Acta Neurochir (Wien) (1992) I17:182-186 =Acta N uro&irurgica 9 Springer-Verlag 1992 Printed in Austria Relationships Between Ki-67 Labelling Index, Amplification of the Epidermal Growth Factor Receptor Gene, and Prognosis in Human Glioblastomas S. H. Torp 1, E. Helseth 2, A. Dalen 3, and G. Unsgaard 2 l Institute of Cancer Research, Medical Technical Centre, Trondheim, 2Department of Neurosurgery, and 3Department of Microbiology, University Hospital of Trondheim, Trondheim, Norway Summary The aim of this study was to determine possible relationships between Ki-67 labelling index (Ki-67 LI), amplification of the epi- dermal growth factor receptor (EGFR) gene, and prognosis in hu- man glioblastomas. Ki-67 LI was determined on cryosections of biopsy specimens of 20 human glioblastomas with a mouse anti- human Ki-67 monoclonal antibody. Amplification of the EGFR gene was determined by slot blot and Southern blot analyses of DNA extracted from the tumour biopsies. The Ki-67 LI was higher in the glioblastoma group with EGFR gene amplification (8 tumours, me- dian value of Ki-67 LI 4.2, range 0.4-24.6) than in those without EGFR gene amplification (12 tumours, median value of Ki-67 LI 0.8, range 0.2-11.8) (0.05 p < 0.1). The glioblastoma patients with Ki-67 LI > 15 (10 tumours) had a statistically significant shorter survival than those with Ki-67 LI < 1.5 (10 tumours) (p < 0.05). The glioblastoma patients with EGFR gene amplification lived shorter time than those without EGFR gene amplification (p > 0.05). Keywords: Glioblastoma; epidermal growth factor receptor; Ki- 67; survival. Introduction In human gliomas the proto-oncogene c-erb-B 1 [ho- mologous to the epidermal growth factor receptor (EGFR) gene] is commonly amplified and o v e r e x p r e s s e d 2, 8, ~2,15, 16, 24. T h e E G F R is a transmem- brane receptor protein with binding specificities for epidermal growth factor (EGF), transforming growth factor a, and amphiregulin 25. Since EGFR gene am- plification occurs mainly in glioblastomas, it is likely that this genetic event is related to the progression of gliomas. So far, EGFR gene amplification has shown no statistically significant correlation to survival of glioma patients 2. The monoclonal antibody Ki-67 has been used to assess the growth fraction in tumour tissue 3. Ki-67 de- tects a nuclear antigen expressed during the G1, S, G2, and M phases of the cell cycles, but absent in the GO phase 9,10. In human gliomas the Ki-67 Labelling Index (Ki-67 LI) has shown a good correlation with histo- logical grade < 6, ,1, 17, ,8, 20, 21, 27 and is comparable to other proliferative markers such as mitoses and bro- modeoxyuridine labelling index (BrdU LI) 14' 18, 21 BrdU LI has been shown to correlate with survival in glioblastoma patients ~4. For Ki-67 LI no overall cor- relation with survival has been shown 22, although five patients with an index below 2.5 lived longer than 40 weeks 27. The aim of this study was to determine possible relationships between Ki-67 LI, EGFR gene amplifi- cation, and prognosis in glioblastoma patients. Materials and Methods Twenty human patients with glioblastomas operated upon at the Department of Neurosurgery, University Hospital of Trondheim, Trondheim, Norway, in the time period 1986-89 were included in the study. No chemotherapy or radiotherapy was given before sur- gery. Immediately after surgical removal one portion of the tumour was fixed in 3.9% formalin for routine histopathology (the patho- logical diagnoses were based on the criteria of WHO28), and the rest was shock frozen in liquid nitrogen and stored at - 70 ~ Acetone fixed frozen sections were incubated 1h with the IgG1 murine monoclonal antibody Ki-67 (Dakopatts, Glostrup, Den- mark) at a dilution of 1 : 50 in PBS. The staining procedure was performed by an avidin-biotin-peroxidase technique (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, U.S.A.). The sec- tions were lightly counterstained with haematoxylin. In each experi- ment frozen sections of human tonsils (rich in Ki-67 positive cen- trocytes) 9 were included as positive controls whereas in the negative controls Ki-67 was omitted. The negative control included regularly an irrelevant mouse monoclonal antibody instead of the primary antibody. The Ki-67 LI was defined as the percentage of Ki-67 positive cells divided by the total number of cells 3. Ten representative areas were evaluated at a magnification of x 400 with an eyepiece grid covering an area of 0.055 mm2. 400-6000 cells were counted per section.