Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques.

New genomic large-scale screening techniques have made the task of establishing an accurate molecular fingerprint of cancer cells feasible. Here, we have used a two-phase strategy for identification of molecular alterations in gliomas. First, cDNA microarrays (Clontech Laboratories, Inc., Research Genetics) were used to pinpoint differentially expressed genes between normal brain and diffuse astrocytomas (grades II-IV), and between a primary tumor and a later tumor reoccurrence in the same patient. More than 200 gene expression alterations were detected from glioblastomas, whereas relatively few changes were seen in grade II and grade III tumors. The most distinct progression-related expression change was the up-regulation of the insulin-like growth factor binding protein 2 (IGFBP2) gene. Second, a high-density tissue microarray of 418 brain tumors was constructed and used for clinical validation of gene expression changes. Strong expression of IGFBP2 was associated with progression and poor patient survival in diffuse astrocytomas (P < 0.0001). Third, comparisons of the data between (a) multiple spots retrieved from one predefined tumor region (IGFBP2 and vimentin immunohistochemistry, 20 tumors) or between (b) standard slides and arrayed tissues (p53 immunohistochemistry, 42 tumors) revealed very little variation. In conclusion, the combined use of DNA microarrays and tissue microarrays offers a powerful strategy for rapid identification and thorough characterization of differentially expressed genes in gliomas.

Grading of diffusely infiltrating astrocytomas by quantitative histopathology, cell proliferation and image cytometric DNA analysis. Comparison of 133 tumours in the context of the WHO 1979 and WHO 1993 grading schemes.

The aim of the study was to evaluate the applicability of quantitative histopathology as an aid for grading diffusely infiltrating astrocytomas. Primary astrocytomas were analysed for parameters (mean nuclear size, mitosis count, area fraction of endothelial cells and tumour necrosis, area fraction of nuclei, and Ki-67 (MIB-1) labelling index), which are closely related to the World Health Organization (WHO) 1979 and WHO 1993 grading criteria. All estimates correlated with the WHO histopathological grade and patient outcome. According to the receiver-operating characteristics curve, the presence of tumour necrosis and mitosis count (cut-off at 3 mitoses/mm2 of neoplastic tissue) showed the best sensitivity and specificity in separating patients with different survival. The multivariate survival analyses confirmed this result. A decision-tree model was constructed based on these two variables: twig I with less than 3 mitoses/mm2, twig II with equal or more than 3 mitoses/mm2 but no necrosis, and twig III with tumour necrosis. This model was found to be more strongly associated with survival than the WHO 1979 or WHO 1993 grading schemes. Low-malignancy astrocytomas (WHO grade II or twig I tumours) could be further divided into two prognostic categories by the image cytometric DNA analysis. The results put an emphasis on astrocytoma grading on mitosis counts (grade II vs. III) and tumour necrosis (grade III vs. IV). To standardize the sampling for mitosis counting, it is suggested that a parallel Ki-67 immunostaining be used for the identification of the most proliferative areas.

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.

Prognostication of astrocytoma patient survival by Ki-67 (MIB-1), PCNA, and S-phase fraction using archival paraffin-embedded samples.

The prognostic power of three proliferation estimation methods, Ki-67 (MIB-1) and PCNA immunohistochemistry, and flow cytometry (S-phase and S + G2/M fractions, respectively), were evaluated in 50 cases of astrocytoma. Each proliferation index showed a strong association with the grade of malignancy (grades I-IV). The MIB-1 labelling index (LI) provided additional information, as it could be used for the discrimination of grade II and grade III astrocytomas (P = 0.0357). All three proliferation estimation methods also had strong prognostic potential (MIB-1 LI: P < 0.0001; PCNA Li: P < 0.0001; S-phase: P = 0.0004; S + G2/M: P = 0.0124). According to the receiver operating characteristics (ROC) curve, the MIB-1 LI showed generally the best sensitivity and specificity in placing the patients correctly into groups of survivors and non-survivors, which was further confirmed in the multivariate analysis. Only 4 per cent of the patients having high MIB-1 scores (> 15.3 per cent) were alive after 2-years' follow-up. In contrast, 72 per cent of patients with tumours of low proliferation activity survived. It appears that Ki-67 (MIB-1) immunolabelling using archival paraffin-embedded samples is of value in predicting prognosis in astrocytic tumours.

Comparison of three quantitation methods for PCNA immunostaining: applicability and relation to survival in 83 astrocytic neoplasms.

Recent studies on astrocytic tumours demonstrated a close association between patient prognosis and neoplastic proliferation estimated by such methods as Ki-67 and bromodeoxyuridine labelling. Novel monoclonal PCNA antibodies and special antigen-retrieval techniques have the advantage of working on routinely fixed and embedded specimens and thus make the estimation of proliferation simpler. In addition to PCNA-positive cell count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells of 83 astrocytomas in two ways: (1) per mm2 of neoplastic tissue (uncorrected PCNA index); and (2) per mm2 of total neoplastic nuclear area (corrected PCNA index). Both of these methods were reproducible and showed a good correlation with PCNA-LI and malignancy grade (I-IV). With quantitation methods 1 and 2, the proliferative status of about 2000 cells could be estimated in about 7-10 min, whereas the PCNA count by PCNA-LI of 200 cells took approximately the same time. The proliferation indices obtained by all three quantitation methods were highly significantly related to patient prognosis. The corrected PCNA index, having a close association with the neoplastic cellularity, even divided the glioblastoma group (grade IV) into two significantly different prognostic groups in which 56 and 17 per cent of the patients were alive after 1-year follow-up. The combination of PCNA immunohistochemistry and morphometry seems to give important prognostic information about astrocytomas independent of the histopathological grade.