Original Article Open Access
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 7, 2014; 20(29): 10050-10061
Published online Aug 7, 2014. doi: 10.3748/wjg.v20.i29.10050
IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions
Takafumi Naito, Katsuhiko Nosho, Miki Ito, Hisayoshi Igarashi, Kei Mitsuhashi, Eiichiro Yamamoto, Yasushi Adachi, Yasuhisa Shinomura, Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan
Shinji Yoshii, Hiroaki Takahashi, Department of Gastroenterology, Keiyukai Sapporo Hospital, Sapporo 003-0027, Japan
Hironori Aoki, Masafumi Nomura, Center for Gastroenterology, Teine-Keijinkai Hospital, Sapporo 006-0811, Japan
Yasutaka Sukawa, Shuji Ogino, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, United States
Masao Hosokawa, Department of Surgery, Keiyukai Sapporo Hospital, Sapporo 003-0027, Japan
Masahiro Fujita, Toshinao Takenouchi, Department of Pathology, Keiyukai Sapporo Hospital, Sapporo 003-0027, Japan
Reo Maruyama, Hiromu Suzuki, Department of Molecular Biology, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan
Yoshifumi Baba, Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto 860-0862, Japan
Kohzoh Imai, Division of Cancer Research, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
Hiroyuki Yamamoto, Division of Gastroenterology and Hepatology, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan
Author contributions: Naito T and Nosho K designed the research, analyzed the data and wrote the manuscript; Sukawa Y, Adachi Y, Yamamoto E, Maruyama R and Suzuki H analyzed the data; Naito T, Ito M, Igarashi H and Mitsuhashi K performed experiments; Yoshii S, Aoki H, Nomura M, Takahashi H, Hosokawa M, Fujita M and Takenouchi T provided the collection of the human material and analyzed the data; Baba Y, Imai K, Yamamoto H, Ogino S and Shinomura Y edited the manuscript; Nosho K and Ogino S helped interpretation and drafting of this manuscript.
Supported by The Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research, grant No. 23790800 (to Nosho K) and 23390200 (to Shinomura Y); A-STEP (Adaptable and Seamless Technology Transfer Program through Target-driven R and D) (to Nosho K); Daiwa Securities Health Foundation (to Nosho K); Kobayashi Foundation for Cancer Research (to Nosho K); Sagawa Foundation for Promotion of Cancer Research (to Nosho K); Suzuken Memorial Foundation (to Nosho K), and Takeda Science Foundation (to Nosho K); and USA National Institute of Health, grant number R01 CA151993 (to Ogino S)
Correspondence to: Katsuhiko Nosho, MD, PhD, Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, S-1, W-16, Chou-ku, Sapporo 060-8543, Japan. nosho@sapmed.ac.jp
Telephone: +81-11-6112111 Fax: +81-11-6112282
Received: January 11, 2014
Revised: March 1, 2014
Accepted: April 21, 2014
Published online: August 7, 2014
Processing time: 207 Days and 22.6 Hours

Abstract

AIM: To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions.

METHODS: To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quantification of the relative methylation levels (scale 0%-100%) of IGF2 DMR0 and LINE-1, we used bisulfite pyrosequencing method. Tumor specimens were analyzed for microsatellite instability, KRAS (codons 12 and 13), BRAF (V600E), and PIK3CA (exons 9 and 20) mutations; MLH1 and MGMT methylation; and IGF2 expression by immunohistochemistry.

RESULTS: The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas (with or without HGD) was as follows: mean 61.7, median 62.5, SD 18.0, range 5.0-99.0, interquartile range 49.5-74.4. The IGF2 DMR0 methylation level was divided into quartiles (Q1 ≥ 74.5, Q2 62.6-74.4, Q3 49.6-62.5, Q4 ≤ 49.5) for further analysis. With regard to the histological type, the IGF2 DMR0 methylation levels of SSAs (mean ± SD, 73.1 ± 12.3) were significantly higher than those of HPs (61.9 ± 20.5), TSAs (61.6 ± 19.6), and non-serrated adenomas (59.0 ± 15.8) (P < 0.0001). The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level (r = -0.21, P = 0.0051). IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas (P < 0.0001). Multivariate logistic regression analysis also showed that IGF2 DMR0 hypomethylation was inversely associated with SSAs (P < 0.0001). The methylation levels of IGF2 DMR0 and LINE-1 in TSAs with HGD (50.2 ± 18.7 and 55.7 ± 5.4, respectively) were significantly lower than those in TSAs (61.6 ± 19.6 and 58.8 ± 4.7, respectively) (IGF2 DMR0, P = 0.038; LINE-1, P = 0.024).

CONCLUSION: IGF2 DMR0 hypomethylation may be an infrequent epigenetic alteration in the SSA pathway. Hypomethylation of IGF2 DMR0 and LINE-1 may play a role in TSA pathway progression.

Key Words: BRAF; Colon polyp; Colorectal neoplasia; Colorectum; Genome; Insulin-like growth factor; Long interspersed nucleotide element-1; Microsatellite instability; Serrated pathway

Core tip: The serrated pathway attracts considerable attention as an alternative colorectal cancer (CRC) pathway. We previously reported the association of insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation with prognosis and its link to LINE-1 hypomethylation in CRC; however, there have been no studies describing its role in the serrated pathway. Therefore, we evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in 351 serrated lesions and 185 non-serrated adenomas. Our results suggest that the IGF2 DMR0 may be an infrequent epigenetic alteration in the sessile serrated adenoma pathway. Moreover, we found that the hypomethylation of IGF2 DMR0 and LINE-1 may play an important role in the progression of traditional serrated adenoma.
  • Citation: Naito T, Nosho K, Ito M, Igarashi H, Mitsuhashi K, Yoshii S, Aoki H, Nomura M, Sukawa Y, Yamamoto E, Adachi Y, Takahashi H, Hosokawa M, Fujita M, Takenouchi T, Maruyama R, Suzuki H, Baba Y, Imai K, Yamamoto H, Ogino S, Shinomura Y. IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions. World J Gastroenterol 2014; 20(29): 10050-10061
  • URL: https://www.wjgnet.com/1007-9327/full/v20/i29/10050.htm
  • DOI: https://dx.doi.org/10.3748/wjg.v20.i29.10050
INTRODUCTION

The serrated neoplasia pathway has attracted considerable attention as an alternative pathway of colorectal cancer (CRC) development, and serrated lesions exhibit unique clinicopathological or molecular features[1-23]. According to the World Health Organization (WHO) classification[24], colorectal premalignant (or non-malignant) neoplastic lesions with serrated morphology currently encompass three major categories: hyperplastic polyp (HP), sessile serrated adenoma (SSA), and traditional serrated adenoma (TSA).

SSA and TSA are premalignant lesions, but SSA is the principal serrated precursor of CRCs[15]. In particular, there are many clinicopathological and molecular similarities between SSA and microsatellite instability (MSI)-high CRC, for example, right-sided predilection, MLH1 hypermethylation, and frequent BRAF mutation[7,15,17-19,25-28]. Therefore, SSAs are hypothesized to develop in some cases to MSI-high CRCs with BRAF mutation in the proximal colon[7,15,17,25,26,28,29].

In contrast, TSAs are much less common than SSAs, and thus, there are fewer data on their molecular profile[15,25]. TSAs typically do not show MLH1 hypermethylation or develop to MSI-high CRCs, but they do commonly have MGMT hypermethylation[15,25,26]. With regard to the PIK3CA gene, a previous study reported that no mutation was found in serrated lesions, and that mutations were uncommonly, but exclusively, observed in non-serrated adenomas (1.4%)[30]. Because some HPs do share molecular features with TSAs (e.g., KRAS mutation)[3,25,26,31], it has been suggested that the TSA pathway (HP-TSA-carcinoma sequence) may diverge from the SSA pathway (HP-SSA-SSA with cytological dysplasia-carcinoma sequence) on the basis of KRAS vs BRAF mutations and/or MLH1 vs MGMT hypermethylation within subsets of HPs[15]. However, a definite precursor of TSA has not been established. In addition, the key carcinogenic mechanism involved in this TSA pathway remains largely unknown.

Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) has been shown to be associated with an increased risk of CRC[32,33], suggesting that it may play a role in colorectal carcinogenesis. The imprinting and expression of IGF2 are controlled by CpG-rich regions known as differentially methylated regions (DMRs)[34-37]. In particular, IGF2 DMR0 hypomethylation has been suggested as a surrogate-biomarker for IGF2 LOI[38]. Previously, we reported that IGF2 DMR0 hypomethylation in CRC was associated with poor prognosis and might be linked to global DNA hypomethylation [long interspersed nucleotide element-1 (LINE-1) hypomethylation][38]. However, to date, there have been no studies describing the role of IGF2 DMR0 hypomethylation in the early stage of colorectal carcinogenesis.

To investigate the role of IGF2 DMR0 hypomethylation in serrated lesions we examined IGF2 DMR0 and LINE-1 methylation levels as well as other molecular alterations using a large sample of 1330 colorectal tumors (351 serrated lesions, 185 non-serrated adenomas, and 794 CRCs).

MATERIALS AND METHODS
Histopathological evaluation of tissue specimens of colorectal serrated lesions

Histological findings related to all colorectal serrated lesion specimens were evaluated by a pathologist (Fujita M) who was blinded to the clinical and molecular information. Serrated lesions (HPs, SSAs, and TSAs) were classified on the basis of the current WHO criteria[24]. HPs were further subdivided into microvesicular HPs and goblet cell HPs.

SSAs are characterized by the presence of a disorganized and distorted crypt growth pattern that is usually easily identifiable upon low-power microscopic examination. Crypts, particularly at the basal portion of the polyp, may appear architecturally distorted, dilated, and/or branched, particularly in the horizontal plane, which leads to the formation of boot, L, or anchor-shaped crypts. The cytology is typically quite bland, but a minor degree of nuclear atypia is allowable, particularly in the crypt bases[15,25,26].

To accurately analyze the association between the histological types and molecular features of each type of serrated lesion we consecutively collected more than 100 formalin-fixed paraffin-embedded (FFPE) tissue specimens of each histological type (HP, SSA, and TSA). In total, 364 tissue specimens of serrated lesions [121 HPs, 122 SSAs, 10 SSAs with cytological dysplasia, 96 TSAs, and 15 TSAs with high-grade dysplasia (HGD)] from patients who underwent endoscopic resection or other surgical treatment at Sapporo Medical University Hospital, Keiyukai Sapporo Hospital or Teine-Keijinkai Hospital between 2001 and 2012 were available for assessment. All of HPs were microvesicular HPs.

The serrated lesions were classified by location: the proximal colon (cecum, ascending and transverse colon), distal colon (splenic flexure, descending, sigmoid colon) and rectum. Informed consent was obtained from all the patients before specimen collection. This study was approved by the institutional review boards of the participating institutions. The term “prognostic marker” is used throughout this article according to the REMARK Guidelines[39].

Tissue specimens of CRC and non-serrated adenomas

FFPE tissue specimens of 827 CRCs (stages I-IV), 85 non-serrated adenomas (i.e., tubular or tubulovillous adenomas), and 110 non-serrated adenomas with HGD from patients who underwent surgical treatment or endoscopic resection at the above hospitals were also collected. The criterion for diagnosing cancer was invasion of malignant cells beyond the muscularis mucosa.

DNA extraction and pyrosequencing for KRAS, BRAF, and PIK3CA and MSI analysis

Genomic DNA was extracted from the FFPE tissue specimens of the colorectal tumors using a QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, United States). PCR and targeted pyrosequencing were then performed using the extracted genomic DNA to determine the presence of KRAS (codons 12 and 13), BRAF (V600E) and PIK3CA (exons 9 and 20) mutations[40,41]. MSI analysis was performed as previously described using 10 microsatellite markers[14]. MSI-high was defined as instability in ≥ 30% of the markers and MSI-low/microsatellite stable (MSS) as instability in < 30% of the markers[14].

Sodium bisulfite treatment and pyrosequencing to measure IGF2 DMR0 and LINE-1 methylation levels

Bisulfite modification of genomic DNA was performed using a BisulFlash™ DNA Modification Kit (Epigentek, Brooklyn, NY, United States).

We measured the relative methylation level at the IGF2 DMR0 using a bisulfite-pyrosequencing assay as previously described[38]. The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as percentage (scale 0%-100%). We calculated the average of the first and second CpG sites in the IGF2 DMR0 as the IGF2 DMR0 methylation level. Likewise, to accurately quantify the LINE-1 methylation levels we utilized a pyrosequencing assay, as previously described[42].

Pyrosequencing to measure MGMT and MLH1 promoter methylation

Pyrosequencing for MGMT and MLH1 methylation was performed using the PyroMark kit (Qiagen). We used a previously defined cut-off of ≥ 8% methylated alleles for MGMT and MLH1 hypermethylated tumors[43].

Immunohistochemistry for IGF2 expression

For IGF2 staining, we used anti-IGF2 antibody (Rabbit polyclonal to IGF2; Abcam, Cambridge, MA, United States) with a subsequent reaction performed using Target Retrieval Solution, Citrate pH 6 (Dako Cytomation, Carpinteria, CA, United States). In each case, we recorded cytoplasmic IGF2 expression as no expression, weak expression, moderate expression, or strong expression relative to normal colorectal epithelial cells. IGF2 expression was visually interpreted by Nosho K, who was unaware of the other data. For the agreement study of IGF2 expression, 128 randomly selected cases were examined by a second pathologist (by Naito T), who was also unaware of the other data. The concordance between the two pathologists (P < 0.0001) was 0.84 (κ = 0.69), indicating substantial agreement.

Statistical analysis

JMP (version 10) software was used for all statistical analyses (SAS Institute, Cary, NC, United States). All P values were two-sided. Univariate analyses were performed to investigate the clinicopathological and molecular characteristics including IGF2 DMR0 and LINE-1 hypomethylation, according to histological type, classified as serrated lesion, non-serrated adenoma, and CRC. P values were calculated by analysis of variance for age, tumor size, and the methylation levels of IGF2 DMR0 and LINE-1 and by χ2 or Fisher’s exact test for all other variables. A multivariate logistic regression analysis was employed to examine associations with IGF2 DMR0 hypomethylation (as an outcome variable), adjusting for potential confounders. The model initially included sex, age, tumor size, tumor location, histological type, and the LINE-1 methylation level, and MSI, BRAF, KRAS, and PIK3CA mutations. In the CRC-specific survival analysis, the Kaplan-Meier method and log-rank test were used to assess the survival time distribution. The Spearman correlation coefficient was used to assess the correlation of the IGF2 DMR0 methylation level and IGF2 expression.

RESULTS
The IGF2 DMR0 methylation level in serrated lesion and non-serrated adenomas

We assessed 559 FFPE tissue specimens of serrated lesions and non-serrated adenomas in the IGF2 DMR0 methylation assay and obtained 536 (96%) valid results. The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas (with or without HGD) was as follows: mean 61.7, median 62.5, SD 18.0, range 5.0-99.0, interquartile range 49.5-74.4 (all on a 0-100 scale) (Figure 1). The IGF2 DMR0 methylation level was divided into quartiles (Q1 ≥ 74.5, Q2 62.6-74.4, Q3 49.6-62.5, Q4 ≤ 49.5) for further analysis.

Figure 1
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Figure 1 Distribution of IGF2 differentially methylated region 0 methylation levels in 351 serrated lesions. Hyperplastic polyp, sessile serrated adenoma (SSA), SSA with cytological dysplasia, traditional serrated adenoma (TSA) and TSA with high-grade dysplasia (HGD) and 185 non-serrated adenomas (tubular adenoma, tubular adenoma with HGD, tubulovillous adenoma and tubulovillous adenoma with HGD). DMR: Differentially methylated region; IGF2: Insulin-like growth factor 2.

We evaluated the IGF2 DMR0 methylation level in serrated lesions (HP, SSA, and TSA) and non-serrated adenomas according to their histological type. The IGF2 DMR0 methylation levels of SSAs (n = 120, mean ± SD, 73.1 ± 12.3) were significantly higher than those of HPs (n = 115, 61.9 ± 20.5, P < 0.0001), TSAs (n = 91, 61.6 ± 19.6, P < 0.0001), and non-serrated adenomas (n = 80, 59.0 ± 15.8, P < 0.0001) (Figure 2).

Figure 2
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Figure 2 IGF2 differentially methylated region 0 methylation level according to histological type. Insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 methylation levels of sessile serrated adenoma (mean ± SD; 73.1 ± 12.3) were significantly higher compared with those of hyperplastic polyp (61.9 ± 20.5, P < 0.0001), traditional serrated adenoma (61.6 ± 19.6, P < 0.0001), and non-serrated adenoma (59.0 ± 15.8, P < 0.0001). P-values were calculated by analysis of variance.

IGF2 DMR0 hypomethylation was associated with larger tumor size in serrated lesions and non-serrated adenomas (Table 1). With regard to the histological type, IGF2 DMR0 hypomethylation was less frequently detected in SSAs than in HPs, TSAs, and non-serrated adenomas (P < 0.0001) (Table 1). Multivariate logistic regression analysis also showed the IGF2 DMR0 hypomethylation was inversely associated with SSAs (P < 0.0001).

Table 1 IGF2 differentially methylated region 0 hypomethylation in serrated lesions and non-serrated adenomas n (%).
Clinicopathological featureTotal nIGF2 DMR0 methylation (quartile)
P value
Q1 (≥74.5)Q2 (62.6-74.4)Q3 (49.6-62.5)Q4 ( ≤49.5)
All cases536134130131141
Sex
Male326 (61)78 (58)80 (62)92 (70)76 (54)0.041
Female210 (39)56 (42)50 (38)39 (30)65 (46)
Age (mean ± SD)61.5 ± 12.259.9 ± 12.360.8 ± 12.063.1 ± 11.662.3 ± 13.00.150
Tumor size (mm) (mean ± SD)14.3 ± 11.49.9 ± 4.013.4 ± 7.414.7 ± 11.119.1 ± 17.6< 0.0001
Tumor location
Rectum70 (13)11 (8.5)14 (11)18 (14)27 (20)0.061
Distal colon161 (31)35 (27)43 (33)37 (29)46 (33)
Proximal colon296 (56)84 (65)72 (56)75 (58)65 (47)
Histological type
Hyperplastic polyp (HP)115 (21)33 (25)25 (19)23 (18)34 (24)< 0.0001
Sessile serrated adenoma (SSA) without cytological dysplasia120 (22)60 (45)39 (30)15 (11)6 (4.3)
SSA with cytological dysplasia10 (1.9)1 (0.8)3 (2.3)6 (4.6)0 (0)
Traditional serrated adenoma (TSA) without high-grade dysplasia (HGD)91 (17)22 (16)21 (16)23 (18)25 (18)
TSA with HGD15 (2.8)2 (1.5)2 (1.5)2 (1.5)9 (6.4)
Non-serrated adenoma (tubular or tubulovillous adenoma) without HGD80 (15)11 (8.2)17 (13)32 (24)20 (14)
Non-serrated adenoma with HGD105 (20)5 (3.7)23 (18)30 (23)47 (33)
Percentage indicates the proportion of patients of each histological type who met the criteria for a specific clinical or molecular feature. P values were calculated by analysis of variance for age and tumor size and by χ2 or Fisher’s exact test for all other variables. The P value for significance was adjusted by Bonferroni correction to 0.010 (= 0.05/5).
Association of IGF2 expression and IGF2 DMR0 methylation level in serrated lesions and non-serrated adenomas

We examined IGF2 overexpression in 168 colorectal serrated lesions and non-serrated adenomas. The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level (r = -0.21, P = 0.0051).

IGF2 DMR0 methylation level in colorectal cancer

A total of 827 paraffin-embedded CRCs (stages I-IV) were subjected to an IGF2 DMR0 methylation assay with 794 (96%) valid results. The distribution of the IGF2 DMR0 methylation level in these 794 CRCs was as follows: mean 54.7, median 55.0, SD 13.7, range 7.5-98.0, interquartile range 46.1-63.0 (all on a 0-100 scale). The IGF2 DMR0 methylation level was divided into quartiles (Q1 ≥ 63.0, Q2 55.0-62.9, Q3 46.1-54.9, Q4 ≤ 46.0) for further analysis.

Colorectal cancer patient survival and IGF2 DMR0 methylation level

The influence of the IGF2 DMR0 methylation level on clinical outcome was assessed in CRC patients. During the follow-up of 398 patients with metastatic CRC (stages III-IV) who were eligible for survival analysis, mortality occurred in 134, including 118 deaths confirmed to be attributable to CRC. The median follow-up period for censored patients was 3.3 years. Kaplan-Meier analysis was performed using categorical variables (Q1, Q2, Q3, and Q4). Slightly but insignificantly higher mortality was observed in patients with IGF2 DMR0 hypomethylation compared with those without hypomethylation in terms of cancer-specific survival (log-rank test: P = 0.13) (Figure 3A). In another Kaplan-Meier analysis, Q4 cases were defined as the “hypomethylated group” and the Q1, Q2, and Q3 cases were combined into a “non-hypomethylated group”; the hypomethylated group (log-rank test: P = 0.038) was found to have significantly higher mortality (Figure 3B). Similar results were observed in terms of overall survival (log-rank test: P = 0.040) (data not shown).

Figure 3
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Figure 3 Kaplan-Meier survival curves for colorectal cancer according to the IGF2 differentially methylated region 0 and long interspersed nucleotide element-1 methylation levels in metastatic colorectal cancers. A: Patients with Insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation had a slightly higher mortality rate than those with IGF2 DMR0 hypermethylation, but this difference was not significant (log-rank test: P = 0.13); B: IGF2 DMR0 hypomethylation (Q4 cases) was significantly associated with unfavorable cancer-specific survival (log-rank test: P = 0.038); C: Significantly higher mortality was observed in patients with long interspersed nucleotide element-1 (LINE-1) hypomethylation compared with those with LINE-1 hypermethylation (log-rank test: P = 0.026); D: LINE-1 hypomethylation (Q4 cases) was significantly associated with unfavorable cancer-specific survival (log-rank test: P = 0.0037).
LINE-1 methylation level and CRC patient survival

The LINE-1 methylation level in CRC was also divided into quartiles (Q1 ≥ 58.7, Q2 54.8-58.6, Q3 50.8-54.7, and Q4 ≤ 50.7). A significantly higher mortality rate was observed among Q4 cases (log-rank test: P = 0.0037) in the Kaplan-Meier analysis (Figure 3C, D).

Association of histological type and IGF2 DMR0 and LINE-1 methylation levels as well as other molecular features of serrated lesions and non-serrated adenomas

Table 2 shows the clinicopathological and molecular features of serrated lesions and non-serrated adenomas. No significant difference was observed between SSAs (69.0 ± 10.8) with cytological dysplasia and SSAs without (73.1 ± 12.3) in IGF2 DMR0 methylation levels (P = 0.32). In contrast, MSI-high was more frequently (P < 0.0001) found in SSAs with cytological dysplasia [40% (4/10)] than in SSAs [0.8% (1/120)]. With regard to the LINE-1 methylation level, no significant difference was observed between the methylation level and histological type in serrated lesions and non-serrated adenomas (P = 0.59).

Table 2 Clinical and molecular features of serrated lesions and non-serrated adenomas (tubular or tubulovillous adenoma) according to histological type n (%).
Clinical or molecular featureTotal nHistological type
P value
Serrated lesion
Non-serrated adenoma
HPSSA without cytological dysplasiaSSA with cytological dysplasiaTSA without high-grade dysplasia (HGD)Tubular adenoma without HGDTubulovillous adenoma without HGD
All cases4161151201091773
Sex
Male263 (63)78 (68)72 (60)5 (50)55 (60)50 (65)3 (100)0.36
Female153 (37)37 (32)48 (40)5 (50)36 (40)27 (35)0 (0)
Age (mean ± SD)60.3 ± 11.857.5 ± 12.157.2 ± 11.674.1 ± 4.760.9 ± 12.366.6 ± 11.466.0 ± 8.9< 0.0001
Tumor size (mm)10.5 ± 5.49.3 ± 3.711.6 ± 5.412.3 ± 6.49.7 ± 4.710.9 ± 7.215.7 ± 13.20.0069
(mean ± SD)
Tumor location
Rectum42 (10)15 (13)0 (0)0 (0)16 (18)10 (14)1 (33)< 0.0001
Distal colon127 (31)44 (39)17 (14)1 (10)39 (44)25 (34)1 (33)
Proximal colon239 (59)54 (48)103 (86)9 (90)34 (38)38 (52)1 (33)
BRAF mutation
Wild-type183 (44)59 (51)16 (13)2 (20)28 (31)75 (97)3 (100)< 0.0001
Mutant231 (55)56 (49)104 (87)8 (80)61 (69)2 (2.6)0 (0)
KRAS mutation
Wild-type357 (87)92 (81)117 (98)10 (100)74 (83)62 (81)2 (67)< 0.0001
Mutant55 (13)21 (19)3 (2.5)0 (0)15 (17)15 (19)1 (33)
PIK3CA mutation
Wild-type406 (99)113 (99)117 (100)10 (100)89 (100)74 (99)3 (100)0.67
Mutant2 (0.5)1 (0.9)0 (0)0 (0)0 (0)1 (1.3)0 (0)
MSI status
MSS/MSI-low408 (98)113 (98)119 (99)6 (60)90 (99)77 (100)3 (100)0.0004
MSI-high8 (1.9)2 (1.7)1 (0.8)4 (40)1 (1.1)0 (0)0 (0)
IGF2 DMR064.5 ± 17.261.9 ± 20.573.1 ± 12.369.0 ± 10.861.6 ± 19.658.9 ± 16.161.0 ± 7.1< 0.0001
methylation level (mean ± SD)
LINE-158.7 ± 5.058.6 ± 3.458.1 ± 5.458.3 ± 8.458.8 ± 4.759.4 ± 6.060.9 ± 1.40.59
methylation level (mean ± SD)
Percentage indicates the proportion of patients of each histological type who met the criteria for a specific clinical or molecular feature. P values were calculated by analysis of variance for age, tumor size, methylation levels of IGF2 DMR0 and LINE-1 and by χ2 or Fisher’s exact test for all other variables. The P value for significance was adjusted by Bonferroni correction to 0.0050 (= 0.05/10). HGD: High-grade dysplasia; HP: Hyperplastic polyp; MSI: Microsatellite instability; MSS: Microsatellite stable; SSA: Sessile serrated adenoma; TSA: Traditional serrated adenoma; IGF2: Insulin-like growth factor 2.

Mutations of BRAF, KRAS, and PIK3CA were detected in 49%, 19%, and 0.9% of HPs, 87%, 2.5%, and 0% of SSAs, 69%, 17%, and 0% of TSAs and 2.6%, 19%, and 1.3% of non-serrated adenomas, respectively (Table 2).

IGF2 DMR0 and LINE-1 hypomethylation in TSAs and non-serrated adenomas with high-grade dysplasia

Tables 3 and 4 show the clinicopathological and molecular features of the TSAs (with or without HGD), non-serrated adenomas (with or without HGD), and CRCs (stages I-IV). The IGF2 DMR0 methylation levels in TSAs with HGD (50.2 ± 18.7) were significantly lower than those in TSAs without (61.6 ± 19.6, P = 0.038) (Table 3). With regard to LINE-1, the methylation levels in TSAs with HGD (55.7 ± 5.4) were significantly lower than those in TSAs without (58.8 ± 4.7) (P = 0.024).

Table 3 Clinical and molecular features of sessile serrated adenomas with cytological dysplasia, traditional serrated adenomas, non-serrated adenomas (tubular or tubulovillous adenoma), and colorectal carcinomas according to disease stage n (%).
Clinical or molecular featureHistological type
P value
SSA with cytological dysplasiaColorectal adenoma
Colorectal carcinoma
TSA without HGDTSA with HGDNon-serrated adenoma without HGDNon-serrated adenoma with HGDStage IStage IIStage IIIStage IV
All cases10911580105171217292114
Sex
Male5 (50)55 (60)9 (60)53 (66)54 (51)107 (63)123 (57)168 (58)73 (64)0.50
Female5 (50)36 (40)6 (40)27 (34)51 (49)64 (37)94 (43)124 (42)41 (36)
Age (mean ± SD)74.1 ± 4.760.9 ± 12.362.7 ± 13.666.6 ± 11.266.3 ± 10.565.1 ± 11.067.4 ± 11.566.6 ± 12.563.4 ± 9.50.0016
Tumor size (mm)12.3 ± 6.49.7 ± 4.712.8 ± 4.311.0 ± 7.429.3 ± 17.326.3 ± 15.853.1 ± 23.550.5 ± 22.750.9 ± 19.6< 0.0001
(mean ± SD)
Tumor location
Rectum0 (0)16 (18)5 (33)11 (14)23 (22)65 (38)73 (34)135 (46)37 (33)< 0.0001
Distal colon1 (10)39 (44)7 (47)26 (34)27 (26)44 (25)64 (29)59 (20)42 (37)
Proximal colon9 (90)34 (38)3 (20)39 (51)54 (52)62 (36)80 (37)98 (34)34 (30)
BRAF mutation
Wild-type2 (20)28 (31)7 (47)78 (98)102 (98)161 (95)204 (94)282 (97)103 (95)< 0.0001
Mutant8 (80)61 (69)8 (53)2 (2.5)2 (1.9)9 (5.3)13 (6.0)9 (3.0)6 (5.5)
KRAS mutation
Wild-type10 (100)74 (83)11 (73)64 (80)48 (46)108 (64)145 (69)202 (70)84 (74)< 0.0001
Mutant0 (0)15 (17)4 (27)16 (20)57 (54)62 (36)66 (31)88 (30)29 (26)
PIK3CA mutation
Wild-type10 (100)89 (100)14 (93)77 (99)99 (94)161 (94)194 (89)249 (85)103 (90)< 0.0001
Mutant0 (0)0 (0)1 (6.7)1 (1.3)6 (5.7)10 (5.9)23 (11)43 (15)11 (9.7)
MSI status
MSS/MSI-low6 (60)90 (99)15 (100)80 (100)105 (100)163 (95)198 (91)276 (95)110 (96)< 0.0001
MSI-high4 (40)1 (1.1)0 (0)0 (0)0 (0)8 (4.7)19 (8.8)16 (5.5)4 (3.5)
IGF2 DMR069.0 ± 10.861.6 ± 19.650.2 ± 18.759.0 ± 15.852.0 ± 13.655.7 ± 15.853.4 ± 13.355.5 ± 12.953.1 ± 12.9< 0.0001
methylation level (mean ± SD)
LINE-158.3 ± 8.458.8 ± 4.755.7 ± 5.459.5 ± 5.956.9 ± 5.555.8 ± 7.253.1 ± 6.255.1 ± 6.554.1 ± 7.6< 0.0001
methylation level (mean ± SD)
Percentage indicates the proportion of patients of each histological type who met the criteria for a specific clinical or molecular feature. P values were calculated by analysis of variance for age, tumor size, methylation levels of IGF2 DMR0 and LINE-1 and by χ2 or Fisher’s exact test for all other variables. The P value for significance was adjusted by Bonferroni correction to 0.0050 (= 0.05/10). HGD: High-grade dysplasia; HP: Hyperplastic polyp; MSI: Microsatellite instability; MSS: Microsatellite stable; SSA: Sessile serrated adenoma; TSA: Traditional serrated adenoma; IGF2: Insulin-like growth factor 2.
Table 4 Clinicopathological and molecular features of fifteen traditional serrated adenomas with high-grade dysplasia.
No.Age/sexLocationSize (mm)KRAS mutationBRAF mutationPIK3CA mutationMGMT methylationMLH1 methylationMSI statusLINE-1 methylation levelIGF2 DMR0 methylation levelIGF2 expression
175/MRectum8c.35G>A (p.G12D)WildWild(-)(-)MSS/MSI-low5870Weak
254/FSigmoid colon20c.35G>A (p.G12D)WildWild(+)(-)MSS/MSI-low53.739.5Strong
362/FTransverse colon15c.38G>A (p.G13D)WildWild(+)(-)MSS/MSI-low53.372.0No expression
484/MRectum5c.35G>A (p.G12D)WildWild(-)(-)MSS/MSI-low65.026.5Moderate
585/MSigmoid colon12Wildc.1799T>A (p.V600E)Wild(-)(-)MSS/MSI-low58.045.5Strong
648/MSigmoid colon20Wildc.1799T>A (p.V600E)Wild(-)(-)MSS/MSI-low53.740.5Moderate
769/MSigmoid colon10Wildc.1799T>A (p.V600E)Wild(-)(-)MSS/MSI-low58.752.0No expression
860/MDescending colon9Wildc.1799T>A (p.V600E)Wild(-)(-)MSS/MSI-low59.041.5Moderate
934/MSigmoid colon18Wildc.1799T>A (p.V600E)Wild(+)(-)MSS/MSI-low57.342.0Strong
1061/MRectum10Wildc.1799T>A (p.V600E)Wild(-)(-)MSS/MSI-low56.729.0Strong
1152/FAscending colon15Wildc.1799T>A (p.V600E)Wild(+)(+)MSS/MSI-low57.057.0Moderate
1270/FRectum13Wildc.1799T>A (p.V600E)Wild(+)(+)MSS/MSI-low63.084.5Weak
1366/FAscending colon12WildWildc.1624G>A (p.E542K)(-)(-)MSS/MSI-low49.728.0Moderate
1452/MSigmoid colon12WildWildWild(-)(-)MSS/MSI-low48.044.5Moderate
1569/FRectum13WildWildWild(+)(-)MSS/MSI-low44.380.0Weak
HGD: High-grade dysplasia; MSI: Microsatellite instability; MSS: Microsatellite stable; TSA: Traditional serrated adenoma.

Similarly, the methylation levels of IGF2 DMR0 (52.0 ± 13.6) and LINE-1 (56.9 ± 5.5) in non-serrated adenomas with HGD were significantly lower than those in non-serrated adenomas without (59.0 ± 15.8, P = 0.0016 and 59.5 ± 5.9, P = 0.0027, respectively) (Table 3).

DISCUSSION

In this study, we examined the IGF2 DMR0 and LINE-1 methylation levels as well as other molecular alterations in 351 serrated lesions, 185 non-serrated adenomas, and 794 CRCs. IGF2 DMR0 hypomethylation was less frequently detected in SSAs than in HPs, TSAs, and non-serrated adenomas. We also found that IGF2 DMR0 and LINE-1 hypomethylation in TSAs and non-serrated adenomas with HGD were more frequently detected in TSAs and non-serrated adenomas without HGD, suggesting that hypomethylation may play an important role in the progression of these tumors.

In the current study, we confirmed that IGF2 DMR0 hypomethylation was associated with poor CRC prognosis, suggesting its oncogenic role and malignant potential. In addition, our data showed that the IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level. Therefore, our findings support the validity of the quantitative DNA methylation assay (bisulfite-pyrosequencing) for examining the IGF2 DMR0 methylation level.

HPs are classified into three subtypes, namely microvesicular HPs, goblet cell HPs, and mucin-poor HPs. Microvesicular and goblet cell HPs are the most common, whereas mucin-poor HPs are rare[44]. Recent studies have reported that microvesicular HPs may be a precursor lesion of SSAs and that borderline lesions between microvesicular HPs and SSAs can occur[25,26,28]. In the current study, we found that the IGF2 DMR0 methylation levels of SSAs were significantly higher compared with those of HPs (microvesicular HPs), TSAs, and non-serrated adenomas. Our data also showed that IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas.

Our current study had some limitations due to its cross-sectional nature and the fact that unknown bias (i.e., selection bias) may confound the results. Nevertheless, our multivariate regression analysis was adjusted for potential confounders including age, tumor size, tumor location, LINE-1 methylation level, and BRAF and KRAS mutation. The results demonstrate that IGF2 DMR0 hypomethylation is inversely associated with SSAs. Moreover, our data have shown that the IGF2 DMR0 methylation levels of SSAs with cytological dysplasia were higher than those of HPs, suggesting that HPs (microvesicular HPs) or SSAs with IGF2 DMR0 hypomethylation may tend not to progress to the typical SSA pathway [HP-SSA-SSA with cytological dysplasia-carcinoma (MSI-high) sequence] but to the alternate pathway. Thus, our finding of differential patterns of IGF2 DMR0 hypomethylation in serrated lesions may be a clue for elucidating the differentiation of serrated lesions.

In the current study, IGF2 DMR0 hypomethylation was found in TSAs and hypomethylation was more frequently detected in TSAs with HGD when compared with TSAs without HGD. These results may imply that IGF2 DMR0 hypomethylation can occur in the early stage of the TSA pathway and that TSAs with IGF2 DMR0 hypomethylation are precursor lesions that progress to TSAs with HGD or CRCs with hypomethylation. In other words, TSAs without IGF2 DMR0 hypomethylation may tend not to progress to TSAs with HGD. Otherwise, TSAs without IGF2 DMR0 hypomethylation may tend to rapidly develop to CRCs; therefore, they are infrequently detected in the stage of TSA with HGD. However, because the number of TSA with HGD samples was small (n = 15), our findings require further confirmation from future independent studies.

Global DNA hypomethylation is associated with genomic instability, which leads to cancer[45-50]. As the LINE-1 or L1 retrotransposon constitutes a substantial portion (ca. 17%) of the human genome, the level of LINE-1 methylation is regarded as a surrogate marker of global DNA methylation[46,51]. We previously reported that LINE-1 methylation is highly variable but is strongly associated with a poor prognosis in CRC[45]. However, no previous study has reported the role of LINE-1 hypomethylation in serrated lesions. In serrated lesions, unlike the IGF2 DMR0 methylation level, no significant difference was observed between the LINE-1 methylation level and histological type. We also found that the LINE-1 methylation levels in TSAs with HGD were significantly lower than those in TSAs. These results suggest that both IGF2 DMR0 hypomethylation and LINE-1 hypomethylation are important epigenetic alterations in the progression of TSAs. Because the carcinogenic mechanism remains unclear, further analyses are needed to clarify the role in the TSA pathway of the hypomethylation of these locations.

Previous studies have reported that SSAs with cytological dysplasia have accumulated genetic abnormalities and are at a high risk of progression to colorectal carcinoma[7,26,28]. Loss of staining for MLH1 leads to MSI and repeat tract mutation in genes such as TGFβRII is restricted to lesions with cytological dysplasia in SSAs[26,27,52,53]. In the current study, MSI-high was more frequently detected in SSAs with cytological dysplasia than in SSAs without. Our data indicate that in SSAs with cytological dysplasia, it is not hypomethylation of IGF2 DMR0 or LINE-1 but rather MSI due to MLH1 hypermethylation that plays an important role in the evolution to colorectal carcinoma.

The RAS-RAF-MEK-ERK signaling pathway is commonly altered in CRC and serrated lesions through oncogenic mutation of either BRAF or KRAS[15,21,25]. Moreover, CRCs with serrated morphology are particularly prone to mutations targeted by anti-epidermal growth factor receptor therapy. Therefore, as the variety of molecularly targeted agents for CRC increases, understanding of molecular alterations is becoming increasingly important[21,40]. BRAF and KRAS mutations are mutually exclusive and demonstrate a subtype specificity in serrated lesions[10,15,17-19,28]; they are most likely initiating events in the majority of HPs[54]. Previous studies have reported that BRAF is mutated with increasing frequency in SSAs (60%-100%)[3-5,9,11,16]. In the current study, BRAF mutations were detected in 49% of HPs and 87% of SSAs, respectively. Therefore, our data relating to the frequency of BRAF mutations in SSAs are consistent with previous reports. The activation of the RAS-RAF-MEK-ERK signaling pathway by BRAF or KRAS mutation is also common in TSAs. Previous studies have reported BRAF mutation rates in TSAs ranging from 27% to 55%[6,8,16,55], compared to KRAS mutation rates of 29%-46%[6,8]. In the current study, BRAF and KRAS mutations were detected in 69% and 17% of TSAs, respectively. Thus, the wide variation in the relative proportion of BRAF vs KRAS mutations in different studies reflects differences in histological classification or small sample size.

In conclusion, we found that IGF2 DMR0 hypomethylation can occur in the early stage of any histological types of serrated lesions; however, hypomethylation may be an infrequent epigenetic alteration in SSAs. These results imply that IGF2 DMR0 hypomethylation may be a key epigenetic event that affects the progression of HPs. Our data also suggest that the hypomethylation of IGF2 DMR0 and LINE-1 may play an important role in the progression of the TSA pathway.

COMMENTS
Background

The serrated pathway attracts considerable attention as an alternative colorectal cancer (CRC) pathway. Authors previously reported the association of insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation with poor prognosis and its link to global DNA hypomethylation [long interspersed nucleotide element-1 (LINE-1) hypomethylation] in CRC; however, to date, there have been no studies describing its role in the serrated pathway.

Research frontiers

Sessile serrated adenoma (SSA) and traditional serrated adenoma (TSA) are premalignant lesions, but SSA is the principal serrated precursor of CRC. In particular, there are many clinicopathological and molecular similarities between SSA and microsatellite instability (MSI)-high CRC, for example, right-sided predilection, MLH1 hypermethylation, and frequent BRAF mutation. Therefore, SSAs are hypothesized to develop in some cases to MSI-high CRCs with BRAF mutation in the proximal colon. In contrast, a definite precursor of TSA has not been established. In addition, the key carcinogenic mechanism involved in this TSA pathway remains largely unknown. To investigate the role of IGF2 DMR0 hypomethylation in serrated lesions they examined IGF2 DMR0 methylation levels as well as other molecular alterations.

Innovations and breakthroughs

This is the first report of an association between histopathological findings and IGF2 DMR0 hypomethylation in serrated lesions. IGF2 DMR0 hypomethylation was less frequently detected in SSAs than in hyperplastic polyps (HPs), TSAs, and non-serrated adenomas. They also found that IGF2 DMR0 and LINE-1 hypomethylations in TSAs and non-serrated adenomas with high-grade dysplasia were more frequently detected in TSAs and non-serrated adenomas, suggesting that such hypomethylation may play an important role in the progression of those tumors. Thus, their finding of differential patterns of IGF2 DMR0 hypomethylation in serrated lesions may be a clue for elucidating the progression of serrated lesions.

Applications

In the current study, authors found that the IGF2 DMR0 methylation levels of SSAs were significantly higher compared with those of HPs (microvesicular HPs), TSAs, and non-serrated adenomas. They also showed that IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas. Therefore, their data challenge the common conception of discrete molecular features of SSAs vs other serrated lesions (TSAs and HPs) and may have a substantial impact on clinical and translational research, which has typically been performed with the dichotomous classification of SSAs.

Terminology

IGF2 DMR: IGF2 expression is controlled by CpG-rich regions known as IGF2 DMRs in CRC. In particular, IGF2 DMR0 hypomethylation has been suggested as a surrogate-biomarker for IGF2 loss of imprinting. LINE-1: Global DNA hypomethylation is associated with genomic instability, which leads to cancer. As the long interspersed nucleotide element-1 or L1 retrotransposon constitutes a substantial portion of the human genome, the level of LINE-1 methylation is regarded as a surrogate marker of global DNA methylation. Serrated pathway: The serrated neoplasia pathway has attracted considerable attention as an alternative pathway of CRC development, and serrated lesions exhibit unique clinicopathological or molecular features. Of the serrated lesions, SSAs are hypothesized to develop in some cases to MSI-high CRCs with BRAF mutation in the proximal colon.

Peer review

The authors investigated the hypomethylations of IGF2 DMR0 and LINE-1; MSI; and mutations of KRAS, BRAF, and PIK3CA in patients with serrated lesions and non-serrated adenomas. The results demonstrated that IGF2 DMR0 hypomethylation can occur in the early stage of any histological types of serrated lesions; however, hypomethylation may be an infrequent epigenetic alteration in SSAs. The authors also revealed that the hypomethylation of IGF2 DMR0 and LINE-1 may play an important role in the progression of the TSA pathway. This article may have a substantial impact on clinical and translational research in the progression of serrated lesions related to malignant transformation.

Footnotes

P- Reviewer: Pescatori M, Yoshimatsu K S- Editor: Gou SX L- Editor: A E- Editor: Ma S

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