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Annals of Surgical Oncology 9:371-379 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Deficient Expression of O6-Methylguanine-DNA Methyltransferase Combined With Mismatch-Repair Proteins hMLH1 and hMSH2 Is Related to Poor Prognosis in Human Biliary Tract Carcinoma

Naohiko Kohya, MD, Kohji Miyazaki, MD, PhD, Shiroh Matsukura, MD, Hiroyuki Yakushiji, MD, PhD, Yoshihiko Kitajima, MD, PhD, Kenji Kitahara, MD, PhD, Masao Fukuhara, MD, PhD, Yusaku Nakabeppu, PhD and Mutsuo Sekiguchi, MD, PhD

From the Department of Surgery (NK, KM, SM, HY, YK, KK), Saga Medical School, Saga, Japan; Department of Biochemistry (MF, YN), Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and Department of Biology (MS), Fukuoka Dental College, Fukuoka, Japan.

Correspondence: Address correspondence and reprint requests to: Kohji Miyazaki, MD, PhD, Department of Surgery, Saga Medical School, Nabeshima 5-1-1, Saga, Japan 849-8501; Fax: 81-952-34-2019; E-mail: iyazak2@ post.saga-med.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is an important step in the induction of mutations in the organism by alkylating agents. The O6-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location.

Methods: We examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining.

Results: MGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types.

Conclusions: Combined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.

Key Words: Biliary tract carcinoma • MGMT • hMLH1 • hMSH2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alkylation of DNA at the O6 position of guanine is an important step in the induction by alkylating agents of mutations in the organism.1 O6-methylguanine is capable of pairing with both thymine and cytosine during DNA replication, resulting in a G:C to A:T transition mutation in DNA.2 O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself to repair the premutagenic base in DNA. The level of MGMT varies among different organs and tissues,3,4 whereas most tumor cells express high levels of MGMT; approximately 20% of human tumor cell lines are deficient in MGMT expression. MGMT-lacking cell lines are hypersensitive to alkylating agents, and these cell lines, termed Mer- or Mex-, show little or no methyltransferase activity.5– 9 The mechanisms responsible for regulating MGMT expression remain unknown. Abnormal MGMT expression may induce either human oncogene activation or tumor suppressor gene inactivation, thereby contributing to carcinogenesis and tumor progression. Therefore, abnormal MGMT expression may correlate with the malignant grade of cancer and the prognosis of patients with neoplastic disease.

O6-Methylguanine on its own is not deleterious to cells, and it does not inhibit DNA enzymatic processes, such as replication and transcription. However, the preferred base pairing during DNA synthesis results in incorporation of thymine opposite O6-methylguanine rather than cytosine, and this in turn results in a G:C to A:T transition mutation if not repaired. The O6-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway of the cell,10 which subsequently excises the errant thymine residue in the daughter strand. However, unless the O6-methylguanine is repaired before the resynthesis step in MMR, there is a high likelihood that thymine will be reinserted opposite the lesion. It is believed that the ensuing repetitive cycle of futile MMR results in the generation of chronic strand breaks, which in turn elicits an apoptotic response.11 Of five or more distinct proteins that are involved in the MMR pathway,12 two—MLH-1 and MSH-2—have frequently been found to be suppressed in tumor cells.13

Biliary tract carcinoma is a relatively rare tumor that is characterized by a poor prognosis. Mortality rates are highest among American Indian women from the southwestern United States and in Chilean and Japanese women.14 Alkylating agents are metabolized and activated in hepatocytes and are released from the bile duct and stored in the gallbladder. The epithelium of the gallbladder and extrahepatic bile duct is highly exposed to alkylating agents because of its physiological function and anatomical location.

Previous studies have observed that genetic abnormalities in biliary tract carcinoma are focused mainly on dominant oncogene K-ras mutations15–18 and on abnormalities of the tumor suppressor genes p5316–19 and p16INK4.20 Abnormalities of these factors are considered to be early changes during carcinogenesis and are not correlated with pathologic factors or patient prognosis.21 The aim of this study was to analyze the association of deficient or reduced MGMT and MMR enzyme (hMLH1 and hMSH2) expression with clinicopathologic factors and prognosis in human biliary tract carcinomas.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Thirty-nine gallbladder carcinoma specimens and 35 extrahepatic bile duct carcinoma specimens were obtained from patients who underwent surgical resection at the Department of Surgery, Saga Medical School, from April 1989 to February 2000.

Antibody Preparation
Polyclonal rabbit antibodies against human MGMT protein were prepared by using tryptophan operon of Escherichia coli (TrpE) fusion protein, as previously described.22 E. coli BL21 (DE3) carrying pET3d:TrpE-hMGMT-1, which encodes the TrpE polypeptide fused to a part of MGMT (residues 1–45) at its C terminus, was used to produce each fusion protein,23 and polyclonal antibodies against the fusion protein were raised in rabbits. First, the serum was applied to the TrpE-hMGMT-1–coupled column, and 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 recover the antibody fraction at a much higher specificity, the eluted fraction was applied to an affinity column with TrpE-mMGMT-1, in which a corresponding part of mouse MGMT (residues 1–58) was fused to TrpE24 as a ligand, and the bound fraction was eluted and dialyzed. This fraction was used as the anti-MGMT antibodies.

Determination of Anti-MGMT Antibody
We determined the specificity of this MGMT antibody by Western blot analysis by using cell-line lysate. HeLa S3 is an MGMT-proficient (Mer+) cell line25,26 of cervical carcinoma. HeLa MR is an MGMT-deficient (Mer-) cell line,25,26 and HeLa MRV-11 is a transfected vector only. HeLa MR5-2 is an MGMT overexpressor that is transfected with human MGMT expression vector. We performed semiquantitative analysis with these cell-line lysates, and the specificity of this antibody was checked.27

Immunohistochemical Analysis
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections of all 39 gallbladder carcinoma and 35 extrahepatic bile duct carcinoma specimens. The primary antibodies used in this study were rabbit polyclonal antibody against MGMT and monoclonal antibody against hMLH1 (clone G168-728; PharMingen, San Diego, CA) and hMSH2 (clone G219-1129; PharMingen). Sections (5 µm) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. The sections were placed in .01 M of citrate buffer (pH 6.0) and exposed to microwaves for 15 minutes. Next, .3% hydrogen peroxidase was applied for 10 minutes to block endogenous peroxidase activity, and the tissues were then incubated in 10% normal goat serum for 10 minutes to abolish nonspecific protein binding. The sections were then incubated with a primary antibody at 4°C overnight. The MGMT antibody was used at a dilution of 1/200. The hMLH1 and hMSH2 antibodies were used at a dilution of 1/50. Immunostaining was performed by the streptavidin-biotin-peroxidase complex method by using a Histofine SAB-PO kit (Nichirei Co., Tokyo, Japan). Staining was visualized with diaminobenzidine tetrachloride. The nuclei were counterstained lightly with hematoxylin.

Positive staining was identified by the presence of brown staining in the cytoplasm or in the nucleus. MGMT, hMLH1, and hMSH2 expression was evaluated as positive if the distribution of stained cells was to >10% of cancer cells. Normal epithelium and lymphocytes within the tumor section were used as positive internal controls. The status of MGMT, hMLH1, and hMSH2 was assessed by two surgical pathologists (N.K and G.E.) without knowledge of the clinical and pathological features of the case or the clinical outcome.

Statistical Analysis
The correlation between MGMT, hMLH1, and hMSH2 expression and clinicopathologic factors was determined by the {chi} 2 test or Student’s t-test at a 5% level of significance. Survival was calculated from the day of the last follow-up. The Kaplan-Meier method was used for the survival analysis, and statistical significance was analyzed by the Wilcoxon method. A P value of <.05 was considered to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of MGMT, hMLH1, and hMSH2 Protein in Biliary Tract Carcinoma
Expression of MGMT, hMLH1, and hMSH2 was localized in the nuclei of neoplastic cells (Fig. 1). In all normal tissue, MGMT protein was expressed and detected on vascular endothelial cells, vascular smooth muscle, smooth muscle, normal epithelium mucosa, lymphocytes, and inflammatory cells (data not shown). Fig. 1A shows positive immunoreactivities for MGMT in the nuclei in a gallbladder carcinoma specimen, and smooth muscle also shows positive immunoreactivities. Fig. 1B shows negative immunoreactivities for MGMT in a gallbladder carcinoma specimen, but vascular smooth muscle and lymphocytes show positive immunoreactivities. Fig. 1C shows positive immunoreactivities for hMLH1 in the nuclei in a gallbladder carcinoma specimen. The positive immunoreactivities for hMLH1 were also shown in vascular endothelial cells, vascular smooth muscle, and lymphocytes (data not shown). Fig. 1D shows positive immunoreactivities for hMSH2 in the nuclei in a gallbladder carcinoma specimen. The positive immunoreactivities for hMSH2 were also shown in lymphocytes (data not shown). Positive nuclear immunohistostaining of MGMT was observed in 16 (41.0%) of 39 gallbladder carcinomas and in 14 (40.0%) of 35 extrahepatic bile duct carcinomas. Of 39 cases of gallbladder carcinoma, positive nuclear immunohistostaining for hMLH1 and hMSH2 was observed in 19 (48.7%) and 16 (41.0%) cases, respectively, whereas for the 35 cases of extrahepatic bile duct carcinoma, these values were 15 (42.9%) and 12 (34.3%).



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  		FIG. 1. Immunohistochemical results of the expression of O6-methylguanine-DNA methyltransferase (MGMT), hMLH1, and hMSH2 protein in gallbladder carcinoma specimens. (A) Strongly positive immunoreactivities for MGMT in the nuclei in gallbladder carcinoma specimen are shown (Mer+). (B) There was no immunoreactivity for MGMT in cancer cells, but positive in lymphocytes or endothelial cells. (C) Positive immunoreactivities for hMLH1 in the nuclei. (D) Positive immunoreactivities for hMSH2 in the nuclei.

 
Correlation Between MGMT Expression and Clinicopathologic Factors
The correlation between MGMT status and clinicopathologic factors is shown in Table 1 for gallbladder carcinoma and in Table 2 for extrahepatic bile duct carcinoma. In gallbladder carcinoma, MGMT-negative staining correlated with hepatic invasion (P < .05), whereas in extrahepatic bile duct carcinoma, MGMT-negative staining had a tendency to correlate with clinicopathologic factors, namely, lymph node metastasis, venous invasion, and perineural invasion.Table 2 for extrahepatic bile duct carcinoma. In gallbladder carcinoma, MGMT-negative staining correlated with hepatic invasion (P < .05), whereas in extrahepatic bile duct carcinoma, MGMT-negative staining had a tendency to correlate with clinicopathologic factors, namely, lymph node metastasis, venous invasion, and perineural invasion.


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  		TABLE 1. Expression of MGMT in gallbladder carcinoma and its correlations with clinicopathologic factors
 

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  		TABLE 2. Expression of MGMT in extrahepatic bile duct carcinoma and its correlations with clinicopathologic factors
 
Correlation Between hMLH1 and hMSH2 Expression and Clinicopathologic Factors
The correlation between hMLH1 and hMSH2 status and clinicopathologic factors is shown in Table 3 for gallbladder carcinoma and in Table 4 for extrahepatic bile duct carcinoma. There was no significant correlation between clinicopathologic factors and hMLH1 and hMSH2 status except for age in hMSH2 in gallbladder carcinoma. There was no significant correlation between clinicopathologic factors and hMLH1 and hMSH2 status in extrahepatic bile duct carcinoma.


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  		TABLE 3. Expression of hMLH1 and hMSH2 in gallbladder carcinoma and its correlations with clinicopathologic factorsa
 

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  		TABLE 4. Expression of hMLH1 and hMSH2 in extrahepatic bile duct carcinoma and its correlations with clinicopathologic factorsa
 
Prognostic Significance of MGMT and MMR Status
The correlation between MGMT protein expression and survival rates of 39 gallbladder carcinoma patients and 35 extrahepatic bile duct carcinoma patients with follow-up data was also examined. Figure 2 shows the Kaplan-Meier survival curves of the patients grouped according to the immunoreactivity of MGMT in their tumors. In both gallbladder and extrahepatic bile duct carcinoma, patients with tumors negative for MGMT had a significantly poorer prognosis than those with tumors positive for MGMT (P < .05).



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  		FIG. 2. Kaplan-Meier analysis of disease-specific survival of patients with (A) gallbladder carcinomas and (B) extrahepatic bile duct carcinomas according to O6-methylguanine-DNA methyltransferase (MGMT) status. Both tumors with MGMT-negative status were significantly correlated with poorer outcome (Wilcoxon test, P < .05).

 
Figure 3 shows the Kaplan-Meier survival curves of combined MGMT status with MMR status. In both tumor types, survival to date among patients with tumors positive for both MGMT and MMR proteins has been 100%, which suggests that the combination is a more significant positive prognostic biomarker than MGMT status alone. However, hMLH1 and hMSH2 were not independently correlated with patient prognosis (data not shown).



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  		FIG. 3. Kaplan-Meier analysis of disease-specific survival of patients with (A) gallbladder carcinomas and (B) extrahepatic bile duct carcinomas according to O6-methylguanine-DNA methyltransferase (MGMT) combined with mismatch-repair (MMR) status. The survival rate of the patients with tumors positive for both the MGMT and MMR proteins has been 100%.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The DNA repair enzyme MGMT plays an important role in protecting cells against alkylating agents that induce O6-alkylylguanine in DNA.28 The occurrence of abnormal MGMT expression in tumors may be accompanied by an accumulation of gene mutations that are critical for tumor progression. Reduced or deficient expression of MGMT protein may occur as a result of genetic mutation, messenger RNA instability, or hypermethylation of the promoter region. However, loss of expression is not commonly due to deletion, mutation, or rearrangement of the MGMT gene5,29,30 or to messenger RNA instability.31 Hypermethylation of the normally unmethylated CpG island in the promoter region of tumor suppressor and MMR genes (e.g., p16, Rb, VHL, E-cadherin, and hMLH1) correlates with loss of transcription,32–35 and the human MGMT gene has a CpG island in the promoter region.36 Recent studies have reported that methylation of discrete regions of the MGMT CpG island is associated with a silencing of the gene in the cell lines.37–39 The tight correlation between MGMT CpG island methylation and loss of expression in these tumors may have accounted for the loss of MGMT activity previously reported in a subset of numerous tumor types.37–45 However, in tumors such as ovarian and pancreatic carcinoma or meningioma, decreased MGMT activity is infrequent.40,41,45,46

Our data demonstrate that deficient expression of MGMT is correlated with tumor progression and a poorer prognosis in gallbladder carcinoma. The possibility is recognized that deficient expression of MGMT is caused mainly by hypermethylation of the promoter region in MGMT. Hypermethylation of MGMT can occur not only in MGMT, but also in numerous other genes with a CpG island in their promoter regions. Hypermethylation of multiple genomes, including oncogenes and tumor suppressor genes, might bring about tumor progression and a poorer prognosis. However, recent studies have found that inactivation of MGMT by promoter hypermethylation is associated with G to A mutations in the K-ras oncogene in colorectal cancer.47 This finding demonstrates that deficient expression of MGMT can strongly induce G:C to A:T mutations in oncogene or tumor suppressor genes. The accumulation of mutations in oncogenes and tumor suppressor genes may lead to tumor progression and a poorer prognosis.

In this study, MGMT-negative staining was correlated with hepatic invasion (P < .05) in gallbladder carcinoma (Table 1) and had a tendency to correlate with lymph node metastasis, venous invasion, and perineural invasion in extrahepatic bile duct carcinoma. This finding indicates that deficient MGMT expression in cancer cells increases tumor progression in biliary tract carcinoma (Table 2). To our knowledge, no previous reports have compared MGMT expression with clinicopathological factors. We also reported a significant correlation between MGMT status and viral infection in hepatocellular carcinomas, depth of wall invasion in gastric cancers, and local recurrence in breast cancers with immunohistochemical staining.27 The definite role of reduced MGMT in tumor progression remains unknown, but our findings suggest that reduced MGMT expression may induce an accumulation of gene mutations in many human genes and could induce oncogene activation or tumor suppressor gene inactivation, thereby contributing to tumor progression.

In only one previous report did MGMT status correlate with patient survival in ovarian cancer. MGMT activity after chemotherapy has been reported to correlate with tumor stage and grade, but not with patient survival.48 Our data show that deficient MGMT expression significantly correlated with a poor prognosis in gallbladder and extrahepatic carcinoma (P < .05). We previously observed in an immunohistochemical study that reduced MGMT expression was correlated with a poor prognosis in hepatocellular carcinomas, gastric cancers, and breast cancers.27 Deficient MGMT expression does not directly shorten patient survival, but the associated accumulation of oncogene activation and tumor suppressor gene inactivation could consequently shorten patient survival.

Many tumors occur in MGMT- mice exposed to alkylating agents, whereas no or few tumors occurred in normal mice treated in the same manner.49,50 Recent studies have found that an acquired resistance of mammalian methyltransferase-deficient Mer- cell lines to alkylating agents is associated with a loss of capacity for MMR, a phenomenon known as methylation tolerance.10,51 The accumulation of alkylated bases in chromosomal DNA may provoke abortive MMR, an event that could lead to cell death.52 In contrast to MGMT-/- MLH1+/+ mice who show a decrease in the size of the thymus and hypocellular bone marrow after administration of alkylating agents, no conspicuous change was found in MGMT-/- MLH1-/- mice treated in the same manner.53 In this study, MMR status alone did not correlate with clinicopathologic factors or prognosis, but when MGMT and MMR status were combined, the prognostic value was more significant than that of MGMT status alone for both gallbladder and extrahepatic bile duct carcinoma. These results suggest that the combination of MGMT and MMR status is a more significant indicator of the malignant potential of biliary tract carcinoma.

In conclusion, deficient MGMT expression was correlated with one pathologic factor—namely, hepatic invasion—as well as with a poorer prognosis in biliary tract carcinoma. Furthermore, combined MGMT and MMR status was a more significant prognostic biomarker. Although the precise role that MGMT status plays in prognosis remains to be determined, MGMT and MMR status represent potential prognostic markers in human biliary tract carcinomas that probably reflect an accumulation of genetic mutations.


   Acknowledgments
 
The authors thank Dr. Genichiro Edakuni (Department of Pathology, Saga Medical School) for technical help.

Received for publication July 9, 2001. Accepted for publication February 9, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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