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Carcinogenesis Advance Access originally published online on December 20, 2006
Carcinogenesis 2007 28(6):1364-1370; doi:10.1093/carcin/bgl246
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Genetic and epigenetic profiling in early colorectal tumors and prediction of invasive potential in pT1 (early invasive) colorectal cancers

Katsuhiko Nosho*, Hiroyuki Yamamoto, Taiga Takahashi, Masashi Mikami, Hiroaki Taniguchi, Nobuki Miyamoto, Yasushi Adachi, Yoshiaki Arimura, Fumio Itoh1, Kohzoh Imai2 and Yasuhisa Shinomura

First Department of Internal Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan
1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, St Marianna University, Kawasaki 216-8511, Japan
2 Sapporo Medical University, Sapporo 060-8543, Japan

* To whom correspondence should be addressed. Tel: +81 11 611 2111 ext. 3211; Fax: +81 11 611 2282; Email: nosho{at}sapmed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphologically, early colorectal tumors are divided into two groups, protruded-type tumors and flat-type tumors. Although some studies have shown genetic alterations in protruded-type tumors, little is known about genetic and epigenetic alterations in flat-type tumors, as well as pT1 (early invasive) colorectal cancers (CRCs). In the current study, we compared the frequencies of genetic and epigenetic alterations of the RAS–RAF and Wnt signaling pathways in flat-type and protruded-type tumors. In addition, we investigated the relationship between those alterations and invasive potential of pT1 CRCs. Methylations of RASSF2, O-6-methylguanine-DNA methyltransferase (MGMT), Wnt inhibitory factor-1 (WIF-1), EPHB2, CDKN2A and MLH1 were detected in 44.3, 30.3, 81.4, 7.5, 43.6 and 13.4% of the 307 early colorectal tumors, respectively. Mutations of KRAS, BRAF, catalytic subunit alpha of phosphatidylinositol 3'-kinase (PIK3CA) and ß-catenin were detected in 25.4, 4.6, 1.6 and 9.4% of those tumors, respectively. Methylations of MGMT, WIF-1 and CDKN2A were detected in significantly higher percentages of protruded-type tumors than in flat-type tumors. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors than in protruded-type tumors. RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation. Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs. In conclusion, since genetic alterations of these pathways have frequently occurred in flat-type tumors, flat-type tumors seem to have a distinct genetic profile different from that of protruded-type tumors. RASSF2 methylation with oncogenic activation is a promising biomarker for predicting invasive potential of pT1 CRCs.

Abbreviations: CRC, colorectal cancer; MGMT, O-6-methylguanine-DNA methyltransferase; MSI-H, high-frequency microsatellite instability; MSI, microsatellite instability; MSP, methylation-specific polymerase chain reaction; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3'-kinase; PIK3CA, catalytic subunit alpha of phosphatidylinositol 3'-kinase; WIF-1, Wnt inhibitory factor-1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Morphologically, early colorectal tumors can be divided into two groups, protruded-type tumors and flat-type tumors (1–3). Previous studies showed that flat-type tumors tended to reach deeper layers earlier and showed higher rates of invasive potential than did protruded-type tumors (2,3). Although some studies have shown genetic alterations in protruded-type colorectal tumors (4–6), little is known about genetic and epigenetic alterations in flat-type colorectal tumors.

The RAS–RAF signaling pathway mediates cellular responses to growth signals not only in advanced colorectal cancers (CRCs) but also in early colorectal tumors. Previous studies have shown that KRAS mutations are rare in flat-type colorectal tumors (4–7). BRAF mutations have also been reported in hyperplastic polyps and serrated adenomas, and they have been shown to be associated with the progression of a hyperplastic polyp to serrated adenoma (8,9). Recently, mutations in catalytic subunit alpha of phosphatidylinositol 3'-kinase (PIK3CA) have been identified in CRCs. The PIK3CA gene encodes the catalytic subunit p110alpha of phosphatidylinositol 3'-kinase (PI3K) belonging to class IA of PI3Ks (10). Since oncogenic RAS activates PI3Ks, activation of the PI3Ks signaling pathway via the RAS–RAF signaling or PIK3CA mutations was considered to be one of the most common mechanisms involved in colorectal carcinogenesis (10,11).

Epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT) has been shown to be associated with the appearance of G-to-A transitions in KRAS mutations during colorectal tumorigenesis (12,13). MGMT is known to encode a DNA repair protein that removes potentially carcinogenic and cytotoxic alkyl adducts from the O-6 position of guanine. Alterations in the MGMT gene impair the ability of the MGMT protein to remove alkyl groups from the O-6 position of guanine. Therefore, the alterations are thought to increase the mutational rate and the risk of CRC (12–15).

Recently, Akino et al. (16) have shown that RASSF2 methylation is associated with its transcriptional inactivation in colorectal tumors. Primary CRCs with KRAS or BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced KRAS-induced oncogenic transformation. Since the activities of RASSF2 include induction of morphologic changes and apoptosis, RASSF2 is thought to act as a novel tumor suppressor gene that regulates the RAS–RAF signaling pathway (16).

On the other hand, it has been reported that ß-catenin acts as a downstream transcriptional activator in the Wnt signaling pathway. Adenomatous polyposis coli dysfunction or abnormalities of the ß-catenin gene result in cytoplasmic accumulation of unphosphorylated ß-catenin (17–19). This stabilized ß-catenin protein translocates into the nucleus, where it modulates gene transcription by interacting with T cell factor-4/lymphoid enhancer factor-1, resulting in transcriptional activation of target genes (20).

Aberrant activation and up-regulation of the Wnt signaling pathway are key features of many cancers (21–23). Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit the Wnt signaling pathway. Previous studies have shown that WIF-1 silencing due to DNA hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in colorectal tumors (21,23). On the other hand, the receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of T cell factor-4/ß-catenin. Although pre-malignant lesions of the colon expressed high levels of EPHB2, the expression of this kinase was reduced or lost in advanced CRCs by mutation or DNA hypermethylation (24).

Thus, it seems important to clarify genetic and epigenetic alterations of the RAS–RAF and Wnt signaling pathways in early colorectal tumorigenesis. In the current study, we compared the frequencies of genetic and epigenetic alterations of those genes in flat-type (n = 131) and protruded-type tumors (n = 73) by using direct DNA sequencing and real-time polymerase chain reaction (PCR) that measures DNA methylation (MethyLight), respectively. In addition, we investigated the relationship between those alterations and invasive potential of pT1 (early invasive) CRCs (n = 103).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and tissue samples
Formalin-fixed, paraffin-embedded tissues of 307 colorectal tumors were obtained from patients who had undergone polypectomy or surgical treatment. These tumor samples consisted of 27 hyperplastic polyps, 5 serrated adenomas, 71 adenomas with low-grade dysplasia, 31 adenomas with high-grade dysplasia, 70 intra-mucosal carcinomas and carcinoma in situ and 103 pT1 (early invasive carcinomas) CRCs (pT1 in the TNM classification of the Union International Against Cancer). Tumor samples were carefully microdissected by expert pathologists. In case of pT1 CRCs, tumor samples were taken from the microscopically visible deepest invading part of the tumor. All samples of pT1 CRCs were obtained by surgical treatment to analyze the presence or absence of lymph node metastasis. No patients fulfilled the criteria for hereditary CRC.

Locations of the tumors were divided into proximal colon (cecum, ascending and transverse colon) and distal colon (descending and sigmoid colon and rectum). Macroscopic types were divided into protruded type (height of tumor ≥2.5 mm) and flat type (height of tumor <2.5 mm) according to the Paris classification (1). It was difficult to divide pT1 CRCs into protruded-type or flat-type tumors because colorectal tumors become thick when they have invaded the submucosal layer. Therefore, macroscopic type was classified only in early colorectal tumors other than pT1 CRCs. Informed consent was obtained from each subject and the institutional review committee approved this study.

Detection of KRAS, BRAF, PIK3CA and ß-catenin exon 3 mutations
KRAS mutations at codon 12 and codon 13, BRAF mutations at codon 600 and PIK3CA mutations in exon 9 and exon 20 were analyzed by direct DNA sequencing as described previously (8,11,12). Exon 3 of ß-catenin was amplified by PCR using the following primer pair: forward, 5'-GAACCAGACAGAAAAGCGGCTG-3' and reverse, 5'-ACTCATACAGGACTTGGGAGG-3'. Products were purified and then sequenced in both directions using BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The sequence reactions were run and analyzed on an ABI 3100 Genetic Analyzer (Applied Biosystems).

Quantitative real-time PCR to measure DNA methylation (MethyLight)
Sodium bisulfite treatment of genomic DNA and MethyLight were performed as described previously (25–28). We used an ABI 7000 (Applied Biosystems) for quantitative real-time PCR (Figure 1A). Using seven sets of primers and probes, we amplified promoters of six genes of interest (RASSF2, MGMT, WIF-1, EPHB2, CDKN2A and MLH1) and ß-actin (ACTB) to normalize for the amount of input bisulfite-converted DNA. These primers and probes were designed specifically for bisulfite-converted DNA. The sequences of primers and probes and PCR conditions are available upon request. The percentage of methylated reference at a specific locus was calculated by dividing the GENE:ACTB ratio of a sample by the GENE:ACTB ratio of universal methylated DNA (fully methylated) (Chemicon International, Temecula, CA) and multiplying by 100 (25,27,28).


Figure 1
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  		Fig. 1. Quantitative real-time PCR to measure DNA methylation (MethyLight) (A) and immunohistochemical staining of MGMT protein in colorectal tumor tissues (B and C). (A) The analysis of RASSF2 methylations. Bisulfite-converted universal methylated DNA was used for percentage of methylated reference calculation. The fluorescence is plotted versus the PCR cycle number for both reactions, and each sample is indicated. (B) A tumor with MGMT methylation showed no staining in tumor cells but clear staining in the nuclei of non-tumor cells (x200). (C) A tumor without MGMT methylation showed staining for the MGMT protein in tumor cell nuclei (x200).

 
Immunohistochemistry of MGMT
Immunohistochemistry with an anti-human MGMT mouse monoclonal antibody (MAB16200, 1:100 dilution; Chemicon International) was done as described previously (29). The sections were examined microscopically by two well-trained pathologists who were blinded to the clinicopathological characteristics. 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 (14).

Microsatellite instability analysis
Early colorectal tumors were analyzed for microsatellite instability (MSI) by using five microsatellite markers (BAT-25, BAT-26, D2S123, D5S346 and D17S250) as described previously (30).

Statistical analysis
Alteration of each target gene was assessed for associations with clinicopathological characteristics using the following statistical tests: Mann–Whitney U test for age and size and the chi-square two-tailed test or Fisher's exact test for the remaining parameters. Logistic regression analysis for invasive potential was performed by using a backward stepwise method (likelihood ratio) (software, SPSS for Windows, version 11; Chicago, IL). Clinicopathological characteristics (age, size, gender, location and invasive potential) and the genetic and epigenetic alterations for the unmatched data were examined using unconditional logistic regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Methylations of RASSF2, MGMT, WIF-1, EPHB2, CDKN2A and MLH1
Methylations of RASSF2, MGMT, WIF-1, EPHB2, CDKN2A and MLH1 were detected in 136 (44.3%), 93 (30.3%), 250 (81.4%), 23 (7.5%), 134 (43.6%) and 41 (13.4%) of the 307 early colorectal tumors, respectively. Methylations of RASSF2 and WIF-1 were correlated significantly with tumors of larger size (P < 0.0001 and P = 0.0290, respectively). WIF-1 methylation was correlated significantly with older age (P = 0.0070). Methylations of MGMT, WIF-1 and EPHB2 were detected in significantly higher percentages of distal colon tumors (38.9, 87.7 and 10.5%, respectively) than in proximal colon tumors (20.7, 74.5 and 4.1%; P = 0.0005, 0.0030 and 0.0347, respectively) (Table I).


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  		Table I. Epigenetic alterations and clinicopathological characteristics in early colorectal tumors

 
CDKN2A methylation was detected in a significantly higher percentage of pT1 CRCs than in non-pT1 CRCs (P = 0.0152). MLH1 methylation was correlated significantly with female gender (P = 0.0005) and pT1 CRCs (P = 0.0059) (data not shown).

KRAS, BRAF, PIK3CA and ß-catenin exon 3 mutations
Mutations of KRAS, BRAF, PIK3CA and ß-catenin were detected in 78 (25.4%), 14 (4.6%), 5 (1.6%) and 29 (9.4%) of the 307 early colorectal tumors, respectively (Table II). Among the 78 tumors positive for KRAS mutations, codon 12 mutation, codon 13 mutation and both mutations were detected in 51, 21 and 6 tumors, respectively. KRAS mutation was correlated significantly with older age (P = 0.0252), tumors of larger size (P < 0.0001), female gender (P = 0.0055) and pT1 CRCs (P = 0.0297). Regarding BRAF mutations, all the tumors positive for mutations demonstrated missense mutations at codon 600. All the PIK3CA mutations occurred in exon 9 (codon 545) or exon 20 (codons 1047 and 1049). Neither BRAF nor PIK3CA mutation was correlated significantly with any clinicopathological characteristics. There was a mutually exclusive relationship between KRAS, BRAF and PIK3CA mutations. In other words, no KRAS mutation was detected in tumors with BRAF or PIK3CA mutation, and vice versa. Exon 3 ß-catenin mutations were single-base substitutions that were located within the critical serine/threonine codons for glycogen synthase kinase-3ß phosphorylation of ß-catenin (codons 29–48). ß-Catenin mutation was correlated significantly with tumors of larger size (P = 0.0034).


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  		Table II. Genetic alterations and clinicopathological characteristics in early colorectal tumors

 
Relationships between morphology and genetic and epigenetic alterations
Methylations of MGMT, WIF-1 and CDKN2A were detected in significantly higher percentages of protruded-type tumors (37.0, 91.8 and 60.3% of 73 tumors) than in flat-type tumors (23.7, 76.3 and 42.0% of 131 tumors; P = 0.0432, 0.0061 and 0.0122, respectively) (Table I). KRAS mutation was detected in a significantly higher percentage of flat-type tumors (26.7% of 131 tumors) than in protruded-type tumors (12.3% of 73 tumors; P = 0.0166) (Table II). KRAS mutation in G-to-A transitions was detected in a significantly higher percentage of flat-type tumors (20.6%) than in protruded-type tumors (8.2%; P = 0.0212). Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors (40.5% of 131 tumors) than in protruded-type tumors (19.2% of 73 tumors; P = 0.0019).

Relationships between genetic and epigenetic alterations
RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation (P = 0.0137). Although MGMT methylation was detected in a higher percentage of tumors with KRAS mutation in G-to-A transition, no significant correlation was found between the methylation and the mutation. Neither WIF-1 methylation nor EPHB2 methylation was correlated significantly with ß-catenin mutation.

Relationships between invasive potential and genetic and epigenetic alterations
Lymphatic invasion, venous invasion and lymph node metastasis were observed in 24 (23.3%), 35 (34.0%) and 13 (12.6%) of the 103 pT1 CRCs, respectively. Relationships between invasive potential (lymphatic invasion, venous invasion or lymph node metastasis) and genetic and epigenetic profiles are shown in Table III.


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  		Table III. Genetic and epigenetic profiles in 43 pT1 CRCs with invasive potential

 
MGMT methylation was correlated with lack of venous invasion (P = 0.0316). WIF-1 methylation was correlated significantly with venous invasion (P = 0.0117). KRAS mutation was correlated significantly with lymphatic invasion (P = 0.0433) and venous invasion (P = 0.0010).

KRAS, BRAF or PIK3CA mutation was correlated significantly with venous invasion (P = 0.0002). RASSF2 methylation with KRAS, BRAF or PIK3CA mutation was correlated significantly with lymphatic invasion (P = 0.0056), venous invasion (P = 0.0001) and lymph node metastasis (P = 0.0303) (Table IV).


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  		Table IV. Relationships between invasive potential and genetic and epigenetic alterations in pT1 CRCs

 
Logistic regression analysis for risk factors of invasive potential
Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs (Table V). Venous and lymphatic invasions were independent risk factors for lymph node metastasis (Table V).


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  		Table V. Unconditional logistic regression analysis for risk factors of invasive potential in pT1 CRCs

 
Immunohistochemical staining of MGMT protein in colorectal tumors with or without KRAS mutation in G-to-A transitions
Expression of MGMT protein was reduced in 11 (64.7%) of the 17 tumors with KRAS mutation in G-to-A transitions. On the other hand, expression of MGMT protein was positive in 26 (65.0%) of the 40 tumors without KRAS mutation in G-to-A transitions (Figure 1B and C). A significant correlation was found between KRAS mutation in G-to-A transitions and reduced MGMT protein expression (P = 0.0387). However, reduced MGMT protein expression was not correlated significantly with invasive potential.

MSI analysis
No high-frequency microsatellite instability (MSI-H) was found in 307 early colorectal tumors (data not shown), and these tumors were classified as microsatellite stable or low-frequency MSI according to the National Cancer Institute's guideline (30).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the current study, we compared the frequencies of genetic and epigenetic alterations of the RAS–RAF and Wnt signaling pathways in flat-type and protruded-type colorectal tumors. In addition, we investigated the relationship between those alterations and invasive potential of pT1 CRCs. As a result, we have shown that genetic alterations frequently occur in flat-type colorectal tumors and that RASSF2 methylation with oncogenic activation is correlated significantly with invasive potential.

We analyzed methylations of RASSF2, MGMT, WIF-1, EPHB2, CDKN2A and MLH1 by using MethyLight. A methylation-specific polymerase chain reaction (MSP) has been used in most previous studies. However, since MSP is not able to distinguish low levels of methylation from high levels of methylation, the frequency of DNA hypermethylation might be overestimated. Therefore, quantitative measurement is needed to clarify the frequency of DNA hypermethylation.

In a previous study using combined bisulfite restriction analysis, RASSF2 methylation was detected in 21 (43%) of 49 colorectal adenomas and in 51 (42%) of 122 advanced CRCs (16). The frequency of RASSF2 methylation in the present study was similar to those in that report. In addition, RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation.

It has been reported that down-regulation of WIF-1 mRNA expression was observed in 32 (72.7%) of 44 colorectal adenomas and 18 (78.2%) of 23 pT1 CRCs (21). The methylation status determined by using MSP was significantly correlated with down-regulation of WIF-1 expression. The frequency of WIF-1 methylation in early colorectal tumors in the present study was similar to the frequencies reported previously. It has been reported that EPHB2 methylation was observed in 54 (53.5%) of 101 advanced CRCs by using MSP (24). Batlle et al. (31) showed that loss of EPHB2 expression strongly correlates with late stage of CRCs. Therefore, the low frequency of EPHB2 methylation in the present study might be due to the fact that our samples were early colorectal tumors and/or the fact that different methods were used for analyzing EPHB2 methylation.

Ogino et al. (25) showed by using MethyLight that methylations of MGMT, CDKN2A and MLH1 occurred in 41, 32 and 15% of advanced CRCs, respectively. Although the frequency of CDKN2A methylation in pT1 CRCs in the present study was similar to that found in the previous study, the frequencies of MGMT and MLH1 methylation in the present study were lower than those reported previously. This discrepancy might be due to the fact that our samples were early colorectal tumors and/or the fact that different primers were used for analyzing methylation.

Since MSI-H due to defective DNA mismatch repair occurs in 10–15% of sporadic CRCs, we analyzed 307 early colorectal tumor tissues for MSI. However, no tumor tissue was classified as MSI-H. Previous studies have demonstrated that sporadic colorectal adenomas and early invasive cancers that show MSI-H are very rare (4,32). Thus, our results might be due to the fact that all tumor samples were early colorectal tumors.

The percentage of KRAS mutation in G-to-A transitions was significantly lower in protruded-type tumors than in flat-type tumors and pT1 CRCs. Maltzman et al. (33) reported that KRAS mutation in G-to-A transitions was detected in 58 (7.9%) of 738 colorectal adenomas. This result is consistent with our data on mutational patterns in protruded-type tumors. Therefore, most of the adenomas analyzed in that previous study might be protruded-type tumors. On the other hand, it was reported that the mutational patterns of KRAS in 51 (21.8%) of 234 sporadic advanced CRCs were transitions from G-to-A at codon 12 and codon 13 (12). This is consistent with our data on mutational patterns in flat-type tumors and pT1 CRCs. Therefore, CRCs with KRAS mutation in G-to-A transitions could largely arise from early flat-type colorectal tumors. Further analysis is needed to clarify this issue.

Although methylations of MGMT, WIF-1 and CDKN2A were detected in a significantly higher percentage of protruded-type tumors than in flat-type tumors, mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors than in protruded-type tumors. Therefore, genetic alterations of the RAS–RAF and Wnt signaling pathways might frequently occur in flat-type tumors. Since early detection of flat-type tumors is sometimes difficult compared with early detection of protruded-type tumors, analysis of these alterations seems to be applicable to a new diagnostic strategy, such as fecal DNA examination for flat-type tumors.

To date, therapy for pT1 CRCs has varied from endoscopic to definitive surgery. The overall frequency of lymph node metastasis in pT1 CRCs may be as low as 10% (34,35), and endoscopic local resection is an effective therapy for most pT1 CRCs without lymph node metastasis. Therefore, it is very important to develop a method to preoperatively predict the metastatic potential of pT1 CRCs. Kurokawa et al. (35) showed by using multiple logistic analysis that tumor matrilysin expression and venous invasion were independent risk factors for lymph node metastasis in pT1 CRCs. In addition, lymphatic invasion and tumor matrilysin expression were shown to be stage-independent risk factors for lymph node metastasis. Ueno et al. (36) showed that the absence of an unfavorable tumor grade, vascular invasion and tumor budding would be the most informative combination of criteria for selecting patients with a low risk of recurrence of pT1 CRCs.

On the other hand, Ahnen et al. (37) showed that KRAS mutations could have a higher rate of subclinical lymphatic involvement and are associated with a poor prognosis in stage II (pT3-T4 N0 M0) CRCs. Thus, not only lymph node metastasis but also lymphatic and venous invasions are thought to be significant risk factors in the early stage of CRCs. In the present study, KRAS mutation and methylations of MGMT and WIF-1 were found to be correlated significantly with invasive potential. In addition, RASSF2 methylation with KRAS, BRAF or PIK3CA mutation was found to be correlated more strongly with invasive potential.

MGMT methylation was correlated reversely with venous invasion. Although we found a significant correlation between KRAS mutation in G-to-A transitions and reduced MGMT protein expression, reduced MGMT protein expression was not correlated significantly with invasive potential. Kohonen-Corish et al. (38) have shown that MGMT defect is unlikely to contribute to the poor prognosis of low-level MSI CRCs. Therefore, our results are consistent with this previous report.

Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs. These results suggest that accumulation of genetic and epigenetic alterations in the RAS–RAF signaling pathway might play an important role in the aggressive biological behaviors of pT1 CRCs.

In summary, combined analysis of genetic and epigenetic alterations is needed to clarify the mechanisms of development and progression of early colorectal tumors. Since genetic alterations of the RAS–RAF and Wnt signaling pathways have frequently occurred in flat-type tumors, flat-type tumors seem to have a distinct genetic profile different from that of protruded-type tumors. In addition, RASSF2 methylation with oncogenic activation is a promising biomarker for predicting invasive potential of pT1 CRCs.


   Acknowledgments
 
This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.Y. and K.I.) and Grants-in-Aid for Cancer Research and for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (H.Y. and K.I.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 29, 2006; revised December 5, 2006; accepted December 5, 2006.


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S. Ogino, A. Hazra, G. J. Tranah, G. J. Kirkner, T. Kawasaki, K. Nosho, M. Ohnishi, Y. Suemoto, J. A. Meyerhardt, D. J. Hunter, et al.
MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer
Carcinogenesis, September 1, 2007; 28(9): 1985 - 1990.
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