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Annals of Surgical Oncology 8:807-816 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Expression and Prognostic Significance ofO6-Methylguanine-DNA Methyltransferase in Hepatocellular, Gastric, and Breast Cancers

Shiroh Matsukura, MD, Kohji Miyazaki, MD, PhD, Hiroyuki Yakushiji, MD, PhD, Akiomi Ogawa, MD, PhD, Katsumi Harimaya, MD, Yusaku Nakabeppu, PhD and Mutsuo Sekiguchi, PhD

From the Departments of Surgery (SM, KM, HY) and Pathology (AO), Saga Medical School, Saga, Japan; Department of Orthopedic Surgery (KH), Faculty of Medicine, Kyushu University, Fukuoka, Japan; Department of Biochemistry (YN), Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Japan Science and Technology Corporation (YN), Japan; and Department of Biology and Frontier Research Center (MS), Fukuoka Dental College, Fukuoka, Japan.

Correspondence: Address correspondence and reprint requests to: Kohji Miyazaki, MD, PhD, Department of Surgery, Saga Medical School, 5-1-1, Nabeshima, Saga 849-8501, Japan; Fax: 81-952-34-2019.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: O6-Methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine, a promutagenic DNA base damaged by endogenous and environmental alkylating agents. There are few reports that describe whether or not abnormal MGMT expression correlates with the prognosis in human solid cancers.

Methods: The expression of MGMT was immunohistochemically evaluated in 60, 62, 105, and 46 paraffin-embedded samples from patients with curatively resected hepatocellular, gastric, colorectal, and breast cancers, respectively.

Results: The expression of MGMT was a positive predictive factor for overall survival in hepatocellular (P = .005) and gastric cancers (P < .001) and for relapse-free survival in breast cancers (P < .001). MGMT-positive gastric tumors (n = 42) were correlated with the absence of serosal invasion (P = .045), lymph node metastasis (P = .006), intestinal type (P = .018), and low pathological tumor, node, metastasis stage (P < .001). All breast tumors that recurred locally after operation were MGMT negative (P = .004). The clinicopathologic characteristics of colorectal cancers with respect to MGMT expression did not significantly differ.

Conclusions: The expression of MGMT is a predictive prognostic marker in patients with hepatocellular, gastric, and breast cancers. These findings may help to establish therapeutic strategies for patients with these types of solid cancer.

Key Words: O6-methylguanine-DNA methyltransferase (MGMT) • Hepatocellular carcinoma • Gastric cancer • Colorectal cancer • Breast cancer • Prognosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquitous and environmental alkylating agents such as N-nitroso compounds are principally metabolized and activated in hepatocytes.1 Because endogenous alkylating compounds are released into bile and the digestive tract, epithelial cells in the biliary and gastrointestinal tract are always exposed to activated alkylating agents.2 Alkylating agents cause gene mutations or cell death in vitro3 and carcinogenesis or apoptosis in vivo.4,5 These biological effects are induced by the promutagenic base, O6-methylguanine, which is produced by the alkylating agents.6 O6-Methylguanine preferentially mispairs with thymine instead of cytosine during DNA replication, leading to a G:C/A:T transition mutation.7 Humans possess O6-methylguanine-DNA methyltransferase (MGMT), which repairs O6-methylguanine to prevent such mispairing.8 Abnormal MGMT expression causes O6-methylguanine to accumulate in cellular DNA,9 and this could result in activation of oncogenes or inactivation of tumor suppressor genes, contributing to carcinogenesis or tumor progression.10–12 Recent findings from animal models and in vitro studies demonstrate that a deficiency in O6-methylguanine repair seems to be one major determinant of susceptibility to carcinogenesis by alkylating agents.13,14 The carcinogenic mechanism induced by disrupting the MGMT gene was convincingly demonstrated by using the transgenic or knockout mouse model.4,5,15,16 If activation of oncogenes or inactivation of tumor suppresser genes arises because of abnormal MGMT expression in humans, alterations in such cancer-related genes accumulate. However, reports describing solid cancers and whether or not abnormal expression of MGMT correlates with the tumor grade or the prognosis are scarce.

Zaidi et al.17 reported that MGMT immunohistochemical staining correlates with protein quantity and activity. Thus, immunohistochemical analysis can determine both the expression and distribution of MGMT protein. This study investigates the relationship between negative expression of MGMT determined by immunohistochemistry and clinicopathologic features, including prognosis, to clarify whether or not abnormal MGMT expression participates in the carcinogenesis and tumor progression of hepatocellular, gastric, colorectal, and breast cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tumor Specimens
Patients with primary hepatocellular carcinoma (HCC), gastric cancer, colorectal cancer, and breast cancer admitted to the Department of Surgery, Saga Medical School Hospital, were considered for inclusion in this study. This study included consecutive series of 60, 62, 105, and 46 patients with primary hepatocellular, gastric, colorectal, and breast cancer from 1991 to 1998, 1991 to 1992, 1990 to 1992, and 1982 to 1997, respectively. We selected patients with HCC who had simultaneously conserved fresh-frozen samples to investigate genomic analysis. For breast cancer patients, those without preoperative systemic chemotherapy were selected. The series of patients with gastric and colorectal cancer were completely consecutive. Follow-up data for retrospective analyses were obtained by reviewing patient records and by contacting patients’ families and physicians. The mean follow-up period of patients with hepatocellular, gastric, colorectal, and breast cancer was 22.0 months (range, 5 to 80), 56.0 months (range, 10 to 82), 56.4 months (range, 10 to 90), and 63.8 months (range, 5 to 168) months, respectively. Representative formalin-fixed, paraffin-embedded tumor specimens from patients with each cancer who underwent curative resection were selected for this study. The clinical and pathologic features of patients with each type of cancer were classified with the UICC TNM Classification of Malignant Tumors.18

Anti-MGMT Antibody
Because the commercialized anti-human MGMT antibody was nonexistent when this study was started, we used the MGMT custom-made antibody. Polyclonal rabbit antibodies against human MGMT protein were prepared by using TrpE fusion protein, as described.19 Escherichia coli BL21 (DE3) carrying pET3d:TrpE-hMGMT-1, which encodes the TrpE polypeptide, fused to a region of MGMT (residues 1 to 45) at the C terminus was used to produce each fusion protein,20 and polyclonal antibodies against the fusion protein were raised in rabbits. The serum was initially applied to a TrpE-hMGMT-1-coupled column, and their bound materials were eluted at pH 2.3 and dialyzed against 10 mM of Tris-HCl (pH 7.4) and 150 mM of NaCl. To increase antibody specificity, the eluted fraction was applied to an affinity column with TrpE-mMGMT-1, in which a corresponding region of mouse MGMT (residues 1 to 58) was fused to TrpE21 as a ligand, and then the bound fraction was eluted and dialyzed. This fraction was used as anti-MGMT antibody.

Immunohistochemical Staining for MGMT
Sections (5 µm) were deparaffinized in xylene and dehydrated. Antigen was retrieved by microwaving the samples three times for 5 minutes in 10 mM of sodium citrate buffer (pH 6.5). Endogenous peroxidase activities were blocked by immersing the slides in methanol containing 3% hydrogen peroxide for 10 minutes. The slides were then incubated with 10% normal goat serum (Nichirei Co., Tokyo, Japan) for 30 minutes to reduce background staining, followed by anti-human MGMT antibodies (1/200) at 4°C overnight. Negative control sections were incubated with normal rabbit serum instead of the antibodies. The slides were then exposed to EnVision+TM polymer reagent (Dako Corporation, Carpinteria, CA), and goat anti-rabbit immunoglobulins were conjugated with peroxidase-labeled dextran polymer at room temperature for 30 minutes. The slides were washed in phosphate-buffered saline twice and developed by using a Histofine DABTM substrate kit (Nichirei) according to the manufacturer’s instructions at room temperature for 8 minutes. Nuclei were counterstained with hematoxylin. Normal epithelia, interstitial fibroblast, vascular smooth muscle, and smooth muscle layers within the sections were used as internal positive controls. Two pathologists without knowledge of clinicopathologic features and clinical outcome of the patients assessed the status of MGMT expression as positive or negative. The sample was considered positive when immunoreactivity was detected in >10% of the cells in nuclei, cytoplasm, or both.22,23

Statistical Analysis
The clinicopathologic characteristics were compared with MGMT-positive and -negative groups, and the significance of associations was determined with the Mann-Whitney U-test or Student’s t-test for continuous data and the {chi}2 test for categorical data. The survival data were used to generate Kaplan-Meier curves that were compared on the basis of MGMT status by using the log-rank test. Statistical significance was judged as P < .05. Multivariate analysis with the Cox proportional hazards regression model was performed by using statistical analysis software (JMP MacintoshTM version; SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of Anti-MGMT Antibody
We determined the specificity of this antibody by Western blot analysis by using cell-line lysate. HeLa S3 is an MGMT-proficient (Mer+) cell line24,25 of cervical carcinoma. HeLa MR is an MGMT-deficient (Mer-) cell line,24,25 and HeLa MRV-11 is transfected vector only. HeLa MR5-2 is an MGMT overexpressor that is transfected with human MGMT expression vector. The band corresponded to MGMT protein (23 kDa; Fig. 1). Semiquantitative analysis is shown in Fig. 1.



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  		FIG. 1. The pecificity of O6-methylguanine-DNA methyltransferase (MGMT) antibody was determined by Western blot analysis by using cell-line lysate. HeLa S3 and HeLa MRV-11 were MGMT-positive and MGMT-negative cell lines, respectively. HeLa MR5-2 was an MGMT overexpressor. The band corresponded to MGMT protein (23 kDa). Semiquantitative analysis is shown. The amount of protein (µg) on loading is in parentheses.

 
Expression of MGMT
MGMT proteins were expressed intensely and uniformly in the nuclei and cytoplasm of cell lines and were detected in normal epithelia, interstitial fibroblasts, vascular smooth muscle, and the smooth muscle layer (data not shown). Tumors positive for MGMT homogeneously expressed the protein in the nuclei of HCC (Fig. 2A), gastric cancer (Fig. 2B), colorectal cancer, and breast cancer (data not shown) cells. Several cancers in which signals for the protein were detected in the cytoplasm were classified as MGMT positive (Fig. 2C, gastric cancer). Tumors without signals for the protein in the nuclei and cytoplasm were defined as MGMT negative (Fig. 2D, gastric cancer). Interstitial fibroblast was shown as an internal positive control in Fig. 2D. MGMT positivity was identified in 50.0% (30 patients) of 60 HCCs, 67.7% (42 patients) of 62 gastric cancers, 33.3% (35 patients) of 105 colorectal cancers, and 54.3% (25 patients) of 46 breast cancers.



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  		FIG. 2. O6-methylguanine-DNA methyltransferase (MGMT) immunohistochemical staining of hepatocellular carcinoma (HCC) and gastric cancer (magnification: A, B, D, x200; C, x100). (A) Representative MGMT-positive HCC; (B, C) MGMT-positive gastric cancer. Gastric cancer with MGMT proteins stained in nuclei (B) or cytoplasm (C) were defined as MGMT positive. No signal for protein was detected in MGMT-negative gastric cancer (D). Interstitial fibroblast was shown as an internal positive control (D).

 
Correlation Between MGMT Expression Status and Clinicopathologic Features
HCC
No significant correlation was found between MGMT expression status and age, sex, viral infection, liver cirrhosis, tumor diameter, grade of differentiation, portal invasion or intrahepatic metastasis, or pathologic tumor, node, metastasis stage (Table 1).


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  		TABLE 1. Correlation of MGMT expression with clinicopathologic features in hepatocellular carcinoma
 
Gastric Cancer
Serosal invasion, lymph node metastasis, histologic type, and pathologic tumor, node, metastasis stage of the gastric cancers were associated with MGMT expression status, with a significant difference (P = .045, P = .006, P = .018, and P < .001, respectively; Table 2). MGMT-negative tumors invaded deeper into the stomach wall, had a higher ratio of the present lymph node metastasis and diffuse type, and were classified at a higher pathologic tumor, node, metastasis stage than MGMT-positive tumors. MGMT expression and the other clinicopathologic features analyzed in the study did not significantly correlate.


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  		TABLE 2. Correlation of MGMT expression with clinicopathologic features in gastric cancer
 
Colorectal Cancer
The MGMT expression status and age, sex, tumor size, tumor location, lymphatic invasion, venous invasion, serosal invasion, lymph node metastasis, grade of differentiation, and pathologic tumor, node, metastasis stage of colorectal cancer did not significantly correlate (Table 3).


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  		TABLE 3. Correlation of MGMT expression with clinicopathologic features in colorectal cancer
 
Breast Cancer
All of the breast cancers were pathologically diagnosed as invasive ductal carcinoma. Because only two patients with breast cancer died, we analyzed not overall survival but relapse-free survival rates. Local recurrence of MGMT-negative tumors was frequent, with a significant difference (P = .004; Table 4). The MGMT expression status and the other clinicopathologic characteristics did not significantly correlate.


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  		TABLE 4. Correlation of MGMT expression with clinicopathologic features in breast cancer
 
Univariate Analysis of Survival
HCC
The overall 5-year survival rate for patients with MGMT-positive tumors was 89.1%, compared with 46.9% for those with MGMT-negative tumors (P = .005; Fig. 3A).



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  		FIG. 3. Overall -year survival of patients with O6-methylguanine-DNA methyltransferase (MGMT)-positive [MGMT(+)] and -negative [MGMT(-)] in hepatocellular (A), gastric (B), and colorectal (C)cancers. 5-year survival probability of MGMT(+) and MGMT(-) was 89.1% and 46.9%, respectively (log-rank test) (A). 5-year survival probability of MGMT(+) and MGMT(-) was 88.0% and 35.0%, respectively (log-rank test) (B). 5-year survival probability of MGMT(+) and MGMT(-) was 82.9% and 76.1%, respectively (log-rank test) (C). Comparison of relapse-free survival of patients with [MGMT(+)] and [MGMT(-)] breast cancers. 10-year relapse-free survival probability was 100.0% and 35.7%, respectively (log-rank test) (D).

 
Gastric Cancer
The overall 5-year survival rates for patients with MGMT-positive and -negative tumors were 88.0% and 35.0%, respectively (P < .001; Fig. 3B).

Colorectal Cancer
The overall 5-year survival rates for patients with MGMT-positive and -negative tumors were 82.9% and 76.1%, respectively, with no significant difference (P = .6521; Fig. 3C).

Breast Cancer
The 10-year relapse-free survival rates for patients with MGMT-positive and -negative tumors were 100.0% and 35.7%, respectively (P < .001; Fig. 3D).

Multivariate Analysis of Survival
To determine the variables affecting the survival of gastric cancer patients, five variables correlated in univariate analysis (serosal invasion; lymph node metastasis; histologic type; pathological tumor, node, metastasis stage; and MGMT status) were analyzed by using the Cox proportional hazards regression model. Analysis showed pathological tumor, node, metastasis stage (P = .0034) and lymph node metastasis (P = .0196) to be significant variables to independently predict postoperative survival (Table 5). Among HCC patients, MGMT status (and likewise, pathological tumor, node, metastasis stage) was an independent prognostic factor (data not shown).


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  		TABLE 5. Multivariate analysis of Cox proportional hazards models of selected clinicopathologic variables in 62 patients with gastric cancer evaluated for prognosis
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The expression of MGMT protein has been studied. Kokkinakis et al.26 reported that the MGMT protein expression level in pancreatic cancer was correlated with malignant potential. They showed that tumors that expressed high levels of MGMT protein had lower grade differentiation, more advanced stage, and a poorer prognosis than the tumors with low expression.26 This study found that tumors with negative expression had a poorer prognosis than those with positive expression in hepatocellular, gastric, and breast cancer. This discrepancy may be due to the different numbers of patients and the study scale. Because Kokkinakis et al. studied only 12 patients with invasive ductal adenocarcinoma and did not include early-stage disease, statistical significance was not demonstrated. Ishibashi et al.24 suggested that the intracellular distribution of MGMT differs among tumor types and in some cancer cell lines. The expression pattern of pancreatic cancer might be different from that of the cancers in our study.

This study of patients with HCC found that negative MGMT expression was significantly correlated with poor prognosis. MGMT status did not significantly differ with respect to clinicopathologic features, and multivariate analysis of survival showed that it is an independent predictive prognostic factor (data not shown). It is unclear why MGMT expression could be an independent prognostic marker. It is speculated that MGMT status might be correlated with potential liver function.27 Major and Collier27 demonstrated that the mean value of MGMT activities determined in the viral cirrhotic liver is much lower than that of normal tissue. However, in our study MGMT status did not differ between the presence and absence of viral infection or liver cirrhosis.

Negative MGMT expression was significantly correlated with tumor progression and a poor prognosis of gastric cancer. Because MGMT-negative gastric cancers had progressive characteristics, such as advanced pathologic tumor, node, metastasis stage, it is obvious why patients with MGMT-negative gastric cancer had a poorer prognosis. Multivariate analysis of the Cox proportional hazards model showed that MGMT expression status was not an independent prognostic factor. Pathological tumor, node, metastasis stage was the strongest prognostic marker. Thus, abnormalities in genes related to cancerous invasion and metastasis, such as adhesion molecules, should be involved in MGMT dysfunction.

In a study of breast cancer, however, Wani and D’Ambrosio28 reported that the tumor grade and metastatic potential of breast cancer were not correlated with negative expression of the messenger RNA for the MGMT gene. This study of patients with breast cancer showed that tumor grade or metastasis and negative MGMT expression did not significantly correlate, whereas local recurrence and the relapse-free survival rate correlated with negative expression. Activation or inactivation of unknown molecules that contributed to the local recurrence may be involved in negative MGMT expression.

Among cancers analyzed in this study, a significant correlation between negative MGMT expression and the prognosis of patients with colorectal cancers was not found. The significance of this enigma may be organ specific, but early-stage cancers were not included in this study of colorectal cancers. For an explanation for the differences in the results for each tumor type, consideration should be given to variations of point mutations for each malignancy. In gene mutations, a large percentage of pancreas29,30 and, to a lesser extent, colorectal31,32 cancers have K-ras mutations. K-ras and other mutations may occur early in colorectal cancers,33 and therefore evaluating MGMT expression may not be prognostic when evaluating more advanced stage-tumors, as was performed in the colorectal group.

Recently, the first evidence that abnormal MGMT expression could result in activation of an oncogene contributing to carcinogenesis was elucidated in human cancer.12 Esteller et al.12 demonstrated that inactivation of MGMT by promoter hypermethylation was associated with a G to A transition mutation in the K-ras oncogene in colorectal tumorigenesis. Thus, accumulation of this type of mutation in oncogenes or tumor suppressor genes would follow as the results of MGMT negative expression. To the best of our knowledge, few reports have described polymorphism or mutation of the human MGMT gene.34–38 However, several recent reports describing negative MGMT expression have indicated that promoter hypermethylation is responsible for silencing MGMT. Aberrant hypermethylation is associated with a loss of MGMT protein, according to Esteller et al.23 Epigenetic inactivation of MGMT may play an important role in primary human neoplasia.39,40 Aberrant promoter hypermethylation is correlated with the loss of many cancer-related genes, such as Rb,41 p16,42,43 TGFbeta-3,44 E-cadherin,45,46 CD44,47 p73,48 and hMLH1.49 Because the same epigenetic mechanism of negative expression could occur in the MGMT gene and in several other genes that play important roles in malignant potential or tumor progression, the close relationship between negative MGMT expression and the poorer prognosis in hepatocellular, gastric, and breast cancer patients could be explained.

Expression of MGMT was a predictive prognostic marker in patients with hepatocellular, gastric, and breast cancers. These findings may help to establish therapeutic strategies for patients with these types of solid cancer.


   Acknowledgments
 
The authors thank Drs. Akihiro Iyama, Kenji Kitahara, and Michito Mori (Department of Surgery, Saga Medical School) and Drs. Eizaburo Sasatomi, Genichiro Edakuni, and Yongxin Chen (Department of Pathology, Saga Medical School) for their helpful advice and assistance.

Received for publication February 9, 2001. Accepted for publication July 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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