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From the Departments of Pathology,*
Leukemia,
and
Biostatistics,
University of Texas M. D.
Anderson Cancer Center, Houston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of colorectal cancers has truncating mutations or deletions of the adenomatous polyposis coli (APC) gene on chromosome 5q6 or mutations of the ß-catenin gene. Point mutations of the K-ras proto-oncogene,7 loss of the deleted in colorectal cancer (DCC) gene and nearby SMAD2 and SMAD4 genes on chromosome 18q,8 and mutations and/or deletions of the p53 gene on chromosome 17p are also common.9
In a second pathway to colorectal neoplasia, microsatellite instability (MSI; also termed DNA replication errors and ubiquitous somatic mutations) is caused by alteration of a nucleotide mismatch repair gene, including hMSH2, hMLH1, PMS1, PMS2, or GTBP.1-5 MSI is characterized by additions and deletions of nucleotides in numerous repeated nucleotide sequences (microsatellites). Germline mutation of a mismatch repair gene causes hereditary nonpolyposis colorectal cancer. Alterations of mononucleotide tracts present in genes such as transforming growth factor-ß type II receptor and BAX genes are commonly found in MSI-positive carcinomas.10,11
Another molecular defect commonly present in colorectal cancer is CpG island methylation. CpG islands are 0.5- to 2-kb regions rich in cytosine-guanine dinucleotides and are present in the 5' region of approximately half of all human genes.12 Methylation of cytosines within CpG islands is associated with loss of gene expression by repression of transcription and is observed in physiological conditions such as X chromosome inactivation13 and aging,14 but also in neoplasia.15 Examples of this process in colorectal cancers include inactivation of the p16 cell-cycle regulator,16 the estrogen receptor growth suppressor,14 the THBS1 angiogenesis inhibitor,17 the TIMP3 metastasis suppressor,18 the O6-methylguanine DNA methyltransferase DNA repair gene,19 and the hMLH1 nucleotide mismatch repair gene.20
Recently, a distinct pathway of colorectal carcinogenesis was described, termed CpG island methylator phenotype (CIMP).21 CIMP-positive colorectal cancers are characterized by a high degree of concordant CpG island methylation of genes in colorectal cancer but not in normal mucosa. CIMP phenotype is also observed in large colorectal adenomas removed with colorectal cancer,22 but CIMP status in adenomas unassociated with cancer has not been reported.
In the present study, we examined methylation status in a prospective study of sporadic colorectal adenomas removed at colonoscopy from patients without cancer. The methylation status was compared with patient and adenoma characteristics including methylation of adenomas, and with other genetic alterations present in adenomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study includes 108 colorectal adenomas that were collected
from 50 patients prospectively enrolled in the endoscopy unit of The
Johns Hopkins Hospital. K-ras mutation, p53 overexpression, 18q loss,
and microsatellite instability status of these adenomas have been
described previously.23
The demographics of the patient
population and adenoma characteristics analyzed in the present study
are summarized in Table 1
. All patients
had given informed consent for the collection of tissue according to
institutional guidelines.
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Genomic DNA was extracted after microdissection.24 Each specimen was treated with 50 µl of buffer containing 0.5% Tween 20 (Boehringer Mannheim, Mannheim, Germany), 20 µg proteinase K (Boehringer Mannheim), 50 mmol/L Trizma base at pH 8.9, and 2 mmol/L ethylenediaminetetraacetic acid as previously described.24 The samples were incubated at 56°C overnight. Proteinase K was inactivated by incubating the samples at 100°C for 10 minutes. The extracted DNA was stored at -80°C.
Bisulfite Treatment of DNA and Methylation-Specific Polymerase Chain Reaction (MSP) Followed by Restriction Enzyme Digest
The methylation status of p16, MINT2, and MINT31 was
determined by bisulfite treatment of DNA followed by
methylation-specific polymerase chain reaction (MSP-PCR), as described,
with modification.25
The selection of these loci was based
on our previous study that showed these loci had high sensitivity and
specificity for the detection of hypermethylation in cancer and
offered excellent discrimination for CIMP status.21
MINT2
corresponds to a CpG island that is in the 5' region of a cDNA with an
open reading frame that has no protein homology (J. P. Issa,
unpublished data). MINT31 is 2 kb upstream of the CACNA1G, a
T-type calcium channel gene.26
In brief, 2 µg of
microdissected genomic DNA was denatured with 2 mol/L NaOH at 37°C
for 10 minutes, followed by incubation with 3 mol/L sodium bisulfite
(pH 5.0) at 50°C for 16 hours in the dark. After treatment, DNA was
purified using the DNA cleanup kit (Promega, Madison, WI) as
recommended by the manufacturer, incubated with 3 mol/L NaOH at room
temperature for 5 minutes, precipitated with 10 mol/L ammonium acetate
and 100% ethanol, washed with 70% ethanol, and finally resuspended in
20 µl of distilled water. Methylation status of p16,
MINT2, and MINT31 was determined using 5 µl of bisulfite-treated DNA
for bisulfite-PCR followed by restriction enzyme digest of PCR product
as described.27
In brief, a 40 µl aliquot of the
amplified products was digested with restriction enzymes that
distinguish methylated from unmethylated sequences, electrophoresed on
5% acrylamide gels, and visualized by ethidium bromide staining.
Primer sequences, conditions for PCR, and restriction enzymes used are
tabulated in Table 2
. DNA from RKO colon
cancer cell line (American Type Culture Collection, Manassas, VA) was
used as a control for methylation. Methylation status was confirmed by
MSP for p16 in 7 adenomas and for MINT31 in 10 adenomas as
previously described.25
In brief, 4 µl of DNA was used
as template for PCR reactions using primers specific for methylated and
unmethylated alleles. For quantitation of methylated alleles, gel
images were digitized using a BioRad imager and quantitated using the
accompanying software (BioRad, Hercules, CA). Both bisulfite-PCR
and MSP provide semiquantitative results. The loci
(p16, MINT2, and MINT31) used in this study are
unmethylated (<1% methylation) in normal tissues. Therefore, any
locus showing 
1% methylation was considered positive.
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CIMP status was determined for adenomas with two or more evaluated loci. Adenomas were classified as CIMP-negative if none of the evaluated loci were methylated; CIMP-low if one locus was methylated; and CIMP-high if two or more loci were methylated. Adenomas with one unmethylated evaluated locus were not classified. These criteria were based on our previous study in which CIMP-positive adenomas were methylated at the majority of MINT loci and p16, but CIMP-negative adenomas were rarely methylated at any MINT loci and never methylated at p16.21 The sensitivity of any MINT locus to predict CIMP is 75% and specificity is 95%. The sensitivity of p16 to predict CIMP phenotype is 65% and specificity is 100%.
K-ras Mutations, p53 Overexpression, MSI, and Loss of Heterozygosity of Chromosome 18q in Adenomas
These alterations were reported previously.23
Statistical Analysis
The primary statistical endpoint of this study was the determination of factors related to methylation status at the p16, MINT2, and MINT31 loci. All adenomas in the study with a successful determination of their methylation status for at least one locus were included in the analysis. Each adenoma was represented by a methylation index (number of loci methylated/number of loci evaluated). Patients with more than one adenoma were represented multiple times in this data set. To correctly model the within- and between-adenoma correlation as well as simultaneously partition out the effects of the various factors considered, marginal logistic regression models for correlated binary data were used to assess associations between methylation status and the various adenoma and patient characteristics. Estimates were obtained using the generalized estimating equation approach of Liang and Zeger.28 An appropriate correlation structure was chosen to account for possible correlations between adenomas within patients, and also within adenomas between observations from different loci. Patient characteristics included age and sex. Adenoma characteristics included site, size, and histology, as well as presence or absence of K-ras mutations, p53 overexpression, MSI, and loss of heterozygosity of chromosome 18q in polyps. A factor was also included in the model to account for locus-specific methylation rates. Because there was no evidence in the data that any patient or adenoma characteristics had effects that differed according to locus, no locus-by-factor interactions were included in the final generalized estimating equation model. Relationships between adenomas within patients and within adenomas between loci were represented as odds ratios, in which an odds ratio of greater than one suggests positive correlation in methylation status within patients and within adenomas, respectively. Comparisons of adenoma size were done using Proc mixed in SAS (SAS Institute, Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows examples of
methylation at p16, MINT2, and MINT31. Figure 2
summarizes the frequency in adenomas of
methylation at p16, MINT2, and MINT31, and at any locus.
Patient and adenoma characteristics are summarized with methylation
status of adenomas in Tables 3 and 4
. Patient and adenoma characteristics
are summarized with methylation status of adenomas in Table 5
.
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Methylation of MINT2 was present in 43% (34 of 79) of adenomas,
of MINT31 in 34% (26 of 76) of adenomas, and p16 in 27 (19
of 71) of adenomas (P = 0.04; Figure 2
). All
three loci were methylated in 8 adenomas, two loci in 11 adenomas, one
locus in 33 adenomas, and no locus in 56 adenomas. Thus, 48% (52 of
108) of adenomas had methylation at one or more loci, and 52 (56 of
108) of adenomas had no methylation at any of the evaluated locus. The
methylation status for different loci of the same adenoma was
positively correlated (odds ratio, 3.60; P = 0.0078),
indicating that some adenomas had CIMP-high. In adenomas with two or
more evaluated loci, 25% (19 of 76) of adenomas were CIMP-high, 32%
(24 of 76) were CIMP-low, and 43% (33 of 76) were CIMP-negative.
Because 78% (84 of 108) of adenomas were from 26 patients with multiple adenomas, we were able to address within-patient correlation of methylation status. We found that different adenomas within the same patient were not correlated (odds ratio, 0.93; P = 0.77). This finding provides evidence against a field defect responsible for the development of methylation.
The age and sex of the patient or site of adenoma and methylation
status of adenomas were not associated (Tables 3 and 4)
. However,
methylation was associated with villous histology and large size of
adenomas. Methylation was present in 73% (17 of 23) of tubulovillous
or villous adenomas versus 41% (35 of 85) of tubular
adenomas (odds ratio, 3.46; P = 0.02). Methylation was
present in 80% (12 of 15) of adenomas >1 cm in size, 40% (11 of 28)
of adenomas between 0.5 cm to 1 cm in size, and 45% (29 of 65)
adenomas <0.5 cm in size. The size of adenomas was not independently
correlated with methylation status of adenoma as size was directly
correlated with villous component (r = -0.49).
CIMP status of adenomas was also associated with the histological type
of adenomas. The prevalence of CIMP-high, CIMP-low, and CIMP-negative
was 37% (7 of 19), 47% (9 of 19), and 16% (3 of 19) in tubulovillous
adenomas compared to 21% (12 of 57), 26% (15 of 57), and 53% (30 of
57) in tubular adenomas (odds ratio, 3.57; 95% confidence interval,
1.14, 11.12; chi-square, 4.51; P = 0.03) (Figure 3
and Table 5
).
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. MSI-high was present in 2% (1 of 46) of adenomas with
methylation, and 13% (6 of 47) of adenomas with no methylation (odds
ratio, 8.48; P = 0.05). MSI-high adenomas without
methylation were from three hereditary nonpolyposis colorectal cancer
patients who had one adenoma each and from three patients without
hereditary nonpolyposis colorectal cancer patient who had one, one, and
two adenomas, respectively. K-ras mutations were present in 37%
(18 of 52) of adenomas with methylation and 47% (25 of 56) of adenomas
with no methylation (odds ratio, 1.69; P = 0.28).
Adenomas with K-ras mutation and methylation were larger in size
compared to adenomas with K-ras mutation but no methylation (1.1
± 0.16 versus 0.58 ± 0.13, F = 6.92,
P = 0.02). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methylation of one or more CpG island(s) was present in 52% of adenomas, and methylation of two or more CpG islands (CIMP-high) was present in 25% of adenomas. In contrast, in our previous study the prevalence of CIMP-high in adenomas was 49%.22 The adenomas in our previous study were from patients with synchronous cancers and were larger. In addition, methylation was studied using an extended set of markers. In both studies, however, CIMP-high was more common in larger adenomas. In the present study, we also show that methylation is more common in adenomas with tubulovillous or villous histology. Thus, methylation is more common in larger adenomas and adenomas with tubulovillous or villous histology, two adenoma characteristics associated with more frequent predisposition to invasive carcinoma.
p16 methylation was present in 27% of adenomas in the present study. We have previously reported that K-ras mutation was the most frequent genetic alteration in colorectal adenomas occurring in 35% of adenomas, but other genetic alterations are infrequent.23 Thus, p16 methylation is one of the most common early genetic alterations in the adenoma-carcinoma sequence.
CpG island methylation of adenomas in our present study was associated with lack of K-ras mutation or MSI. Colorectal cancers with methylator phenotype have a distinct genetic profile with frequent K-ras mutation, microsatellite instability because of methylation of hMLH1, but less frequent p53 mutation.21,22,30,31 K-ras mutation was also frequent in large (>1.5 cm) colorectal adenomas with CpG island methylation in our previous study.22 In our present study, the colorectal adenomas with CpG island methylation and K-ras mutation are larger as compared to colorectal adenomas with K-ras mutation but no CpG island methylation. O6-Methylguanine DNA methyltransferase, a DNA repair protein that removes alkyl groups and adducts at the O6 position of guanine, is frequently hypermethylated in colorectal carcinomas and adenomas.19,32 Hypermethylation of O6-methylguanine DNA methyltransferase is associated with G to A transitions in the K-ras gene. Hypermethylation of O6-methylguanine DNA methyltransferase is also present in smaller adenomas but G to A transitions in K-ras gene are not present in smaller adenomas.32 The prevalence of smaller adenomas (94%) in our present study may explain these paradoxical results.
MSI-high was present in six adenomas with no methylation and only one adenoma with methylation from three patients with and three without hereditary nonpolyposis colorectal cancer in our study. MSI is more frequent in sporadic colorectal carcinomas than colorectal adenomas,17,23,31,33-35 and MSI in sporadic colorectal carcinomas is principally because of methylation of hMLH1.17,31,33 Although, based on a limited number of samples, our data suggest that most of MSI in colorectal adenomas is because of mutation or allelic loss of hMLH1 or hMSH2 in contrast to sporadic colorectal carcinomas, and methylation of hMLH1 is a late event in the adenoma-carcinoma sequence.
There was no correlation between the methylation status of adenomas within a given patient. These data suggest that in the majority of patients with colorectal adenomas, methylation is an epigenetic alteration in an adenoma and is not because of a field defect. We have previously shown lack of intrapatient correlation for K-ras in colorectal adenomas, but weak intrapatient association for MSI and p53 overexpression.23
In conclusion, our study demonstrates the early role of methylation in colorectal tumorigenesis. Methylation is not correlated in multiple adenomas from an individual patient. Factors related to villous histology and absence of K-ras mutations or MSI are involved in the methylation of colorectal adenomas. The mechanism of methylation of multiple CpG islands is not known. It is postulated that the defect could be either aberrant de novo methylation because of mutation in a DNA-methyltransferase or loss of protection against de novo methylation through the loss of a trans-activating factor.36-38 This latter defect is exemplified by the adenine phosphoribosyltransferase gene in mice, in which an unidentified protein that binds to the Sp1 sites seems to prevent methylation of the CpG island. It has been proposed that other such proteins may prevent methylation of multiple CpG islands, and expression and/or action of such proteins may be regulated in a tissue-specific manner. Loss of such a protein in cancer may result in hypermethylation of multiple CpG islands.
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Footnotes |
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Accepted for publication May 25, 2001.
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