Drug resistance is one of the important factors that determine tumor response to chemotherapy. Several candidates for resistance to various chemotherapeutic agents have been elucidated. O^sup 6^-methylguanine-DNA methyltransferase (MGMT) removes methylation damage induced by nitmsourea from the O^sup 6^ position of DNA guanines before cell injury. Glutathione-S-transferase (GST) [pi] is also involved in nitmsourea resistance. We examined the expression of MGMT and GST [pi] in 18 glioblastomas (GBM) using immunohistochemistry and compared the results with patients' survival after administration of 1-(4amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU)-based chemotherapy. According to the Kaplan-Meier's method, although median progression free survival (PFS) of eight patients whose tumors retained high MGMT (3 + ~2 + ), and W patients whose tumors showed low MGMT expression (1 + ~0) were nine and 15 months, respectively (p = 0.09), median overall survival (OS) of the two groups were 12 and 22 months, respectively, which were significantly different (p = 0.01). GST [pi] expression in GBM was not a prognostic factor. It is suggested that GBM with strong staining of MGMT activity may show more resistance to ACNU-based chemotherapy compared to that with low MGMT. The simple immunohistochemical analysis of MGMT in GBM can be a useful method to determine whether ACNU or another treatment regimen should be recommended. [Neural Res 2003; 25: 241-248]
Keywords:: Glioblastoma; drug resistance; MGMT; nimustine
For patients with malignant glioma, multimodality treatments including surgical removal, radiotherapy, and chemotherapy are performed. In Japan, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride, nimustine hydrochrolide (ACNU), is mainly used for malignant astrocytic tumors as a first line chemotherapeutic agent because of its high permeability across blood-brain barrier (BBB) and good cytotoxic activity for gliomas.
Drug resistance is one of the important factors that determine the treatment outcomes. Many drug resistance related factors have been elucidated. DNA repair enzyme, O^sup 6^-methylguanine-DNA methyltransferase (MGMT) removes methylation damage from the O^sup 6^ position of DNA guanines before cell death which is induced by chloroethylnitrosourea. Recent reports suggested that MGMT expression in glioma cell lines correlated well with the resistance of the cell to nitrosourea in vitro1, and that the degree of MGMT expression in gliomas could be indicative of tumor progression after nitrosourea-based chemotherapy2-8. Also, glutathione (GSH), and glutathione-S-transferase (GST) [pi] reportedly take part in the resistance to nitrosourea9. In these reports, the methods to measure MGMT or GST [pi] were complexes, and often needed frozen sections.
In this report, the authors performed simple immunohistochemical staining of paraffin-embedded tumor specimens using monoclonal anti-MGMT, GST [pi] anti-bodies and compared the results with the patients' overall survival and progression free survival. The objective of this preliminary retrospective study was to obtain information about whether the immunohistochemical analysis of drug resistance associated proteins, such as MGMT and GST [pi], can be an indicator for drug selection for patients with malignant astrocytic tumor.
MATERIALS AND METHODS
Patient population and tumor samples
Patients with GBM were chosen for this study, because GBM was the most frequent glioma and was usually treated with a uniform regimen consisting of surgical removal, radiotherapy, and nitrosourea-based chemotherapy. Histological diagnosis was classified by the pathologists according to the morphological criteria of the World Health Organization. Tumor specimens excised at the Department of Neurosurgery, Nagasaki University Hospital from 1990 to 2001, and corresponding patient data were obtained.
Immunohistochemical staining of MGMT, GST [pi], Ki-67 antigen
The deparaffinized rehydrated tissue sections were pre-treated with 1OmM citrate buffer (pH 6.0) in a microwave oven for 15 min at 500 watts. The sections were cooled to room temperature, then treated with 0.01 % tripsin at 37[degrees]C for 1 5 min and rinsed with PBS for 5 min. For GST [pi] staining, the above-mentioned two steps were omitted. The sections were placed in methanol containing 3% hydrogen peroxide for 10 min and washed in 0.05 M phosphate-buffered saline (PBS) (pH 7.4) for 10 min. Tissue nonspecific activity was blocked by 3% bovine serum albumin (Sigma Chemicals, St. Louis, MO, USA) for 30 min at 37[degrees]C, and sufficient primary antibody monoclonal mouse antiMGMT (Neomarkers, Fremont, CA, USA), GST [pi] (Dako, Carpinteria, CA, USA), and mouse monoclonal MIB-1 antibody (Immunotech S.A., Marseille, Cedex, France) diluted in PBS was applied and incubated overnight at 4[pi]C. The ratio of each antibody to PBS was 1 : 50, 1 : 25, and 1 :50, respectively. Subsequently, biotinylated goat antimouse immunoglobulin (diluted 1:100 in PBS) (Vector Laboratories Inc., Burlingame, CA, USA), and avidin-biotin complex (Vector Laboratories) were applied and incubated at room temperature for 1 h and 30 min, respectively. After each incubation, sections were washed in PBS for 15 min. Then, 0.02% 3,3'diaminobenzidine-4 hydrochloride (Dojindo, Kumamoto, Japan) dissolved in Tris-HCI buffer solution was applied to the sections. The sections were counterstained in Mayer's hematoxylin, dehydrated, and mounted. all sections were examined under microscope. The expression of MGMT or GST [pi] was arbitrarily graded according to the percentage of antigen positive cells as Grade O when there were no positive cells, Grade 1 when there were up to 19% positive cells, Grade 2 when there were 20%-49% positive cells, and Grade 3 when there were 50% or more positive cells (Figures 1 and 2). For MIB-1, depending on the size of the section, 5-10 fields from the area of maximal staining were chosen for counting. Both immunopositive and immunonegative tumor cells were counted under light microscopic high-power fields (x400) set with video-monitoring screen. The total numbers of cells counted per section were at least 500. The MIB-1 staining index was defined as the percentage of immunopositive cells divided by the total cells in the evaluated area.
Chemotherapeutic effect and Statistical Analysis
The tumor responses to therapy were evaluated by periodic radiological findings after the induction of chemotherapy. Progressive disease (PD) was defined as an increase of 25% or more in the size of tumor or an appearance of new lesions. Kaplan-Meier methods of progression free survival (PFS) and overall survival (OS) were used throughout the analysis. Progression free survival was calculated from the date of operation to date of diagnosis of tumor progression which was confirmed by radiological finding as PD, or last followup. Overall survival was calculated from the date of operation to date of death or last follow-up. The final outcome of PFS and OS were obtained on March 31, 2002. For statistical analysis, the StatView for Windows was used. P values were determined by log rank test.
Among 61 patients with GBM who were treated from January 31, 1990 to December 31, 2001 in the institution, 32 were excluded because insufficient specimens were available. Eleven patients were not treated with nitrosourea, and also excluded from the study. Therefore, 18 patients with GBM were included in the study. all patients were newly diagnosed cases. Age ranged from 30 to 74 years, and the mean age was 52.1 years. Thirteen were male, and five were female. All underwent surgical procedures. There were three patients who had gross total removal of the tumor, six patients who had subtotal removal, eight patients who had partial removal, and one patient who had biopsy. After operation, all patients were given ACNU intravenously at a dose of 100 mg m^sup 2^ of the body surface area on the day when local irradiation was started (day 1), and some also received a second ACNU injection on day 36, in combination with daily intravenous administration of IFN-B as the initial treatment. all but one patient received irradiation to the local field (50-60 Gy in 25-30 fractions, 58.1 Gy on average), and the remaining patient received a stereotactic radiotherapy with a total dose of 69 Gy, fractions of which were 23. After initial treatment, some patients received maintenance chemotherapy using intravenous administration of ACNU at a dose of 100 mg m^sup 2^ every 2-3 months. The cumulative dose of ACNU ranged from 80-700 mg (1-5 courses), 213.9 mg on average. Two patients underwent a second removal of the tumor at the time of recurrence. Seven patients received additional stereotactic irradiation when tumors remained radiologically after initial treatment, or tumor recurrences were verified by radiological imaging, the dose of which ranged from 1044.4 Gy. Median overall survival and progression-free survival according to the Kaplan-Meier's method were 20 and 9 months, respectively. Clinicopathology of all patients are presented in Table 1.
The staining of MGMT was predominantly nuclear, and the proportion of positive cases was 72.2%. Semiquantitatively, five tumors were classified as grade 0, five tumors were grade 1, seven tumors were grade 2, and one tumor was grade 3 for MGMT expression (Figure 7). There was some degree of heterogeneity in MGMT staining of the tumor cells in one specimen. In some specimens, endothelial cells were positive (Figure 3), but the classification was carried out according to the degree of staining of the tumor cells regardless of endothelial staining.
The GST [pi] staining was both nuclear and cytoplasmic, and the heterogeneity of GST [pi] staining was not as marked as that of MGMT, and the proportion of positive cases was 83.3%. Three tumors were classified as grade 0, three tumors were grade 1, five tumors were grade 2, and seven tumors were grade 3 for GST [pi] expression (Figure 2). The MIB-1 indices ranged from 10.6 to 53.4, 25.9 on average.
Median overall survival of the eight patients whose tumors retained high MGMT expression (3 + ~2 +) was 12 months, and that of 10 patients whose tumor showed low MGMT expression (1 + ~0) was 22 months, respectively, which was significantly different (log-rank test, p = 0.02). Median progression free survival (PFS) of each group was nine, and 15 months, respectively, and there was a trend in which the PFS of the latter was long (log-rank test, p =0.09) (Figure 4). There was no significant difference in patient's age, proliferative potential of tumors verified by MIB-1 index, and treatment modalities between the two groups which were divided according to the degree of MGMT expression (Table 2). On the other hand, the median survival of 12 patients whose tumors showed high GST [pi] expression (3 + ~2+) was 13 months, and that of six patients whose tumors showed low GST [pi] expression (1 +~0) was 22 months, with median progression free survival of each group at four and nine months, respectively, not significantly different (Figure 5).
For the patients with GBM, multimodality treatments are performed. In general, chemotherapy is administered, and the chemosensitivity may be an important factor that determines the survival of the patients. The nitrosoureas such as ACNU, BCNU and CCNU, alkylating agents, are internationally used for patients with malignant glioma because of high permeability through the blood-brain barrier and cytotoxic activity to tumor cells. However, a certain proportion of gliomas have intrinsic or acquired resistance to nitrosoureas, and there are many patients whose tumors grow after the multimodality treatment including nitrosourea-based chemotherapy. So it is important to predict the response of the tumor before nitrosourea treatment, because other drugs can be selected if necessary.
DNA repair enzyme, O^sup 6^-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O^sup 6^ position of DNA guanine which are induced by nitrosoureas, preventing the formation of the lethal interstrand DNA cross-links, and thus reduces the effectiveness of the drugs.
Also [alpha]-L-glutamyl-L-cysteinylglycine (glutathione) has been shown to inactivate nitrosourea-compounds like BCNU, and glutathione-S-transferase (GST) [pi] would catalyze the process of nitrosourea detoxification. The expression of GST [pi] in glioma cell lines reportedly has the highest association with the nitrosourea resistance of the cells9.
In this retrospective study, the authors examined the expression of the above mentioned factors by means of immunohistochemistry, and compared the results with tumor responses to therapy including ACNU administration, in order to investigate whether there were correlations between the degree of MGMT or GST [pi] expression in tumors and the survival of the patients following ACNU treatment.
There are some reports which mention a reverse-correlation between the degree of MGMT expression in gliomas and the patients' survival following nitrosourea treatment2-8. Popular measurements of MGMT protein and MGMT mRNA, such as restriction endonuclease assay3, DNA adduct removal assay10-12, Western blotting13, northern blotting1,2, and semiquantitative RT-PCR4,8 need frozen specimens, in which complex and careful treatment is essential. The contamination of nontumoral elements, including neurons, astrocytes, endothelial cells, and necrotic debris, also potentially affects the results.
Immunohistochemistry is a simple method for the detection of many cellular proteins in which we can use paraffin embedded sections. Although it seems to be beneficial, since we can evaluate not only the degree of staining, but also the localization of the target factors by comparing the slide with hematoxylin-eosin stained slide, the authors can find only a limited number of reports in which immunohistochemistry or immunofluorescence assay were used to detect MGMT in gliomas5,6,14-17. The antibody chiefly used in the reports was MT3.1, monoclonal anti-human MGMT antibody, and proportion of positive cases reportedly ranged from 63% to almost 100%15,16, which is consistent with the result of this report. It is reported that immunohistochemical expression of MGMT in the specimens is heterogenous14,16, although in our study there were some specimens in which the MGMT staining in the tumor cells were somewhat heterogenous or only endotherial cells had positive staining (Figure 3). Thus, the classification according to the staining seemed to be difficult in a few cases. In this paper, the authors applied the MGMT positivity of tumor cells alone in the maximal staining area. It would be better that a few examiners determine the classification.
Among 18 GBMs, eight tumors (44.4%) showed moderate to high MGMT expresssion which were classified as grade 2 to 3. The remaining 10 tumors (55.6%) showed no or only weak MGMT expression. Median overall survival of seven patients whose tumors retained high MGMT expression (3 + ~2 +) was 12 months, and that of 10 patients whose tumors showed low MGMT expression (1 + ~0) was 22 months, respectively, which was significantly different (p=0.01). Although there was no significant difference because of small samples, median progression free survival of each group was nine and 15 months, respectively. Although we could not find reports in which immunohistochemistry alone was used to detect MGMT in gliomas and the result was compared with the patients' survival after nitrosourea treatment, the immunohistochemistry of MGMT expression in GBM would be important in order to predict the response of patients with GBM after ACNU treatment, while it remains to be evaluated whether the endothelial expression of MGMT is important or not.
Recently, MGMT expression was found to be under epigenetic control. A report mentions that the hypermethylation of the promotor gene results in down regulation of MGMT protein7. In another report, the hypermethylation of the promotor gene of MGMT may be accompanying p53 mutation18. These findings are in agreement with another report, in which the mRNA of MGMT in secondary GBM which is often accompanied by p53 mutation is reduced compared to that in primary GBM whose p53 is wild type17. Although it is interesting to investigate further the relationship between p53 status and the expression of MGMT in GBM, the authors did not refer to it in this paper.
On the other hand, as far as GST [pi] is concerned, we could not find any relationship between the immunohistochemical expression of GST [pi] and the patients' survival following ACNU administration. The result differs to a previous in vitro study using the glioma cell lines9. It may be due to our small sample size, or due to the difference between in vitro study and the clinical investigation, or to the differences in detection methods. Further investigation could determine whether or not the immunohistochemical method for GST [pi] estimation in GBMs is valuable for prediction of patients' survival following ACNU-based chemotherapy.
It is suggested that simple immunohistochemical study of the specimen using monoclonal anti-MGMT antibody will be effective for predicting the response of personal patient with GBM after ACNU-based chemotherapy.
The authors wish to thank Mrs Reiko Yamasaki for skilled technical assistance.
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Takeo Anda*[dagger], Hamisi Kimaro Shabani*, Keishi Tsunoda*, Yoshiharu Tokunaga*, Makio Kaminogo*, Shobu Shibata*[double dagger], Tomayoshi Hayashi[sec] and Masachika Iseki[para]
* Department of Neurosurgery, Nagasaki University School of Medicine, Nagasaki
[dagger] Department of Neurosurgery, National Nagasaki Medical Center, Nagasaki
[double dagger] Department of Neurosurgery, Miyazaki Hospital, Miyazaki
[sec] Department of Pathology, Nagasaki University Hospital, Nagasaki
[para] Department of Pathology, Institute of Tropical Medicine Nagasaki University, Nagasaki, Japan
Correspondence and reprint requests to: Takeo Anda, MD, Department of Neurosurgery, National Nagasaki Medical Center, 2-1001-1 Kubara, Omura, Nagasaki 856-8562, Japan, [email@example.com] Accepted for publication November 2002.
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