Geminin is a nuclear protein that belongs to the DNA replication inhibitor group. It inhibits DNA replication by preventing Cdt1 from loading minichromosome maintenance protein onto chromatin, as is required for DNA replication. For this study, the authors investigated geminin expression in high-grade astrocytic tumors, including anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM), with a view to predicting clinical outcomes on this basis in patients with these malignant brain tumors.
Immunohistochemistry was used to detect geminin expression in 51 patients with high-grade astrocytic tumors (19 AA and 32 GBM). Samples were categorized by taking the median value as the cut-off point for constructing Kaplan-Meier curves. The relation of geminin expression to clinical outcome in these malignant brain tumors was analyzed by using the Kaplan-Meier method and a Cox proportional hazards regression model.
Geminin was expressed in all high-grade astrocytomas (mean geminin labeling index [LI], 24.90%). Kaplan-Meier curves showed that the group with higher geminin LI (≥22.50%) had a better prognosis than the group with lower LI (<22.50%; P = .0296). Similarly, the Cox regression analysis showed that geminin expression has a significant correlation with survival in patients with high-grade astrocytoma (P = .0278), especially in an early stage.
Human malignant gliomas grow diffusely and invade surrounding normal brain tissue. Surgical removal of the entire tumor often is difficult, and irradiation often is administered as adjuvant therapy for the treatment of incompletely resected tumors. However, to date, the clinical effectiveness of irradiation has been limited. In almost all patients, these high-grade tumors are refractory to treatment, and the patients die from brain herniation caused by unrestrained tumor growth.1 The median survival of patients with malignant glioma is <2 years despite extensive and multidisciplinary treatment.
Geminin is a 25-kDa nuclear protein and has a DNA replication-inhibitory function. Its action is mainly through negatively regulating the function of Cdt1, which helps in the formation of prereplication complex (pre-RC) by loading minichromosome maintenance protein (Mcm2-Mcm7) onto chromatin.2–10 In addition, DNA polymerase binds to this complex and initiates DNA synthesis in S-phase. This process repeats constantly and limits DNA replication to once per cycle. To maintain this process, the level of geminin fluctuates at different stages of the cycle; geminin level is at its lowest and is almost absent in G1-phase but is high in S-phase and G2-phase and in the initial stage of M-phase.
Therefore, geminin is responsible not only for the regulation of the cell cycle but also for the genomic integrity of cells thus formed. From these observations, it was believed that geminin likely would have tumor-suppressive functions. However, previous reports have demonstrated that the expression of geminin rises with increasing tumor grade,11–16 leading to a poor prognosis in a variety of cancer patients, eg, breast cancer and renal cell carcinoma.17, 18 Thus there has been a discrepancy between normal molecular function and the outcomes of clinical studies of geminin in many cancers.
Although the effect of geminin expression on clinical prognosis in a number of malignant tumors has been studied, its effect on astrocytic tumors has not yet been investigated. Moreover, previous studies have not related geminin expression to different treatment modalities, for example, radiotherapy. All of our patients received radiotherapy after surgery as a standard treatment protocol for high-grade astrocytomas.19, 20 For the current study, therefore, we retrospectively examined the expression of geminin in high-grade astrocytic tumors (anaplastic astrocytoma [AA], World Health Organization [WHO] grade III; and glioblastoma multiforme [GBM], WHO grade IV) in relation to clinical prognosis, and we also analyzed at the relation between geminin expression and radiosensitivity.
MATERIALS AND METHODS
The tissue samples were collected during surgery, and all tumor specimens were fixed routinely in 10% buffered formalin and embedded in paraffin, which were obtained from the research laboratory of the Department of Neurosurgery, Hiroshima University, Hiroshima, Japan. Samples were used with the prior informed consent, which was obtained before surgery, of the patients or their guardians.
To standardize the study and to make it more specific, only patients with high-grade astrocytoma were investigated. Specimens from 51 patients with high-grade astrocytoma were obtained (WHO grades III and IV). In addition, geminin expression in 20 specimens of diffuse astrocytoma (low-grade glioma, WHO grade II) was examined purely for comparison with geminin expression in high-grade gliomas. Pilocytic astrocytomas (WHO grade I) and astrocytomas of the infratentorial region were excluded from the study. We also excluded other types of gliomas, such as oligodendroglioma, ependymoma, ganglioglioma, etc, and gliomas that originated in the thalamus. The included patients underwent surgery between September 1990 and May 2005. Treatment for all patients was primary surgery and radiation therapy. Chemotherapy was not taken into consideration, because as it was not administered uniformly in all patients; and, unlike radiotherapy, it has not yet been proven that chemotherapy prolongs survival in patients with these tumors.19, 20 Details of the 51 patients are shown in Table 1. The mean survival was 29.81 months (range, 3.07–156.63 months).
Table 1. Clinical Details of 51 Patients with High-grade Astrocytoma
No. of patients
SD indicates standard deviation.
Mean ± SD
47.98 ± 18.78
Mean ± SD
29.30 ± 35.57
Histologic diagnoses and other information on all tumors were obtained from hospital records. Histologic diagnostic subtypes were defined and categorized according to the WHO criteria and guidelines by 1 of the authors (K.S.). Four-micrometer tissue sections were deparaffinized with xylene, and antigen retrieval was carried out by using the heat-induced epitope retrieval method with citrate buffer solution, pH 6.0. Endogenous peroxidase blocking was carried out by dipping the slides into a solution made by mixing 10 mL of 30% H2O2 and 90 mL of 99% methanol for 30 minutes. After each step, the slides were rinsed and washed with phosphate-buffered saline solution, pH 7.5, 3 times for 5 minutes each. An indirect method of immunostaining, the labeled streptavidin biotin (SAB) method, was employed for antibody incubation, using the histofine SAB kit (Nichirei Company. Tokyo, Japan). The antibody used for geminin was geminin (FL-209), which is a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) raised against geminin of human origin, at 1:200 dilution. Similarly, the primary antibody for Ki-67 was mouse monoclonal antibody (MIB-1; Immunotech, Marseille, France) at 1:50 dilution. Primary antibody incubation was performed overnight at 4°C, followed by incubation for 30 minutes in secondary antibody (biotinylated secondary antibody, SAB kit; Nichirei Company). Slides were treated with Mayer hematoxylin as a counterstain for better cytoplasmic visualization and then mounted with coverslips for storage purposes.
The slides were evaluated for geminin labeling index (LI) by 2 of the authors, who had no any prior knowledge of the histologic diagnosis or other clinical records: The numbers of positive cells per 1000 were counted in each sample at ×400 magnification under a microscope. A third author estimated the MIB-1 LI by counting the positive nuclei per 1000 tumor cells in each sample in the same manner.
Statistical analyses were performed using the software package SPSS (Statistical Package for the Social Sciences; version 11.5 for Windows). After calculating the geminin LI for all samples by simple counting, as described above, the mean and median values were calculated.
Differences in geminin expression at different stages of tumor progression (grades II, III, and IV) in astrocytic brain tumors were assessed by using 1-way analysis of variance (ANOVA). Post-hoc tests were performed using the Scheffe test.
Survival was measured in months from the date of first surgery to the date of death for patients who died or the date of last follow-up for patients who remained alive. The Kaplan-Meier (KM) method was used to assess survival against geminin expression. The median geminin LI was taken as the cut-off point for dividing the samples into 2 categories (less than and greater than the median). Survival and life/death status were plotted against the geminin LI; then, the KM curves were prepared. The log-rank test was used in a univariate analysis to evaluate statistical significance.
A multivariate analysis with 95% confidence intervals (95% CIs) was performed comparing several prognostic variables for survival using the Cox proportional hazards regression model. The prognostic continuous variables were age, geminin LI, MIB-1 LI; and the prognostic categorical variables were sex, tumor grade, and extent of tumor resection. All P values <.05 were considered statistically significant. A correlation analysis between the 2 factors age and geminin expression was carried out as a bivariate correlation analysis using the Pearson test.
All 51 clinical samples (100%) showed positive staining for geminin. The mean geminin expression (LI) was 24.90% per high-power field (range, 1.94–68.75% per high-power field), and the median geminin LI was 22.50% per high-power field. Details of the immunohistologic findings are provided in Table 2. Among the 20 patients who had low-grade (grade II) astrocytic tumors, 5 patients (25%) did not have a single positive cell, and the remaining 15 patients (75%) had positive staining. The mean geminin expression (LI) for these low-grade tumors was 11.27% (median, 3.01%). Figure 1 displays representative photomicrographs of geminin immunostaining.
Table 2. Immunohistochemical Findings in 51 Patients with High-grade Astrocytoma
Geminin Expression and Age
Correlation analysis between the factors age and the geminin expression was done by using the bivariate Pearson correlation test, which showed no significant correlation (P = .187), suggesting that there is no significant correlation between age and geminin expression.
Geminin Expression and Tumor Grade
Changes in geminin expression over the course of tumor progression were analyzed and are shown in Figure 2. Geminin expression increased significantly as tumors progressed through each step from 1 grade to another grade (from grade II to grade III and from grade III to grade IV; P = .007; ANOVA between groups). The Scheffe post-hoc test showed a significant difference in geminin expression between grade II and III tumors and grade II and IV tumors (P = .017 and P = .024, respectively). However, we observed no significant differences in geminin expression between grade III tumors and grade IV tumors (P = .879). Therefore, our current results demonstrated that higher grade tumors have higher geminin expression in astrocytic brain tumors, especially in the early stage of tumor progression.
Tumor Grade and Prognosis
We tried to perform the survival analysis of grade III tumors versus grade IV tumors without noting geminin expression. Our analysis showed, as expected, that the survival of patients with AA was significantly better than the survival of patients with GBM (P = .0080) (Fig. 3). Therefore, the correlation between geminin expression and prognosis was studied separately in the AA group and the GBM group.
Geminin Expression and Prognosis
We examined the cumulative survival of 2 groups of patients according to the geminin LI (higher than the median value [>22.50%] and lower than the median value [<22.50%]) in the patients with high-grade astrocytic brain tumors (AA and GBM) by using the KM method. The group that had higher geminin expression had a significantly better survival rate compared with the group that had lower geminin expression (P = .0296) (Fig. 4a). To test the significance of geminin expression further in the prediction of the clinical outcome of these malignant astrocytomas, Cox multiple regression analysis was used to compare the contribution of the geminin LI with that of the other variables mentioned above. The geminin LI (P = .0278) and tumor grade (P = .0436) were identified as significant factors for predicting clinical outcomes, as shown in Table 3.
Table 3. Cox Regression Multivariate Analysis for Survival
To confirm the prognostic significance of geminin expression, we compared patients with AA and patients with GBM separately by using KM curves. The group that had higher geminin expression had a better survival rate than the group that had lower geminin expression in both patients with AA and patients with GBM, whereas a statistical significance was observed only in patients with AA (Fig. 4b) (P = .0006) and not in patients with GBM (Fig. 4c) (P = .5696). Therefore, we observed that higher geminin expression resulted in better survival rates in patients with high-grade tumors, especially in the early stage of malignant progression of astrocytic brain tumors.
Previous studies have found that geminin is expressed highly in a number of malignant conditions, including breast cancer, colorectal cancer, cervical cancer, renal cell carcinoma, oligodendroglioma, and others.11–16 Geminin is a protein that blocks the re-replication of the gene in the same cycle. It is present in the S-, G2-, and M-phases of the cell cycle but is absent in G1-phase; thus, it is the S/G2/M-phase marker. Its absence indicates a lack of cell cycle progression, and its high expression indicates that cells are progressing rapidly through the S-, G2-, and M-phases and are engaged in DNA synthesis and, thus, cell division. It was mentioned above that, in highly proliferating cells, G1-phase becomes very short, and many cells in this state are in S-, G2-, or M-phase. Thus, more geminin is detected in immunostaining of the tissue of highly proliferating cells, ie, tissues from higher grade tumor. Furthermore, previous studies have shown that the replication licensing factors, like minichromosome maintenance factor and geminin, are up-regulated in the early stages of tumorigenesis and malignant progression.11, 16 Therefore, it has been pointed out that geminin has a causal effect on the carcinogenesis of different tumors, especially in the early stages of carcinogenesis. In patients oligodendroglioma, it also was shown that geminin over-expression had an influence on the progression of tumors from low grade to high grade.12 These results are consistent with our data, which show that higher grade tumors have higher geminin expression levels, especially during the stage of early progression of malignancy.
Another previous study examined the effect of geminin expression on the overall survival of patients with breast cancer17; and those authors demonstrated that the higher expression of geminin was a poor prognostic marker in patients with breast cancer. Yet another study examined the relation of geminin expression to the clinical outcome of patients with renal cell carcinoma and showed the same trend18: higher geminin expression was a poor prognostic marker. Surprisingly, the data from our investigation on the relation between geminin expression and survival in patients with high-grade astrocytic tumors contradict those previous findings: Greater frequency of geminin expression is associated with longer survival and, thus, with a better prognosis compared with lower geminin expression (Fig. 4a, Table 3). This trend was similar in both patients with AA and patients with GBM when they were analyzed separately, whereas statistical significance was observed only in patients with AA but not in patients with GBM, suggesting that the geminin LI was a significant predictor of better survival outcomes in patients with high-grade astrocytomas, especially in the early stage of malignant transformation (grade III) (Fig. 4b,c).
A possible reason for these opposing results may lie in differences in the treatments provided. All of our patients both underwent surgery and received radiotherapy as the standard treatment protocol for high-grade astrocytoma. This suggests the possibility that over-expression of geminin may increase radiosensitivity, leading to a better prognosis after radiotherapy, even though geminin expression rises with increasing tumor grade.
In a previous investigation of geminin expression in a series of human normal and cancer cell lines and human primary cancers, it was observed that geminin over-expression stimulated cell cycle progression and proliferation in both normal cells and cancer cells.13 In that study, in 5 of 6 patients with rectal cancer, the expression of geminin was reduced significantly by chemoradiotherapy, thus suggesting that cells expressing high levels of geminin may be more sensitive to radiotherapy. However no direct evidence for this heightened sensitivity was presented. We believe that our current study may be the first to suggest an explicit effect of geminin expression on the radiosensitivity of a cancer.
In both normal cells and cancer cells, as discussed above, the geminin level is lowest and almost absent in G1-phase but is high in S-phase and G2-phase and in the initial stage of M-phase. In cancer cells, however, it has been reported that the over-expression of geminin accelerates the cell cycle from the G1-phase to the S-phase, shortening G1-phase and reducing the number of cells in this phase, thus leading to an accumulation of cells in S-phase.13 The results of clonogenic survival assays clearly indicate that cells synchronized at the M-phase or at the early S-phase are the most radiosensitive, and cells in G1-phase are comparatively radioresistant.21, 22 From these observations, we propose that the higher the expression of geminin, the higher the proportion of cells in phases other than G1 of the cell cycle, resulting in higher radiosensitivity. Therefore, patients who have high-grade astrocytoma patients with over-expressed geminin have favorable outcomes after surgery and radiation therapy.
Some limitations of this study should be acknowledged. First, we used a retrospective design, and a prospective study will be needed to confirm our findings. Second, this was a small study that included only 51 patients with high-grade glioma. Given the postulated effect of a treatment modality, the relation between geminin expression and outcomes when other treatment modalities for brain tumor are involved needs to be explored. Furthermore, we examined only the expression of geminin and MIB-1, and the expression of other molecules, such as p16, p53, epidermal growth factor receptor, etc, may modify the effect of geminin expression on these high-grade tumors. In addition, we have not determined whether the geminin in high-grade astrocytoma is a mutant type or a wild type, and the effect of geminin on the cell cycle of these malignancies may be altered depending on this determination. Finally, it would not be fair to conclude that geminin expression improves radiosensitivity without using a comparison group. We recommend radiotherapy for all of our patients with high-grade glioma after they have undergone surgery, according to the standard protocol for brain tumor management. Therefore, we did not have a control group of patients who did not receive radiotherapy for a comparison study.
In conclusion, the current results indicated that, like what has been observed in other malignancies, geminin is expressed highly in high-grade astrocytic tumors compared with low-grade astrocytic tumors. However, higher expression of geminin in high-grade astrocytomas indicates a more favorable outcome and longer survival in patients who have received radiotherapy after surgery as a standard treatment protocol.
A Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.