Affiliations of authors: Departments of Leukemia (LS, YK, NSH, JV, J-PJI), Biostatistics and Applied Biomathematics (GLR, LX), and Pathology (PSH, SRH), The University of Texas at M. D. Anderson Cancer Center, Houston, TX; Departments of Surgery (RSK) and Pathology (ARP), University of Arizona, Tucson, AZ, and Southern Arizona Veterans Affairs Health Care System, Tucson, AZ; Arizona Cancer Center (JGE, JB, DSA), University of Arizona, Tucson, AZ
Correspondence to: Jean-Pierre Issa, MD, Department of Leukemia, M. D. Anderson Cancer Center, Unit 428, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: jpissa{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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In principle, the molecular abnormalities that are responsible for a field defect should be detectable at high frequency in patients with cancer but at low frequency in patients without neoplasia. Furthermore, these abnormalities should be detectable in healthy individuals who have conditions that put them at risk for the cancer as well as in the neoplastic lesions themselves, and they should occur early in the neoplastic process. Ideally, the abnormalities should also be functionally involved in neoplasia. Age-related epigenetic defects have been proposed as potential sources of the field defect in colon carcinogenesis (3,4). However the identification of genes that clearly identify individuals at (high) risk for colon cancer has been lacking.
The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer. The DNA repair protein encoded by the MGMT gene is involved in defending cells against alkylating agents (5,6). Alterations in the MGMT gene impair the ability of the MGMT protein to remove alkyl groups from the O6-position of guanine, thereby increasing the mutation rate and the risk of cancer (7). To date, no published studies have reported an association between genetic defects in the MGMT gene, such as mutations and/or deletions, and human cancer. However, several studies have reported that transcriptional silencing of this gene in multiple tumor types is associated with hypermethylation of the CpG island in its promoter (8,9). Silencing of MGMT has been shown to be associated with and to precede the appearance of G-to-A point mutations in the KRAS gene during colorectal tumorigenesis (10,11). We hypothesized that MGMT methylation could be one of the mediators of field cancerization in the colon mucosa. To test this hypothesis, we studied MGMT methylation quantitatively in the neoplastic tissues, adjacent mucosa, and distal mucosa of patients with colorectal cancer, as well as in the colonic mucosa from patients without evidence of malignancies.
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PATIENTS, MATERIALS, AND METHODS |
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We collected samples of primary colorectal tumors and samples of the corresponding adjacent normal-appearing tissue from 95 patients who had undergone surgery at the Johns Hopkins Hospital (n = 54; Baltimore, MD), the M. D. Anderson Cancer Center (n = 8; Houston, TX), the University of Arizona (n = 29; Tucson, AZ), and the Southern Arizona Veterans Affairs Health Care System (n = 4; Tucson, AZ) in accordance with institutional policies. All patients provided written informed consent. Tumors were selected solely on the basis of availability. All samples of normal-appearing mucosa were derived from sites adjacent to, but at least 1 cm away from, the tumors. We also obtained two additional samples of normal-appearing colonic mucosa from 36 patients: The two samples originated from tissue located 1 cm and 10 cm away from the cancer. Clinicopathologic data were available for most of the 95 colorectal cancer patients; for some patients we were missing information on sex (n = 4), age at surgery (n = 5), the location of the tumor in the colon (n = 12), and tumor stage (n = 22). We also obtained colonic biopsy specimens from 33 individuals who had no family history of colorectal cancer and who had no colonic lesions at screening colonoscopy. These healthy control specimens were selected on the basis of availability. All tissue samples were fresh-frozen and stored at 80 °C. CpG island methylator phenotype (CIMP) and p53 mutation status were available for 54 and 51 patients respectively from previous studies (12,13).
Cell Lines and Culture Conditions
We isolated DNA from five colon cancer cell lines: SW48, RKO, HCT116, SW480, and CaCO2. HCT116, CACO2, and RKO cells were grown in high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. SW48 and SW480 cells were grown in L-15 medium containing 10% fetal bovine serum in plastic tissue culture plates in a humidified atmosphere containing 5% CO2 at 37 °C. Cell lines were obtained from the American Type Culture Collection (Manassas, VA). Media were purchased from Invitrogen (Carlsbad, CA).
Bisulfite Polymerase Chain Reaction Analysis of DNA Methylation
Genomic DNA was extracted from patient samples and from the cell lines using a standard phenolchloroform method. Bisulfite treatment of 2 µg of genomic DNA was performed as previously described (14). We used combined bisulfite restriction analysis (COBRA) and methylation-sensitive polymerase chain reaction (PCR) (MSP) to examine MGMT promoter methylation in all DNA samples (15). For COBRA, we used the following oligonucleotide primers to amplify a 161-bp region of the promoter cytosine guanine dinucleotide (CpG) island: 5'-TTGGTAAATTAAGGTATAGAGTTTT-3' (sense primer) and 5'-CTAAAACAATCTACGCATCCTC-3' (antisense primer). PCRs were carried out in a volume of 50 µL. Each reaction contained 2 µL of bisulfite-treated DNA,1.25 mM deoxynucleoside triphosphate, 6.7 mM MgCl2, 5 µL of PCR buffer, 1 nmol of each primer, and 1 U Taq polymerase. For PCR amplification, we used both hot-start and touchdown PCR. PCR cycling conditions were 95 °C for 5 minutes, followed by five cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, followed by five cycles of 95 °C for 30 seconds, 57 °C for 30 seconds, and 72 °C for 30 seconds, followed by five cycles of 95 °C for 30 seconds, 54 °C for 30 seconds, and 72 °C for 30 seconds, followed by 20 cycles of 95 °C for 30 seconds and 51 °C for 30 seconds, and a final incubation at 72 °C for 4 minutes. The restriction enzyme TaqI (New England Biolabs, Beverly, MA) was used to examine the methylation status of the amplified region. In brief, 2040 µL of the amplified products were digested with the restriction enzyme Taq
I, which digests methylated DNA but not unmethylated DNA. The digested DNA was then ethanol precipitated, electrophoresed on 6% acrylamide gels, and visualized by ethidium bromide staining. We used a Geldoc 2000 imager (BioRad, Hercules, CA) to perform densitometric analysis of all the bands. Methylation level (%) was defined as the sum of the density of the shifted bands divided by the density of all bands in each lane. The identity of the amplified fragment was verified by restriction enzyme digestion. Each PCR assay included positive and negative controls (DNA from SW48 and RKO cells, respectively). Mixing experiments were performed to rule out PCR bias. All experiments were repeated twice to assess the reproducibility of results.
For MSP, we used the following oligonucleotide primers: 5'-GTAGGTTGTTTGTATGTTTGT-3' (sense primer) and 5'-AACCAATACAAACCAAACA-3' (antisense primer) for amplification of unmethylated DNA (PCR product size 121 bp) and 5'-GGTCGTTTGTACGTTCGC-3' (sense primer) and 5'-GACCGATACAAACCGAACG-3' (antisense primer) for amplification of methylated DNA (PCR product size 118 bp). PCR cycling conditions for both methylated and unmethylated primers were 95 °C for 5 minutes, followed by 35 cycles or 40 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, and finally, 72 °C for 4 minutes. MSP provides qualitative data, and a sample was called positive for methylation if a band was seen in DNA amplified by the methylated reaction primers. MSP PCR products were also visualized on acrylamide gels as described above.
Bisulfite Sequencing
In six normal-appearing mucosa samples (four adjacent to methylated tumor and two adjacent to unmethylated tumor), bisulfite sequencing (performed at the M. D. Anderson Core Sequencing Facility) of cloned PCR products was used to confirm methylation of CpG sites within the MGMT promoter. For this analysis, we cloned the 161-bp PCR products into the TA vector pCR2.1 (Invitrogen) and extracted plasmid DNA from the resulting clones with the use of a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA).
KRAS and TP53 Gene Mutations
In 91 tumors and 89 adjacent normal mucosa samples (DNA was no longer available for four tumors and six normal samples), we used mutant allelespecific PCR amplification of genomic DNA to analyze samples for activating mutations in codons 12 or 13 of the KRAS oncogene as previously described (16). This method allows the detection and verification of a single mutant allele in a background of 106107 copies of the wild-type allele. Data on TP53 gene mutations for 51 of the tumor samples, which were available from a prior study (12), had been obtained by using single-stranded conformational analysis followed by sequencing of shifted bands.
Immunohistochemistry
We performed immunohistochemical staining for MGMT protein on paraffin-embedded tissue sections as previously described (17). Briefly, 5-µm-thick sections were deparaffinized, rehydrated, incubated with 0.3% H2O2 to block endogenous peroxidase activity, and incubated with normal mouse serum to block nonspecific antibody binding. The sections were incubated at 4 °C overnight with a monoclonal antibody against human MGMT protein (MAB16200, 1 : 100 dilution; Chemicon, Temecula, CA). The sections were incubated with biotinylated goat antimouse IgG antibodies (AP124B, 1 : 500 dilution; Chemicon, Temecular, CA). The antigenantibody complexes were visualized using streptavidinhorseradish peroxidase conjugate (LSAB kit, DAKO, Los Angeles, CA) and diaminobenzidine as a chromogen. The slides were counterstained with hematoxylin. Normal-appearing epithelium and stromal cells in each section provided positive internal controls for binding of the primary antibody. MGMT expression in nuclei was scored as present or absent.
Genomic Sequencing
Eight colorectal cancer samples and adjacent normal-appearing tissues and three cancer cell lines (SW48, SW480, and HCT116) in which the MGMT promoter had been found to be methylated were further examined for the presence of genetic defects in the promoter region of MGMT. The promoter region was amplified by PCR from genomic DNA by using the forward primer (5'-GGGCCCACTAATTGATGGCT-3') and the reverse primer (5'-CTCACCAAGTCGCAAACGG-3'). The 951-bp PCR product was directly sequenced using the same primers in the M. D. Anderson Core Sequencing Facility.
Statistical Analysis
All statistical analyses were done using S-Plus software for Windows (version 6.0; Insightful Corporation, Seattle, WA). Methylation status of MGMT as determined by COBRA was analyzed initially as a categorical variable (negative: methylation level <3%, positive: methlyation level 3%). The 3% cutoff was selected because lower values could not be easily distinguished from background staining of the gels. Associations between MGMT methylation status and clinicopathologic variables were analyzed by Fisher's exact test. In parallel, for samples classified as methylation positive, we also analyzed methylation level as a continuous variable and computed means, medians, and ranges. Associations between methylation level analyzed continuously and clinicopathologic variables were analyzed by a Wilcoxon rank-sum test. We examined possible correlations between MGMT methylation levels in normal-appearing mucosa and patient age at diagnosis expressed on a log scale by calculating Pearson's and Spearman's correlation coefficients (r and
, respectively). All reported P values were two-sided, and P
.05 was considered statistically significant.
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RESULTS |
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We measured MGMT promoter methylation in colorectal cancer samples and samples of the corresponding adjacent nonneoplastic mucosa from 95 patients. The locations of CpG sites within the MGMT promoter region and the regions of the promoter that were amplified by the COBRA and MSP primers are shown in Fig. 1, A. By using the quantitative COBRA assay, we found that the mean level of MGMT promoter methylation in the colorectal cancer samples was higher than that in the corresponding adjacent mucosa (20.0% versus 4.3%; difference = 15.7%, 95% CI = 11.2% to 20.3%; P<.001). Among the 95 paired samples, the MGMT promoter was methylated (i.e., methylation level of at least 3%) in 46% of colorectal tumor samples and in 26% of the corresponding samples of adjacent normal-appearing mucosa (see examples in Fig. 1, B).
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Next, we examined the association between MGMT promoter methylation and clinicopathologic features of patients with primary colorectal cancer. We initially analyzed the data using methylation status as a categorical variable; samples with detectable methylation by COBRA (i.e., those with a methylation level 3%) were considered positive for MGMT promoter methylation (Table 1). We found that the MGMT promoter methylation status of cancer samples was not associated with patient age at diagnosis, with the location of the tumor in the colon, or with tumornodemetastasis (TNM) stage. However, statistically significantly more tumors from women than from men had MGMT promoter methylation (16/24 [67%] versus 26/67 [39%], P = .03). We had previously determined the CpG island methylator phenotype (CIMP) status of 54 of the 95 tumors in this study (13). CIMP is a molecular property of some colorectal cancers whereby many genes are hypermethylated simultaneously. Of the 54 tumors whose CIMP status had been determined, 24 were CIMP negative; of these, four (17%) were MGMT promoter methylation positive. Of the 30 CIMP-positive tumors, 20 (67%) were MGMT promoter methylation positive (P<.001). This difference suggests that the MGMT promoter can be affected by CIMP in colorectal cancer. We also analyzed MGMT methylation level as a continuous quantitative variable, limiting the analysis to the 44 tumors that were MGMT promoter methylation positive by the categorical classification. In this group of 44 cancers, there was no association between the degree of methylation and patient age, patient sex, tumor stage, or the location of the tumor in the colon (data not shown).
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Patients who had MGMT promoter methylationpositive cancers also had detectable methylation of the MGMT promoter in the apparently normal-appearing mucosa adjacent to the cancer (Fig. 3). When analyzed as a categorical variable, methylation in normal colon was positive in 22 (50%) of 44 normal-appearing mucosa samples taken from regions adjacent to MGMT promoter methylationpositive cancers compared with only three (6%) of 51 normal-appearing mucosa samples taken from regions adjacent to MGMT promoter methylationnegative cancers (P<.001 by Fisher's exact test). We also examined the methylation status of the MGMT promoter in colonic biopsy specimens obtained at screening colonoscopy from 33 patients who had no family history of colorectal cancer and no colonic lesions (mean age of these patients was 54 years) and found that four specimens (12%) had detectable MGMT promoter methylation. When we used the more sensitive MSP assay at 35 PCR cycles, MGMT promoter methylation was detected in 66%, 19%, and 16% of the samples, respectively. When we used the MSP assay at 40 PCR cycles to increase sensitivity, MGMT promoter methylation was detected in 94%, 34%, and 26% of the samples, respectively. Similar results were obtained when methylation level was analyzed as a continuous variable. That is, MGMT methylation levels as measured by COBRA in the normal-appearing mucosa adjacent to an MGMT promoter methylationpositive cancer were statistically significantly higher than levels in the normal-appearing mucosa adjacent to an MGMT promoter methylationnegative cancer (8.8% versus 0.4%; difference = 8.4%, 95% CI = 5.1% to 12.1%; P<.001, Fisher's exact test).
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Finally, to rule out genetic mutations as a cause of the observed methylation, we sequenced the entire promoter region of the MGMT gene for eight patients with MGMT promoter methylation. We found no mutations or deletions in either their tumor DNA or in the paired normal mucosa DNA, suggesting that the observed methylation was not caused by genetic abnormalities.
MGMT Promoter Methylation and Molecular Characteristics of the Cancers
We examined the association between MGMT promoter methylation level and the presence of mutations in KRAS and TP53, two genes that are frequently mutated in colorectal cancer. We examined these associations in parallel analyses that used MGMT promoter methylation data as a continuous variable and as a categorical variable, with identical results. We found that MGMT promoter methylation positivity in the tumor samples was associated with the presence of KRAS gene mutations, similar to what has previously been observed in a different set of tumors (10). This association between MGMT methylation positivity and KRAS mutations was largely a result of a difference in G-to-A transitions, which were present in 10 (24%) of 42 cancers with MGMT promoter methylation versus three (6%) of 49 cancers without MGMT promoter methylation (P = .03) (Table 1). The frequency of KRAS gene mutations was 12% (3/25; one specimen had a G-to-A mutation and two specimens had G-to-T mutations) in mucosal specimens with detectable MGMT promoter methylation and only 3% (2/64; both specimens had G-to-T mutations) in mucosal specimens that lacked MGMT promoter methylation; however, the difference in proportions was not statistically significant (difference = 9%, 95% CI = 1% to 19%; P = .13; Table 2). It is interesting that the tumors and their corresponding mucosas did not have identical KRAS gene mutation patterns, suggesting multiple independent mutational events.
We used data obtained in a previous study (6) of these tumor and corresponding mucosa samples to examine the association between MGMT promoter methylation status and the presence TP53 gene mutations. In contrast with our findings for the KRAS gene, we found an inverse association between the presence of TP53 gene mutations and MGMT promoter methylation in the tumor samples. The frequency of TP53 gene mutations was 30% (7/23; two cancers had a G-to-A mutation) among the MGMT promoter methylationpositive cancers, and 54% (15/28; nine cancers had a G-to-A mutation) among the MGMT promoter methylationnegative cancers; however, the difference was not statistically significant (P = .13). No TP53 gene mutations were detected in the corresponding normal mucosa samples.
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DISCUSSION |
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One limitation of our study is that we were not able to analyze single colonic crypts for MGMT expression and promoter methylation simultaneously. Much of the tissue we currently have available for these studies is paraffin embedded, and we found that the quality of the immunohistochemistry varied substantially. Thus, although examining cancers in which large tracts of cells may express or lack expression of MGMT protein is relatively straightforward, it is difficult to use immunohistochemistry to examine MGMT protein expression in normal tissues, where an absence of expression in single cells or crypts could be related to promoter methylation or to technical issues. Given the large body of literature describing the association between promoter hypermethylation and gene silencing, it is reasonable to infer that the MGMT promoter methylation we observed (as confirmed by bisulfite sequencing) was associated with MGMT gene silencing in some normal mucosas. Another potential limitation of our study is the possibility that the normal tissues were contaminated with malignant cells. We think this possibility is unlikely given that 1) normal tissue was confirmed histologically, 2) promoter methylation was present in tissue located 10 cm away from the tumor, and 3) promoter methylation was detectable in some patients who did not have tumors.
A field defect is an area of abnormal tissue that precedes and predisposes to the development of cancer (1). Identification of such abnormal fields is important because they provide insight into the earliest stages of cancer and may provide markers for risk assessment. Accumulating evidence suggests that alterations in DNA methylation represent epigenetic phenomena that appear to be early events in tumorigenesis (18,19). For example, it has been noted (4), initially in the colon (3), that normal-appearing tissues, in general, have aberrant methylation of promoter-associated CpG islands, albeit at low levels. Age-related increases in such methylation have been proposed to mark the field defect that accompanies sporadic colorectal tumorigenesis (20,21), but clear markers to identify patients at risk of this disease have been lacking. In this study, we found that MGMT promoter methylation occurred frequently in the normal-appearing colonic mucosa of colorectal cancer patients whose tumors had MGMT promoter methylation and was much less frequent in the normal-appearing colonic mucosa of colorectal cancer patients whose tumors did not have MGMT promoter methylation and of healthy control subjects. Interestingly, we also observed an association between age and promoter methylation levels; among patients who had MGMT promoter methylation in their normal-appearing colonic mucosa, increasing age was associated with increased methylation density.
Because a loss of MGMT protein function is a plausible predisposing factor for cancer through the increased occurrence of mutations (such as G-A mutations in the KRAS gene, as shown herein), our data indicate that MGMT promoter methylation may qualify as a marker of the field defect in colorectal cancer. MGMT promoter methylation has also been detected in the sputum of patients who are at risk for lung cancer (22), and it may represent a marker of the field defect in lung cancer as well. MGMT promoter methylation has been associated with G-to-A mutations in TP53 in lung cancer (23), a finding that is also consistent with the field defect hypothesis. However, we found no association between MGMT promoter methylation and G-to-A mutations in TP53 in colon tumorsa reflection, perhaps, of different mechanisms of mutagenesis in these two tissues. It is interesting that MGMT promoter methylation is a frequent feature of a newly recognized evolutionary pathway for colon carcinogenesis, the hyperplastic polyp/serrated adenoma route (11,24). Patients who have multiple hyperplastic polyps have a high degree of concordance in the methylation patterns of the different tumors (25), supporting the existence of a field defect preceding such lesions. Our data indicate that MGMT promoter methylation in normal colon is a possible risk factor for developing tumors along the serrated adenoma pathway.
In this study, we found that MGMT promoter methylation can be clearly detected in the normal mucosa of healthy individuals. Variations in MGMT enzyme activity in the colon have been reported previously (26), and it is possible that epigenetic inactivation of the MGMT gene, as described here, contributes to this variability. Given the high lifetime risk of colorectal tumor development in the U.S. population, it is reasonable to propose testing to determine whether healthy persons with MGMT promoter methylation in normal colorectal mucosa are at higher risk of developing a colon tumor than those without such methylation.
The causes of MGMT promoter methylation remain unclear. No mutations or deletions were apparent when we sequenced the MGMT promoter region in methylated samples, although a larger region would likely need to be sequenced to rule out the possibility of mutations or deletions. As noted above, we observed an association between age and MGMT promoter methylation that, unlike the relationship between age and the methylation status of other genes (3,27), did not appear to be linear, implying that other factors accelerate MGMT promoter methylation. Chronic inflammation has previously been shown to accelerate DNA methylation in normal tissues (28), and it would be interesting to examine whether inflammation plays a role in MGMT promoter methylation in colorectal carcinogenesis.
In conclusion, our data indicate that some sporadic colorectal cancers may arise from a field defect that is molecularly defined by epigenetic inactivation of MGMT and an increased rate of mutations in multiple genes, including KRAS. The discovery of a marker of a field defect, such as MGMT promoter methylation, in normal-appearing mucosa could be of great use, both for early detection of and risk assessment in colon cancer. Prospective clinical trials using this potential marker of risk are indicated.
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NOTES |
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Manuscript received August 27, 2004; revised July 1, 2005; accepted July 20, 2005.
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