Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polyps
Article first published online: 16 SEP 2008
Copyright © 2008 Wiley-Liss, Inc.
International Journal of Cancer
Volume 123, Issue 11, pages 2587–2593, 1 December 2008
How to Cite
Kim, Y. H., Kakar, S., Cun, L., Deng, G. and Kim, Y. S. (2008), Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polyps. Int. J. Cancer, 123: 2587–2593. doi: 10.1002/ijc.23840
- Issue published online: 24 SEP 2008
- Article first published online: 16 SEP 2008
- Manuscript Accepted: 24 JUN 2008
- Manuscript Received: 18 APR 2008
- Department of Veterans Affairs Medical Research Service
- Theodora Betz Foundation
- Oberkotter Foundation
- serrated polyp;
- tubular adenoma;
- DNA methylation;
- CpG island methylator phenotype;
- BRAF mutation
A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis. © 2008 Wiley-Liss, Inc.
Hypermethylation of CpG islands in the promoter region of tumor suppressor genes and tumor-associated genes may cause transcriptional silencing, with subsequent loss of protein expression in colorectal cancers.1–3 Moreover, a high degree of concurrent promoter methylation in multiple genes, called the CpG island methylator phenotype (CIMP), has been described in a subset of colorectal cancer.4–6 CIMP positive colorectal cancers have been reported to have distinct clinicopathological features such as proximal location, microsatellite instability (MSI), BRAF V600E mutation and wild-type p53.5–9 CpG island methylation has also been reported to occur in precursor lesions such as aberrant crypt foci and colorectal polyps.10–19 Although a higher frequency and the level of promoter methylation and CIMP in serrated colorectal polyps such as sessile serrated adenomas (SSA) and serrated adenomas (SA) has been reported,11–14 there are also reports that indicate that CIMP occurs in 6–30% of tubular adenomas (TA).15–17 Higher frequency of methylation has been associated with large size (>1 cm) and presence of high-grade dysplasia in TA.18, 19 In addition, a higher frequency of promoter methylation of MGMT and CDKN2A has been reported in TA (49 and 34%) compared with hyperplastic polyps (HP) (5 and 10%).16 However, in these studies, different sets and numbers of genes were used to assess the CIMP status and the frequency and the level of promoter methylation, making the comparison of these results difficult. Recent studies have also identified occurrences of BRAF V600E mutation and a close association of BRAF mutation with promoter methylation and MSI in colorectal cancer.7, 9, 13, 14, 20BRAF V600E mutation was rarely observed in TA but was frequently found in serrated polyps.14 Among serrated polyps, BRAF V600E mutation occurred more frequently in SSA (75%) and mixed polyps (89%) compared with HP (19%) and SA (20%).13 However, the association of BRAF V600E mutation with promoter methylation in these polyps was not examined in these studies.
In our study, we compared the frequency of promoter methylation and CIMP in serrated polyps and TA by examining the methylation status of 7 commonly used tumor-associated genes. We also compared the incidence of BRAF V600E and KRAS mutations and their relationship with promoter methylation of 7 genes and CIMP status in these polyps. Finally, we performed multivariate analysis of the frequencies of promoter methylation and BRAF V600E and KRAS mutations and MSI and their relationship to clinicopathological parameters such as location, size and histology of these polyps.
Material and methods
Formalin-fixed paraffin-embedded tissue blocks were obtained from the Department of Pathology, University of California, San Francisco and the San Francisco Veterans Affairs Medical Center. Forty-eight samples of HP, 32 SSA, 30 SA and 32 TA were used in our study. We excluded polyps from patients who had familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer or hyperplastic polyposis. Our study was undertaken with approval of the Institutional Review Board for Protection of Human Subjects at the University of California, San Francisco and the San Francisco Veterans Affairs Medical Center.
Serial sections (5-μm thick) were cut from the paraffin blocks and were used for H&E staining and immunostaining. All H&E stained sections were reviewed by an experienced gastrointestinal pathologist (S.K.). Only cases with typical morphological features were recruited for the study, and polyps with indeterminate features were not included. Polyps with dysplasia without serrated architecture were classified at TA. Polyps with serrated architecture without dysplasia were classified as HP. Serrated polyps without morphological dysplasia and with features of abnormal maturation or abnormal proliferation (basal crypt dilation, crypt branching, deep serrations, mitoses in the upper half of crypts, dystrophic goblet cells and horizontal crypts) were classified as sessile serrated adenoma.21 Polyps with dysplasia and serrated architecture were classified as SA based on the definition of Longacre and Fenoglio-Preiser.22
Microdissection and DNA extraction
Polyps were microdissected from formalin-fixed, paraffin- embedded 7-μm thick histological sections stained with hematoxylin-eosin using a surgical scalpel under microscopic guidance. For genomic DNA isolation, microdissected samples were incubated overnight at 56°C with 0.5% Tween 20 (Sigma, St. Louis, MO), 100 mM of Tris HCl buffer (pH 7.6), 1 mM of EDTA and 20 μg of proteinase K (Sigma). Samples were incubated at 56°C overnight. Proteinase K was then inactivated by incubating at 95°C for 10 min, and the extracted DNA was stored at −20°C until use.
To determine the expression of MLH1 protein in tumors, paraffin sections were stained with anti-MLH1 antibody (1:100; clone G168-15, Pharmingen, Palo Alto, CA). After deparaffinization, sections were subjected to heat-induced antigen retrieval in 10 mM sodium citrate buffer in a pressure cooker (Biocare Medical, Walnut Creek, CA), pH 6.0 for 20 min. Nonspecific protein binding was blocked by incubating sections with 10% goat serum blocking solution (Zymed Laboratories, South San Francisco, CA) for 10 min. Anti-MLH1 antibody was applied and incubated at 4°C overnight. Sections were rinsed in PBS followed by incubation of biotinylated secondary antibody (Zymed) for 10 min at room temperature. After a brief rinse, streptavidin enzyme conjugate (Zymed) was applied and incubated for 10 min. Sections were rinsed, followed by incubation with diaminobenzidine (Zymed) for 2–3 min. After counterstaining with hematoxylin, sections were dehydrated in graded ethanol and cleared in xylene before mounting. Distinct nuclear staining was interpreted as positive staining for MLH1.
The MSI status of each sample was analyzed by examining the polymerase chain reaction (PCR) patterns at the polymorphic repetitive loci, BAT25 and BAT26. Tumor tissue samples and adjacent normal samples were amplified with these markers. Amplifications were carried out in a PTC-100 thermal cycler (MJ Research, South San Francisco, CA), with HotStarTaq DNA polymerase (Qiagen, Valencia, CA). Thermal cycling included initial denaturation at 95°C for 15 min, 45 cycles of 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, followed by a final extension at 72°C for 10 min. Amplified PCR product was electrophoresed on 6% acrylamide gels and visualized by ethidium bromide staining. Samples are scored for allelic shifts between tumor and adjacent normal DNA. Polyps were classified as MSI if allelic shifts were observed.
Bisulfite treatment of DNA, methylation-specific PCR and determination of CIMP status
The methylation status of CpG sites in the promoters of tumor suppressor gene p16, a member of ras-associated domain family gene RASSF2, methylated in tumor genes MINT1 and MINT31, hypermethylated gene in cancer HIC1, mismatch repair gene MLH1 and DNA repair enzyme MGMT was determined by methylation-specific PCR (MSP) as described previously.7, 23 For MLH1 methylation analysis we analyzed the methylation status of region C in the promoter as described previously.7 Bisulfite-modified DNA templates were amplified by PCR using either methylation-specific primers or unmethylation-specific primers of 7 loci (Table I). CIMP status was classified as CIMP-negative if no locus was methylated, CIMP-low if 1 or 2 loci were methylated and as CIMP-high if 3 or more loci were methylated.
|Gene locus||Forward primer||Reverse primer||Annealing temperature (°C)||Length (bp)|
Detection of mutations of BRAF and KRAS genes
Genomic DNA from microdissected polyps was amplified by PCR using primers of the exon 15 of BRAF gene (5′-CTTTACT TACTACACCTCAG and 5′-TAACTCAGCAGCATCTCAGG) and the exon 1 of KRAS gene (5′-ACCTTATGTGTGACAT GTTCTAATATAG and 5′-GAATGGTCCTGCACCAGTAA). The PCR products were sequenced with an ABI PRISM 3100 automated sequencer.
Chi-square test and Fisher's exact test were used for statistical analysis. p values of <0.05 were considered significant.
Methylation status and MSI in serrated polyps and tubular adenomas
No significant difference was observed in the frequency of CpG island methylation at any locus among HP, SSA, SA and TA (81, 91, 90 and 100%, respectively) (Table II, Fig. 1). The frequency of CIMP-H status was also similar among these polyps when compared as subtypes (33, 44, 43 and 28%, respectively) (Table II, Fig. 1), or when compared as serrated polyps as a group with TA (43/110, 39% vs. 9/32, 28%) (Table III). However, CpG island methylation tended to involve different loci in serrated polyps and TA. The frequency of CpG island methylation of HIC1 was significantly higher in serrated polyps compared to TA (42/110, 38% vs. 4/32 13%; p = 0.006) (Table III). The higher frequency of methylation of HIC1 in serrated polyps was largely due to a higher methylation frequency in SSA (22/32, 69%) than in HP (10/48, 21%) and SA (10/30, 33%, p < 0.01) (Table II, Fig. 1). MLH1 was also more frequently methylated in serrated polyps compared to TA, though not statistically significant (16/110, 15% vs. 2/32, 6%, p = 0.06) (Table III). Among serrated polyps, MLH1 was methylated more frequently in HP (10/48, 21%) and SSA (5/32, 16%) compared to SA (1/30, 3%, p = 0.01) (Table II). Although MLH1 was methylated in 12.7% (18/142) of all polyps examined, MLH1 protein was expressed in all cases by immunohistochemistry. By contrast, CpG island methylation of MGMT was observed significantly more frequently in TA compared to serrated polyps (11/32, 34% vs. 15/110, 14%, p = 0.017) (Table III). No significant difference was observed in the frequencies of CpG methylation of RASSF2, p16, MINT1 and MINT31 between serrated polyps and TA.
|Polyp type No. of case||HP 48||SSA 32||SA 30||TA 32||p|
|Small||33 (69)||13 (41)||11 (37)||21 (66)|
|Large||15 (31)||19 (59)||19 (63)||11 (34)|
|Proximal||21 (44)||18 (56)||8 (27)||16 (50)|
|Distal||27 (56)||14 (44)||22 (73)||16 (50)|
|BAT 26||0 (0)||0 (0)||1 (3)||0 (0)|
|RASSF2||26 (54)||20 (63)||19 (63)||21 (66)||ns|
|P16||24 (50)||14 (44)||16 (53)||21 (66)||ns|
|MINT1||21 (44)||9 (28)||13 (43)||9 (28)||ns|
|HlCl||10 (21)||22 (69)||10 (33)||4 (13)||<0.001|
|MINT31||4 (8)||4 (13)||4 (13)||1 (3)||ns|
|MLH1||10 (21)||5 (16)||1 (3)||2 (6)||ns|
|MGMT||5 (10)||4 (13)||6 (20)||11 (34)||0.04|
|Negative||9 (19)||3 (9)||3 (10)||0 (0)||ns|
|Low||23 (48)||15 (47)||14 (47)||23 (72)||ns|
|High||16 (33)||14 (44)||13 (43)||9 (28)||ns|
|KRAS||4 (9)||2 (8)||5 (17)||3 (9)||ns|
|BRAF||40 (85)||26 (81)||22 (76)||0 (0)||<0.001|
|Genes||Serrated polyps||p*||Tubular adenomas||p*||p**|
|Total (n = 110)||P (n = 47)||D (n = 63)||Total (n = 32)||P (n = 16)||D (n = 16)|
|RASSF2||65 (59)||34 (72)||31 (49)||0.019||21 (66)||12 (75)||9 (56)||ns||ns|
|P16||54 (49)||29 (62)||25 (40)||0.034||21 (67)||13 (81)||8 (50)||ns||ns|
|MINT1||43 (39)||28 (60)||15 (24)||<0.001||9 (28)||7 (44)||2 (13)||ns||ns|
|HIC1||42 (38)||27 (57)||15 (24)||0.001||4 (13)||2 (13)||2 (13)||ns||0.006|
|MINT31||12 (11)||5 (11)||7 (11)||ns||1 (3)||0 (0)||1 (6)||ns||ns|
|MLH1||16 (15)||13 (28)||3 (5)||0.001||2 (6)||0 (0)||2 (13)||ns||ns|
|MGMT||15 (14)||9 (19)||6 (10)||ns||11 (34)||2 (13)||9 (56)||0.012||0.017|
|CIMP-H||43 (39)||29 (62)||14 (22)||<0.001||9 (28)||4 (25)||5 (31)||ns||ns|
Among HP, there was no significant difference in the frequency of CpG island methylation at any locus between microvesicular and goblet cell type (83% vs. 81%). However, CIMP-H was more frequently observed in microvesicular than goblet cell types (13/31, 41% vs. 1/12, 8%; p = 0.04) (Table IV). The number of mixed polyp types were too small for proper statistical analysis.
|Subtypes No. of cases||Total||GC 12||MV 32||MP 4||p*|
|Small||33 (69)||8 (67)||21 (66)||4 (100)||ns|
|Large||15 (31)||4 (33)||11 (34)||0 (0)||ns|
|Distal||27 (56)||8 (67)||16 (50)||3 (75)||ns|
|Proximal||21 (44)||4 (33)||16 (50)||1 (25)||ns|
|RASSF2||26 (54)||5 (42)||18 (56)||3 (75)||ns|
|P16||24 (50)||6 (50)||16 (50)||2 (50)||ns|
|MINT 1||21 (44)||3 (25)||17 (53)||1 (25)||ns|
|HlC1||10 (21)||1 (8)||8 (25)||1 (25)||ns|
|MINT 31||4 (8)||1 (8)||3 (9)||0 (0)||ns|
|MLH1||10 (21)||1 (8)||9 (28)||0 (0)||ns|
|MGMT||5 (10)||1 (8)||4 (13)||0 (0)||ns|
|Negative||9 (19)||2 (17)||6 (19)||1 (25)||ns|
|Low||23 (48)||9 (75)||13 (41)||1 (25)||ns|
|High||16 (33)||1 (8)||13 (41)||2 (50)||ns|
|KRAS||4 (8)||1 (8)||2 (6)||1 (25)||ns|
|BRAF||40 (83)||9 (75)||28 (88)||3 (75)||ns|
Site of polyp and CpG island methylation
When a methylation profile of individual locus was compared, HP in the proximal colon showed more frequent CpG island methylation of RASSF2, p16, MINT 1, HIC 1 and MLH1, no difference was observed between SSA, SA and TA, while in the proximal colon compared to those in the distal colon (Table V). Among 10 HP with MLH1 methylation, 8 (80%) were in the proximal colon (Table V, Fig. 1). Interestingly, CpG island methylation of MGMT occurred more frequently in TA in the distal colon compared to those in the proximal colon (9/16, 56% vs. 2/16, 13% p = 0.012) (Table V). No significant regional difference in the frequency of MGMT methylation was observed in HP, SSA and SA. CIMP-H was more frequently observed in the proximally located HP and SA, but SSA or TA did not show regional differences. Serrated polyps as a group showed more frequent CpG island methylation of RASSF2, p16, MINT 1, HIC 1 and MLH1 and CIMP-H (p = 0.019, p = 0.034, p < 0.001, p = 0.001 and p < 0.001, respectively) in the proximal colon compared to those in the distal colon, but TA showed no regional difference (Table III).
|Polyp type No. of case||HP 48||p||SSA 32||p||SA 30||p||TA 32||p|
|Site No. of case||P 21||D 27||P 18||D 14||P 8||D 22||P 16||D 16|
|RASSF2||18 (86)||8 (30)||<0.001||11 (61)||9 (64)||ns||5 (63)||14 (64)||ns||12 (75)||9 (56)||ns|
|p16||15 (71)||9 (33)||0.009||8 (44)||6 (43)||ns||6 (75)||10 (46)||ns||13 (81)||8 (50)||ns|
|MINT1||17 (81)||4 (15)||<0.001||6 (33)||3 (21)||ns||5 (63)||8 (36)||ns||7 (44)||2 (13)||ns|
|HIC1||8 (38)||2 (7)||<0.012||15 (83)||7 (50)||ns||4 (50)||6 (27)||ns||2 (13)||2 (13)||ns|
|MINT31||2 (10)||2 (7)||ns||3 (17)||1 (7)||ns||0 (0)||4 (18)||ns||0 (0)||1 (6)||ns|
|MLH1||8 (38)||2 (7)||0.012||4 (22)||1 (7)||ns||1 (13)||0 (0)||ns||0 (0)||2 (13)||ns|
|MGMT||4 (19)||1 (4)||ns||3 (17)||1 (7)||ns||2 (25)||4 (18)||ns||2 (13)||9 (55)||0.012|
|Negative||0 (0)||9 (33)||0.003||0 (0)||3 (21)||ns||0 (0)||3 (14)||ns||0 (0)||0 (0)||ns|
|Low||7 (33)||16 (59)||ns||9 (50)||6 (43)||ns||2 (25)||12 (55)||ns||12 (75)||11 (69)||ns|
|High||14 (67)||2 (8)||<0.001||9 (50)||5 (36)||ns||6 (75)||7 (32)||0.03||4 (25)||5 (31)||ns|
|Low & High||21 (100)||18 (67)||0.003||18 (100)||11 (79)||0.04||8 (100)||19 (86)||ns||16 (100)||16 (100)||ns|
|KRAS||2 (10)||2 (7)||ns||1 (6)||1 (7)||ns||3 (38)||2 (9)||ns||0 (0)||3 (19)||ns|
|BRAF||17 (81)||23 (85)||ns||15 (83)||11 (79)||ns||6 (75)||16 (73)||ns||0 (0)||0 (0)||ns|
Size of polyp and CpG island methylation
Among HP, the frequency of CpG island methylation at all loci was similar in small and large polyps (25/33, 76% vs. 14/15, 93%, Table VI). However, CIMP-H was more frequently observed in large HP (9/15, 60% vs. 7/33, 21%, p = 0.026). Among 10 HP with MLH1 methylation, the majority was large (60%). RASSF2, MINT1 and MLH1 were also more frequently methylated in large HP (p = 0.016, 0.001 and 0.037, respectively). No difference in the frequency of CpG methylation at any locus or CIMP-H status based on the size of polyps was observed in SSA, SA and TA (Table VI).
|Polyp type No. of case||HP 48||p||SSA 32||p||SA 30||p||TA 32||p|
|Size No. of case||S 33||L 15||S 13||L 19||S 11||L 19||S 21||L 11|
|RASSF2||14 (42)||12 (80)||0.016||8 (62)||12 (63)||ns||7 (64)||12 (63)||ns||12 (57)||9 (82)||ns|
|P16||49 (49)||8 (53)||ns||6 (46)||8 (42)||ns||7 (64)||9 (47)||ns||8 (50)||6 (55)||ns|
|MINT1||9 (27)||12 (80)||0.001||2 (15)||7 (37)||ns||5 (46)||8 (42)||ns||6 (29)||3 (27)||ns|
|HIC1||5 (15)||5 (33)||ns||9 (69)||13 (68)||ns||3 (27)||7 (37)||ns||2 (10)||2 (18)||ns|
|MINT31||4 (12)||0 (0)||ns||2 (15)||2 (11)||ns||2 (18)||2 (11)||ns||1 (5)||0 (0)||ns|
|MLH1||4 (12)||6 (40)||0.037||2 (15)||3 (16)||ns||0 (0)||1 (5)||ns||2 (10)||0 (0)||ns|
|MGMT||3 (9)||2 (13)||ns||3 (23)||5 (1)||ns||3 (27)||3 (16)||ns||9 (29)||5 (46)||ns|
|Negative||8 (24)||1 (7)||ns||1 (8)||2 (11)||ns||1 (9)||2 (11)||ns||0 (0)||0 (0)||ns|
|Low||18 (55)||5 (33)||ns||6 (46)||9 (47)||ns||5 (46)||9 (47)||ns||16 (76)||7 (64)||ns|
|High||7 (21)||9 (60)||0.026||6 (46)||8 (42)||ns||5 (46)||8 (42)||ns||5 (24)||4 (36)||ns|
|KRAS||3 (9)||1 (7)||ns||0 (0)||2 (11)||ns||2 (18)||3 (16)||ns||1 (5)||2 (18)||ns|
|BRAF||26 (79)||14 (93)||ns||11 (85)||15 (79)||ns||9 (82)||13 (68)||ns||0 (0)||0 (0)||ns|
BRAF and KRAS mutation
BRAF V600E mutation was frequently observed in HP (40/48, 85%) SSA (26/32, 81%) and SA (22/30, 76%), but none of 32 TA showed BRAF mutation (p < 0.001) (Table II, Fig. 1). The frequency of BRAF V600E mutation in serrated polyps as a group was 80% (88/110). KRAS mutations were detected at low frequency in all types of polyps; HP (4/48, 9%), SSA (2/32, 8.3%), SA (5/30, 17%) and TA (3/32, 9.3%). BRAF and KRAS mutations were mutually exclusive in all cases except in one case each of SSA and SA where both mutations were present in the same polyp (Fig. 1). There was no unique histological feature in these polyps compared to other SSA or SA. No site- or size-specific difference was observed in the frequencies of BRAF and KRAS mutations among all histological types of polyps (Tables V and VI). Among HP, no difference in the frequency of BRAF and KRAS mutations was observed between microvesicular and goblet cell type (Table IV). BRAF V600E mutation occurred frequently in all 3 types of serrated polyps (ranging from 64 to 93%) regardless of CIMP status (Table VII).
|Subtype||No. of case||CIMP status||No. of case||BRAF mutations||p|
Recent studies indicate that multiple molecular pathways involving genetic and epigenetic changes are involved in colorectal carcinogenesis.24–26 In addition to well-characterized genetic changes observed in chromosomal instability and MSI pathways, additional pathways involving epigenetic changes such as hypermethylation of CpG islands in the promoter of tumor-associated genes and global DNA hypomethylation have been described.2–6, 27 Colorectal polyps have recently been classified into 2 major groups, conventional adenomas and serrated polyps that include HP, SSA, sessile adenomas and mixed polyps.21 Because of the observed differences in the frequency of genetic and epigenetic changes between serrated polyps and conventional adenomas, serrated polyp carcinoma-sequence has been suggested as an alternative pathway of colorectal carcinogenesis in addition to the traditional adenoma-carcinoma sequence.13, 14, 28, 29 Although a higher frequency of promoter methylation and CIMP has been reported to occur in serrated polyps,11–14 recent studies indicate that conventional adenomas also show frequent promoter methylation and CIMP.17–19 Furthermore, the frequent association of BRAF mutation with promoter methylation observed in colorectal cancers7, 13, 14 has not been examined in the subtypes of serrated polyps and conventional adenomas.
The present study was carried out to clarify these issues in the subtypes of serrated polyps and TA. Using 7 gene loci commonly used for the assessment of promoter methylation, we observed that promoter methylation was a frequent event in all types of polyps and that no significant difference in the frequency of promoter methylation at any locus or CIMP status was observed among all histological subtypes of serrated polyps and TA. Our results differ from the previous studies that reported more frequent occurrences of CIMP in serrated polyps compared to TA, but are consistent with the reports of frequent occurrences of CIMP in adenomatous polyps.11–14, 17–19 While the reasons for these differences are not clear, our results together with those of others suggest that promoter methylation is an early and frequent event in colorectal carcinogenesis10, 17, 30 and that CIMP-H occurs in comparable frequency in both serrated polyps-carcinoma and adenoma-carcinoma sequences. Among HP, microvesicular type tended to show a higher frequency of CIMP than goblet cell type (41% vs. 8%), though not statistically significant, supporting the hypothesis that microvesicular type of hyperplastic polyp may be the predominant early precursor of CIMP positive colorectal cancers.
Interestingly, methylation of MLH1 and HIC1 promoters (p < 0.06 and p = 0.006, respectively) were observed more frequently in serrated polyps while more frequent methylation of MGMT promoter (p = 0.017) occurred in TA in our study. These results suggest that distinct gene locus specific methylation occurs predominantly in each type of polyps and underscores the existence of considerable variation in the frequency of methylation among different gene loci in different histological types of polyp and colorectal cancer.7, 14 MSI in sporadic colorectal cancer is generally associated with MLH1 promoter methylation with loss of MLH1 protein expression.31, 32 Surprisingly, although methylation of MLH1 promoter occurred in 21% of HP and 16% of SSA, no MSI or loss of MLH1 protein expression was observed in these polyps. The reasons for this discrepancy in our results are not clear but it is possible that we may have underestimated the incidence of MSI since we used only 2 markers, BAT25 and BAT26, to assess the MSI status. However, very rare occurrences of MSI in polyps have previously been reported.12 Alternatively, hypermethylation of MLH1 that we observed may have represented hemimethylation of alleles which may not lead to loss of protein expression and MSI. Furthermore, a rare occurrence of MSI in serrated polyps and TA observed in our study is consistent with the idea that MSI is a relatively late event in the development of colorectal cancer.8, 26
MGMT is a DNA repair enzyme that removes alkyl DNA adducts from the O6 position of guanine33 and its alterations increase the mutation rate and the risk of cancer.34MGMT promoter methylation has been reported in equal frequency in small adenomas (43%), large adenomas (52%) and carcinomas (40%), indicating that this change occurs early in neoplastic progression.35 In our study, we observed that MGMT methylation occurs more frequently in TA than in serrated polyps (34% vs. 13.6%), though not statistically significant. Positive correlation between MGMT methylation and CIMP-L has recently been reported in sporadic colorectal cancers.36 However, in our study no such relationship was observed in TA and serrated polyps with MGMT methylation, which tend to be associated more frequently with CIMP-H. In sporadic colorectal cancer, MGMT methylation and silencing has been associated with G > A mutations in KRAS and p53 genes.35–37 In our study, KRAS mutations occurred at low frequency in all type of polyps and no association between MGMT methylation and KRAS mutations was observed.
Increasing evidences support the idea that proximal and distal colorectal cancers differ considerably in the incidence according to geographic location, age and molecular biological characteristics.38–40 In high incidence area, about 60% of colorectal cancer is found in the distal location, while proximal tumors predominate in low incidence areas.38 MSI positive colorectal cancer has been reported to occur preferentially in proximal colon and in older women.39 A higher incidence of CIMP positive tumors in proximal colon and the prevalence of chromosomal instability in distal colorectal cancer have also been reported.40, 41 Different histological types of polyps also show varying colonic localization. Adenomas occur equally in proximal and distal colon. SA and HP occur more frequently in the left colon and rectosigmoid, respectively, whereas SSA occur more commonly in proximal colon.42 Our observation of more frequent methylation of 5 of 7 gene loci and CIMP-H in proximally located serrated polyps compared to those in distal colon but not in TA suggest that there may be gene locus-, colonic site- and histological type-specific methylation pattern among polyps. The observation that MGMT methylation was significantly higher in distal TA compared to proximal TA but not in serrated polyps further supports this possibility. Among serrated polyps, only hyperplastic polyps showed size-dependent increase in the frequency of CIMP. Although statistically not significant, the trend for more frequent CIMP-H (36% vs. 24%) and MGMT methylation (46% vs. 29%) observed in larger TA compared to smaller TA observed in our study is consistent with the previous studies that reported association of CIMP-H with more advanced adenomas such as larger adenomas and adenomas with villous component.14, 17–19
RAS/RAF/MAP kinase cascade is an important pathway mediating the cellular response to external signals that regulate cell growth, differentiation and apoptosis.43 Activating mutations of KRAS, a member of RAS family occur in 30–40% of cancers while activating mutations of BRAF, a member of RAF family occur in 5–15% of sporadic colorectal cancers but not in HNPCC.44, 45 Since both are involved in activation of mitogen-activated phosphokinase (MAP kinase) pathway, one would expect a similar biological effect. However, cancers with BRAF V600E mutation have been reported to be associated more frequently with MSI and CIMP and also have more aggressive behavior and poor prognosis.7, 9, 43BRAF V600E mutation in colon epithelial cells has been reported to contribute to a gain in resistance toward apoptotic stimuli as well as playing a role in the development and maintenance of transformed and invasive phenotypes.46 In our study, BRAF V600E mutation was observed in very high frequency (75–88%) in all types of serrated polyps but in none of TA. Rare occurrences of BRAF V600E mutation in adenomas and more frequent BRAF V600E mutations in serrated polyps have been reported previously.13, 14 Although a previous study reported a high frequency of BRAF V600E mutation mainly in SSA and not in HP and SA, our study showed that all types of serrated polyps showed frequent BRAF V600E mutation.13 Unlike colorectal cancers, which showed an association of BRAF V600E mutation with CIMP.7, 9, 13BRAF mutation was not correlated to CIMP-H status in serrated polyps. BRAF V600E mutation was frequently observed even in serrated polyps without methylation at any gene locus. These results together with the recent report of the frequent occurrence of BRAF V600E mutation in serrated hyperplastic aberrant crypt loci indicate that BRAF V600E mutation occurs very early in colorectal carcinogenesis independently of CIMP and is a distinct marker of both serrated polyps and serrated pathway.47
To summarize, our results demonstrate that promoter methylation is a very common event in both serrated polyps of all histological subtypes and TA indicating that it is an early event in both serrated polyp-carcinoma and adenoma-carcinoma sequences. CIMP-H that occurs in a subset of colorectal cancer was observed in 29 and 39% of TA and serrated polyps, respectively, but proximally located serrated polyps were more likely to be CIMP-H. A distinct difference in gene-specific and colonic site-specific-methylation profiles are observed between serrated polyps and TA. BRAF V600E mutation occurred predominantly in serrated polyps independently of CIMP and MSI status and appears to be a distinct marker of both serrated polyps and serrated pathway of colorectal carcinogenesis.
We would like to thank Ms. Rita Burns and Ms. Esther Choi for the preparation of this manuscript.