothelioma cell lines was detected in variable amounts. These studies indicate that (1) there is no correlation of syndecan-1 with either bFGF expression or IMD in mesotheliomas in vivo; (2) syndecan-1 is strongly expressed in the epithelial type of mesothelioma and in the epithelial component of biphasic mesotheliomas and the expression is reduced or lost in sarcomatoid differentiation; together with the finding that (3) syndecan-1 correlates with WT1 immuno-expression, this suggests that syndecan-1 might relate to the differentiation state of mesothelial/mesothelioma cells; and (4) syndecan-1-positive tumours are associated with a longer survival (p = 0.02) than mesotheliomas with no or little syndecan-1 expression, on univariate analysis. These findings therefore indicate that syndecan-1 can be an important prognostic indicator in mesotheliomas and its loss may be important in the epithelial-mesenchymal transformation of mesothelioma cells.     

Prognostic value of p53, bcl-2, and c-erbB-2 protein expression in phaeochromocytomas

Many studies have tried to discriminate malignant from benign phaeochromocytomas, but until now no widely accepted histological, immunohistochemical, or molecular methods have been available. In this study of 29 malignant and 85 benign phaeochromocytomas from 102 patients, immunohistochemistry was performed with antibodies to the tumour suppressor gene product p53 and the proto-oncogene products bcl-2 and c-erbB-2, using the avidin–biotin complex method. Malignant phaeochromocytomas showed a statistically significant higher frequency of p53 (p=0·042) and bcl-2 (p=0·037) protein expression than their benign counterparts. The combination of both markers showed an even higher significance (p=0·004), to which both markers contributed equally. Overexpression of c-erbB-2 was associated with the occurrence of familial phaeochromocytomas (p=0·001), but no difference was found between benign and malignant cases. In conclusion, p53, bcl-2, and c-erbB-2 all appear to be involved in the pathogenesis of a proportion of phaeochromocytomas. Immunoreactivity to p53 and bcl-2 proteins may help to predict the clinical behaviour of phaeochromocytomas. Copyright © 1999 John Wiley & Sons, Ltd.     

SV40-like DNA sequences in pleural mesothelioma,bronchopulmonary carcinoma,and non-malignant pulmonary diseases

Pleural and pulmonary malignancies are usually associated with well-known carcinogen exposure. Recently, the presence of simian virus 40 (SV40)-like DNA sequences has been detected in brain and bone-related human cancers and in pleural mesothelioma. In order to determine whether SV40-like DNA sequences are also present in bronchopulmonary carcinoma and non-malignant lung samples, 125 frozen pleural and pulmonary samples (including 21 mesotheliomas, 63 bronchopulmonary carcinomas, 8 other tumours, and 33 non-malignant samples) and 38 additional samples distant from tumours were studied for the occurrence of SV40-like DNA sequences by polymerase chain reaction (PCR) amplification followed by hybridization with specific probes. Sequences related to SV40 large T antigen (Tag) were present in 28·6 per cent of bronchopulmonary carcinomas, 47·6 per cent of mesotheliomas, and 16·0 per cent of cases with non-neoplastic pleural and pulmonary disease. No statistically significant difference in the occurrence of these DNA sequences was found between malignant mesothelioma and bronchopulmonary carcinoma, but a significantly higher number of mesothelioma cases exhibited SV40-like DNA sequences in comparison with cases of non-malignant pleural or pulmonary disease (P<0·04). Among cases positive for SV40-like DNA sequences, a history of asbestos exposure was found in 3 out of 12 bronchopulmonary carcinomas and 8 out of 10 mesotheliomas. Immunohistochemistry using monoclonal antibodies directed against Tag did not demonstrate nuclear staining. The DNA sequences were not related to BK virus sequences, but three samples were positive with probes hybridizing with JC virus DNA sequences. In conclusion, this study demonstrates the presence of SV40-like DNA sequences in pulmonary neoplasms and in non-malignant lung tissues. It appears that the presence of SV40-like DNA is not unique to cancer. © 1998 John Wiley & Sons, Ltd.     

Immunohistochemical distribution patterns of epidermal growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium

Summary An immunohistochemical study of the epidermal growth factor (EGF) receptor in non-neoplastic pleural mesothelium (35 cases) and in human malignant mesothelioma (36 cases) was made, using a murine monoclonal antibody OM-11-951. All malignant mesotheliomas and non-neoplastic pleural biopsies exhibited a strong cytoplasmic immunoreactivity in mesothelial cells. Nuclear immunoreactivity was detected in mesothelial cells of all specimens of both malignant and non-neoplastic pleura. No statistically significant differences were found between malignant mesothelioma and non-neoplastic pleural mesothelium. There were differences, between the three subtypes of mesothelioma, in the number of cells that exhibited nuclear staining. Statistically significant differences were noted between the epithelial subtype and the mesenchymal subtype (P< 0.005), epithelial subtype versus the mixed cell type (P< 0.005) and between the mesenchymal component of the mixed cell type and the mesenchymal type (P<0.0005). We conclude that there is strong expression of EGF receptor in both malignant mesothelioma and in non-neoplastic pleural mesothelium. Different staining patterns are seen when comparing the different subtypes of mesotheliomas with each other. EGF receptor expression cannot be used to distinguish between malignant and benign mesothelium.     

Apoptosis suppressing protein bcl-2 is expressed in well-differentiated breast carcinomas with favourable prognosis

The expression of bcl-2 protein was analysed immunohistochemically in 202 female breast carcinomas. The intensity of bcl-2 expression was inversely related to tumour grade (P<0·0001), tumor necrosis (P<0·0001), mitotic index (P<0·0001), oestrogen receptor content (P<0·0001), progesterone receptor content (P=0·0007), S-phase fraction (P=0·00047), and apoptotic index (P=0·087). A high fraction of bcl-2-positive cells was related to ductal or lobular type (P=0·03) and slight nuclear pleomorphism (P=0·03). Heterogeneous expression of bcl-2 protein was associated with high grade (P=0·02), severe nuclear pleomorphism (P=0·02), DNA aneuploidy (P=0·018), high S-phase fraction (P=0·05), and early metastasis (P=0·03). Intense expression of bcl-2 protein was significantly related to favourable outcome in the entire cohort (P=0·0013), as well as in axillary lymph node-negative (ANN) tumours (P=0·0124). Long recurrence-free periods in the entire cohort (P=0·037) and in ANN tumours (P=0·08) were confined to cases with intense expression of bcl-2 protein. In multivariate analysis, bcl-2 expression had no independent prognostic value in the entire cohort or in axillary lymph node-negative breast carcinomas, whereas it was a weak independent prognostic factor in axillary lymph node-positive breast carcinomas.     

Characterization of Fibroblast Growth Factor-2 Binding to Ribosomes

Abstract

We have examined the binding of FGF-2 to ribosomes and have found that in NIH 3T3 cells that synthesize high amounts of all FGF-2 forms, both 18 kDa and HMW forms of FGF-2 bind to ribosomes. Ribosomes purified from these cells were treated with RNase or puromycin to identify the binding site of FGF-2 on the ribosome. Neither RNase nor puromycin treatment affected the in vivo binding of FGF-2 to ribosomes suggesting that FGF-2 binds ribosomal protein or rRNA, but not mRNA. The stoichiometry of binding in these cells was approximately 1 FGF-2 molecule bound per 1 ribosome. Binding was unaffected by high salt treatment indicating that FGF-2 tightly associates with polysomes. An in vitro binding experiment performed with purified ribosomes and recombinant FGF-2 suggested that the binding site is saturable. HBNF, a protein with similar charge and size to FGF-2, bound 15-fold less than FGF-2 to purified ribosomes. These results indicate that the binding of FGF-2 to ribosomes is specific.     

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non-small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-N0,1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P=0.04) and both were inversely associated with microvessel density (P<0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P<0.01). A significant co-expression of c-erbB-2 with TP was noted (P=0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P<0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P=0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c-erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c-erbB-2-related immune response remains to be further investigated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of CD44 standard and variant-v6 proteins in transitional cell bladder tumours and their relation to prognosis during a long-term follow-up

The expression of the standard CD44 (CD44s) and its v6 isoform (CD44v6) was analysed immunohistochemically in 173 cases of transitional cell bladder cancer. The results of immunohistochemical analyses were related to established prognostic factors and clinical follow-up data. The expression intensity of CD44s in non-basal tumour cells was significantly related to TN classification, S-phase fraction (SPF), mitotic index, grade, density of tumour infiltrating lymphocytes. The expression intensity of CD44v6 in non-basal tumour cells was inversely related to DNA ploidy, SPF, and mitotic index. The expression intensity of CD44v6 in basal tumour cells was also inversely related to T-category, grade, papillary status, DNA ploidy, SPF, and mitotic index. Strong expression of CD44s in non-basal tumour cells was related to unfavourable outcome in univariate analysis (p=0·008), whereas the strong expression of CD44v6 in both non-basal cells (p=0·005) and basal cells (p=0·0008) was related to high survival probability. In multivariate survival analysis, the expression intensity of CD44v6 was independently related to favourable outcome in muscle invasive tumours, while in superficial tumours, CD44s was an independent prognostic factor. The results suggest that the expression of CD44s and CD44v6 is associated with malignant features and prognosis in bladder cancer. Copyright © 1998 John Wiley & Sons, Ltd.     

Evaluation of membrane‐bound and soluble forms of human leucocyte antigen‐G in systemic sclerosis

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen‐G (HLA‐G) is a non‐classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA‐G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA‐G is also detectable in soluble form (sHLA‐G) deriving from the shedding of surface isoforms (sHLA‐G1) or the secretion of soluble isoforms (HLA‐G5). Several immunosuppressive functions have been attributed to both membrane‐bound and soluble HLA‐G molecules. The plasma levels of sHLA‐G were higher in SSc patients (444·27 ± 304·84 U/ml) compared to controls (16·74 ± 20·58 U/ml) (P < 0·0001). The plasma levels of transforming growth factor (TGF)‐β were higher in SSc patients (18 937 ± 15 217 pg/ml) compared to controls (11 099 ± 6081 pg/ml; P = 0·003), and a significant correlation was found between TGF‐β and the plasma levels of total sHLA‐G (r = 0·65; P < 0·01), sHLA‐G1 (r = 0·60; P = 0·003) and HLA‐G5 (r = 0·47; P = 0·02). The percentage of HLA‐G‐positive monocytes (0·98 ± 1·72), CD4⁺ (0·37 ± 0·68), CD8⁺ (2·05 ± 3·74) and CD4⁺CD8⁺ double‐positive cells (14·53 ± 16·88) was higher in SSc patients than in controls (0·11 ± 0·08, 0·01 ± 0·01, 0·01 ± 0·01 and 0·39 ± 0·40, respectively) (P < 0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA‐G molecules and the membrane expression of HLA‐G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA‐G molecules in the immune dysregulation of SSc.     

An immunohistochemical examination of the expression of E-cadherin, α- and β/γ-catenins,and α2- and β1-integrins in invasive breast cancer

This study examines the expression of the cell–cell adhesion molecules E-cadherin and its associated proteins, the catenins and the matrix–cell adhesion molecules β1- and α2-integrins, in primary invasive breast carcinoma. Expression was assessed immunohistochemically on frozen sections by semi-quantitative scoring of the intensity and proportion of immunoreactivity in 55 cases. Associations with each other and with other histological and prognostic features and survival were sought. There was a significant association between loss of E-cadherin expression and loss of α- and β/γ-catenin immunostaining. In 20 per cent of cases, membranous immunoreactivity with E-cadherin antibody was absent. Absent cytoplasmic expression of α- and β/γ-catenins was seen in 24 and 22 per cent of breast cancers, respectively. The intensity of reactivity with E-cadherin showed a significant association with histological grade (p = 0·002) and tumour type (p < 0·001). Lobular carcinomas frequently showed loss of expression of E-cadherin, as reported elsewhere; loss of catenin expression was also found in these tumours. α-Catenin intensity also showed a relationship with grade (p = 0·008) and with oestrogen receptor (ER) status (p = 0·006). β/γ-Catenin expression was not associated with other known prognostic factors. Forty-nine per cent and 42 per cent of cases showed no membrane immunostaining with β1- and α2-integrin, respectively, and co-ordinated loss of β1- and α2-integrin expression was found. Both β1- and α2-integrin expression were associated with histological grade (p = 0·003 and p = 0·031, respectively) and β1 immunoreactivity with tumour type (p = 0·010). None of the variables examined showed a statistically significant association with tumour size or lymph node stage, or with overall survival, although a trend was seen (p = 0·087) towards poorer survival of patients with tumours with absent or weak expression of β1-integrin. The expression of these markers is of biological interest, but appears to be of little additional use in predicting clinical behaviour. Copyright © 1999 John Wiley & Sons, Ltd.     

In vivo patterns of Bcl-2 family protein expression in breast carcinomas in relation to apoptosis

The expression of the pro-apoptotic proteins (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bcl-X, Mcl-1) was studied by immunohistochemistry in 110 invasive ductal breast carcinomas. The results were correlated with tumour grade, expression of oestrogen receptor (ER) and p53 protein, and the apoptotic index by combined morphology, immunohistochemistry, and a terminal UTP nick end labelling (TUNEL) procedure. Overall, Bcl-2, Bcl-X, Mcl-1, Bax, Bak, ER, and p53 were detected in 62, 75, 68, 75, 60, 68 and 26 per cent of the cases respectively, but at different levels in each case. A high apoptotic index was correlated with high tumour grade (p < 0·001), overexpression of p53 (p < 0·001), Bak expression (p < 0·001), and low expression of Bcl-2 (p < 0·001) and ER (p < 0·001). No correlation was found between the apoptotic index and Bax, Bcl-X, and Mcl-1 immunostaining results. The expression of Bcl-2 and Bcl-X was correlated to that of ER. Overall, the results of this study strongly suggest that Bcl-2 and Bak expression is critical in regulating apoptosis in breast carcinomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Serum cytokine and chemokine profiles in patients with myasthenia gravis

Myasthenia gravis (MG) is an autoimmune‐mediated inflammatory disease of the neuromuscular junction. Previous studies of animal MG models have suggested important roles of cytokines in MG pathogenesis, but adequate studies on cytokines in human MG are lacking. Using a multiplex suspension array system, we measured the serum levels of 27 cytokines/chemokines in 47 anti‐acetylcholine receptor antibody‐positive patients with MG and 20 normal controls (NC) to investigate the contribution of cytokines/chemokines toward MG pathogenesis. Correlations between clinical parameters and cytokine/chemokine levels in patients with MG were also examined. The serum levels of interleukin (IL)‐15 (mean ± standard deviation: 6·85 ± 6·97 pg/ml) and vascular endothelial growth factor (VEGF) (96·21 ± 71·60 pg/ml) significantly increased, whereas IL‐4 levels (3·57 ± 0·86 pg/ml) decreased in patients with MG compared with NC (IL‐15: 4·42 ± 1·55 pg/ml; VEGF: 63·51 ± 32·95 pg/ml; IL‐4: 4·15 ± 0·81 pg/ml, P < 0·05). In addition, eight cytokines (IL‐4, IL‐8, IL‐15, eotaxin, macrophage inflammatory protein‐1α, macrophage inflammatory protein‐1β, VEGF and IL‐1b) were significantly changed among MG patients with thymoma, MG patients without thymoma and NC (P < 0·05). Some cytokines, such as IL‐4, IL‐15, and VEGF, may play roles in the pathogenesis of MG.     

Alterations of expression of Rb,p16INK4A and cyclin D1 in non-small cell lung carcinoma and their clinical significance

Inactivation of the Rb pathway in non-small cell lung carcinoma (NSCLC) occurs mostly through inactivation of the cyclin-dependent kinase inhibitor p16^INK4A and/or up-regulation of cyclin D1. In order to assess the frequency and the prognostic value of these abnormalities in NSCLC, immunohistochemical analysis of Rb, p16^INK4, and cyclin D1 has been performed on 168 cases of NSCLC including 77 squamous cell carcinomas, 43 adenocarcinomas, and 48 basaloid carcinomas. The reduced survival rate of basaloid carcinoma (stage I–II) compared with other histological types of NSCLC was confirmed (p = 0·008). Loss of protein expression of Rb and p16^INK4A was observed in 12 per cent and 58 per cent of NSCLC cases respectively and cyclin D1 overexpression in 43 per cent. There was an inverse correlation between Rb and p16 expression ( p < 0·0001) and a direct correlation between Rb and cyclin D1 expression ( p = 0·0007). In univariate analysis, Rb-negative adenocarcinomas at stages I–II had a significantly shorter survival than Rb-positive cases ( p = 0·04) and stages I–II p16-positive cases had a shorter survival than p16-negative cases ( p = 0·02), which was more significant in basaloid carcinoma ( p = 0·003). p16 status retained its influence on survival in multivariate analysis at stage I–II for all cases ( p = 0·01) and for basaloid carcinoma ( p = 0·005). Cyclin D1 overexpression did not influence survival. Combined Rb/p16/cyclin D1 phenotypes in univariate analysis showed a shorter survival for Rb-negative/p16-positive/cyclin D1-negative tumours ( p = 0·002). These results, linked to previous data, indicate that the Rb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 per cent), but could not confirm an unfavourable role for each individual event (p16^INK4A loss or cyclin D1 up-regulation) in prognosis. Copyright © 1999 John Wiley & Sons, Ltd.     

Cyclin D1 expression in astrocytomas is associated with cell proliferation activity and patient prognosis

An important positive regulator of the cell cycle, cyclin D1, is often amplified and overexpressed in malignancies. Cyclin D1 aberrations were analysed in grade II–IV astrocytomas by fluorescence in situ hybridization (FISH), mRNA in situ hybridization and immunohistochemistry. Proliferation activity was determined by Ki-67^MIB-1 immunolabelling and mitotic counting. High cyclin D1 expression was observed in grade IV astrocytomas (grades II–III versus grade IV; mRNA expression: p < 0·001; immunoexpression: p = 0·013), and correlated with poor patient survival (p < 0·001, n = 46). Upregulated cyclin D1 expression was also closely associated with poor patient prognosis in grade II–III astrocytomas (p < 0·001, n = 30). Cyclin D1 gene was not found to be amplified (n = 7). Cell proliferation activity was significantly increased in tumours exhibiting high cyclin D1 mRNA levels (Ki-67^MIB-1: p < 0·001; mitotic count: p < 0·001) and high cyclin D1 protein expression (Ki-67^MIB-1: p = 0·002; mitotic count: p = 0·012). These results indicate that increased production of cyclin D1 is closely associated with high cell proliferation activity and aggressive behaviour in diffusely infiltrating astrocytomas. Copyright © 1999 John Wiley & Sons, Ltd.