LINE-1 hypomethylation is neither present in rectal aberrant crypt foci nor associated with field defect in sporadic colorectal neoplasia
© Quintanilla et al.; licensee BioMed Central Ltd. 2014
Received: 14 August 2014
Accepted: 17 October 2014
Published: 10 November 2014
Aberrant crypt foci (ACF) are considered the first identifiable preneoplastic lesion in colorectal cancer (CRC), and have been proposed as a potential biomarker for CRC risk. Global DNA hypomethylation is an early event in colorectal carcinogenesis, and long interspersed nuclear element-1 (LINE-1) methylation status is a well-known surrogate marker for genome-wide DNA methylation levels. Despite the gradual increase in DNA hypomethylation in the adenoma–carcinoma sequence, LINE-1 methylation in ACF has never been studied. Moreover, recent studies have reported a field defect for LINE-1 hypomethylation, suggesting that LINE-1 methylation status in normal mucosa could be used to stratify CRC risk and tailor preventive strategies. Thus, we assessed LINE-1 status by pyrosequencing in rectal ACF and paired normal colorectal mucosa from individuals with sporadic colon cancer (CC) (n = 35) or adenoma (n = 42), and from healthy controls (n = 70).
Compared with normal mucosa, LINE-1 in ACF were hypermethylated across all groups (P < 0.0001). Furthermore, LINE-1 methylation status in normal colorectal mucosa was independent of the presence of adenoma or CC (P = 0.1072), and did not differ depending on the distance to the adenoma or CC. Interestingly, when we compared the LINE-1 methylation status in normal mucosa from different segments of the colorectum, we found higher hypomethylation in the rectum compared with the descending colon (P < 0.0001).
Overall, our results suggest that global hypomethylation is not present in rectal ACF and argues against the existence of LINE-1 methylation field defect in sporadic colon cancer.
KeywordsColorectal cancer Aberrant crypt foci LINE-1 Methylation Prevention
Aberrant crypt foci (ACF) are considered the first identifiable preneoplastic lesion in CRC. ACF show features that support their potential role as premalignant lesions, such as dysplasia, monoclonality, and mutations, which are present in adenomas and carcinomas but not in normal colon mucosa[2, 3]. High-magnification chromoscopic colonoscopy can detect ACF in vivo, and several studies using chromoendoscopy have shown a higher rate of ACF occurrence in patients with CRC and adenomas compared with healthy individuals. Accordingly, ACF (especially those located in the rectum) have been proposed as a potential biomarker for CRC risk. Histologically, ACF can be classified as hyperplastic or dysplastic. Data from single-center studies have suggested that dysplastic ACF may correlate with CRC risk, as there is an increased frequency of dysplastic ACF in individuals with a normal colon, patients with adenoma, and patients with CRC. However, other studies have shown conflicting findings regarding the clinical value of dysplastic ACF[7–9], and thus, the role of ACF in colorectal carcinogenesis is still under debate. Although rectal ACF have been suggested as potential biomarkers to identify individuals at high risk for CRC because of their specific histological and molecular features, this is still an area of uncertainty.
Methylation of cytosine residues at CpG dinucleotides is a major epigenetic modification strongly associated with transcriptional silencing. Long interspersed element-1 (LINE-1) elements are a type of genomic repetitive elements that in their full-length form encode the retrotransposon machinery. LINE-1 methylation status is a well-known surrogate marker for genome-wide DNA methylation level. In normal cells, LINE-1 is heavily methylated, whereas cancer cells show LINE-1 hypomethylation, leading to chromosomal instability. This phenomenon occurs early in the colorectal carcinogenesis[14, 15]. However, despite the gradual increase of hypomethylation in the adenoma–carcinoma sequence, LINE-1 methylation in ACF has never been studied. Moreover, recent studies have reported a field defect in LINE-1 hypomethylation[14, 17, 18], suggesting that LINE-1 methylation status in normal mucosa could be used to stratify CRC risk and tailor preventive strategies.
The aims of this study were to analyze the levels of LINE-1 methylation in rectal ACF compared with paired normal colorectal mucosa, and to investigate the putative field defect of LINE-1 hypomethylation by analyzing the normal mucosa of patients from three different CRC risk groups. The analysis was confined to rectal ACF because of their previously reported higher frequency compared with other colonic segments, in addition to their usefulness as CRC biomarkers based on the total number and histological characteristics.
Subjects and methods
The project was granted approval from institutional review board of the Hospital Clinic of Barcelona, and written informed consent was obtained from all participants.
ACF and normal mucosa biopsies were preserved at −80°C optimal cutting compound (OCT) and phosphate-buffered saline (PBS), respectively. DNA was extracted using an All Prep DNA/RNA Mini Kit (catalog number 80204; Qiagen, Hilden, Germany) following the manufacturer’s recommendations. DNA and RNA were quantified using a NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific).
Bisulfite pyrosequencing and LINE-1 methylation analysis
Bisulfite treatment was performed using the EpiTect Bisulfite kit (catalog number 59104; Qiagen) following manufacturer’s instructions. Methylation analysis of LINE-1 promoter was investigated using pyrosequencing-based methylation analysis. PCR was carried out in a 25 μl PCR mixture containing 12.5 μl of GoTaq Colorless Master Mix (catalog number M7142, Promega, WI, USA) (Reaction Buffer pH 8.5, 400 μM of each dNTP, and 3 mM MgCl2), 1 μl of Taq polymerase, 2 μl of forward primer (5′-TTTTGAGTTAGGTGTGGGATATA-3′), 2 μl of reverse biotinylated primer (5′-AAAATCAAAAAATTCCCTTTC-3′), and 100 ng of bisulfite-treated genomic DNA. PCR cycling conditions were 95°C for 15 minutes; 45 cycles of 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 45 seconds; and finally, 10 minutes at 72°C and 4°C forever. The biotinylated PCR product was purified and made single-stranded to act as a template in a pyrosequencing reaction as recommended by the manufacturer using Pyrosequencing Vacuum Tool (Qiagen, Hilden, Germany). In brief, the PCR product was bound to a Streptavidin Sepharose HP column, and the sepharose beads containing the immobilized PCR product were purified, washed, denatured using 0.2 M NaOH solution, and washed again. Then, 10 μM of pyrosequencing primer (5′AGTTAGGTGTGGGATATAGT-3′) was annealed to the purified single-stranded PCR product, and pyrosequencing was performed using a PSQ 96MA Pyrosequencing System (Qiagen). CpGenome Methylated DNA (catalog number S7821; Millipore, Darmstadt, Germany) was used as a positive control. Methylation level of LINE-1 elements was calculated as the mean percentage of the four CpG sites analyzed, as previously described.
Distribution of LINE-1 methylation levels was assessed with the Shapiro–Wilks test, which showed that the data were not normally distributed (P = 0.017). Accordingly, the Wilcoxon signed-rank test was used for comparing average methylation levels of paired ACF and normal mucosa. When comparing global methylation between two independent groups and between more than two groups, the Mann–Whitney U-test and Kruskal–Wallis test, respectively, were used. LINE-1 methylation levels are expressed as median and interquartile range (IQR). All statistical analyses were performed using SPSS software (v20; IBM Inc., Armonk, NY, USA) and results were considered significant at P < 0.05.
Baseline characteristics of individuals and aberrant crypt foci in each colon cancer risk group
Control (n = 70)
Adenoma (n = 42)
Colon cancer (n = 35)
Sex, n (%)
Age, years, mean ± SD
58.1 ± 15.3
68.6 ± 9.9
66.8 ± 8.6
Colon cancer location, n (%)
Colon cancer TNM stage, n (%)
Adenoma features (per patient)a
– Tubular, n (%)
– Tubulovillous, n (%)
– Villous, n (%)
– High-grade dysplasia, n (%)
– Adenoma size, mean (mm) ± SD
12.3 ± 9.8
8.4 ± 5
– Number of adenomas per patient, mean ± SD (range)
2.4 ± 2 (1 to 9)
1.2 ± 1.2 (1 to 4)
Advanced adenomab, n (%)
Adenoma location (per patient)c, n (%)
Serrated polyps features (per patient)d
– Recto-sigmoid hyperplastic polyps, n (%)
– Proximal serrated polyps, n (%)e
– Serrated polyps ≥10 mm, n (%)
– Serrated polyps per patient, n, mean ± SD (range)
1.6 ± 0.8 (1 to 3)
3.1 ± 3.4 (1 to 5)
– Hyperplastic polyp, n (%)
– Sessile serrated adenoma, n (%)
–Traditional serrated adenoma, n (%)
ACF for molecular study, n
Dysplastic ACF, n (%)
Hyperplastic ACF, n (%)
Rectal ACF do not display LINE-1 hypomethylation
LINE-1 methylation levels in normal colorectal mucosa are similar in all CC risk groups
LINE-1 methylation levels in normal mucosa and aberrant crypt foci for each of the three groups of subjects
Healthy controls (n = 70)
Adenoma (n = 42)
Colon cancer (n = 35)
Normal rectal mucosa, n/N
76.73% (73.23 to 78.22)
75.34% (73.7 to 77.2)
77.06% (75.04 to 79.23)
Normal descending colon mucosa, n/N
77.67% (75.84 to 80.18)
78.45% (77.37 to 79.92)
80.30% (77.19 to 81.39)
Aberrant crypt foci, n/N
79.41% (77.24 to 81.58)
80.32% (77.45 to 82.82)
81.37% (77.48 to 83.14)
Dysplastic aberrant crypt foci, n/N
80.58% (78.54 to 82.37)
80.99% (79.15 to 83.07)
82.74% (79.83 to 83.43)
Hyperplastic aberrant crypt foci, n/N
79.32% (76.94 to 81.54)
79.59% (76.80 to 82.22)
80.02% (76.50 to 82.55)
LINE-1 methylation levels in normal mucosa stratifying by location of the neoplastic lesion
Patients with proximal adenoma (n = 10)a
Patients with distal adenoma (n = 30)a
Patients with proximalacolon cancer (n = 11)
Patients with distal colon cancer (n = 24)a
Normal rectal mucosa
73.87% (72.47 to 75.90)
76.06% (74.78 to 77.33)
77.59% (72.78 to 80.12)
77.40% (75.70 to 79.79)
Normal descending mucosa
80.72% (76.62 to 82.68)
78.38% (76.55 to 80.01)
77.14% (75.59 to 80.06)
78.59% (76.90 to 79.41)
LINE-1 methylation levels in normal mucosa differ between colonic segments
Interestingly, when we compared the LINE-1 methylation status in normal mucosa from different segments of the colorectum, we found a higher degree of hypomethylation in the rectum compared with the descending colon [76.13% (73.75 to 78.06) versus 78.42% (76.51 to 80.33); P < 0.0001], suggesting different genome-wide methylation profiles between normal colon and rectal mucosa (Figure 3B). Stratification by sex did not vary the results (see Additional file2: Table S1).
Overall, our results argue against the hypothesis that global hypomethylation occurs at the earliest stages of colorectal carcinogenesis. Overexpression of β-catenin and KRAS mutations may indicate that ACF constitute a premalignant stage. Moreover, LINE-1 hypomethylation is already present at the adenoma stage. Our data suggest, for the first time, to our knowledge, that the formation of ACF is not triggered by hypomethylation in LINE-1 elements, and that genome-wide hypomethylation occurs after ACF formation, most likely during the transition from non-adenomatous to adenomatous epithelia. In fact, hypermethylation of LINE-1 elements in ACF suggests that reversal epigenetic changes occur in these lesions regardless of their pathogenic progression.
Despite the fact that two previous reports have suggested the existence of LINE-1 hypomethylation field defects in CRC, our results indicate the absence of this phenomenon in our sample set. Kamiyama and collaborators recently showed that normal colonic mucosa from patients with synchronous CRC displayed higher hypomethylation of LINE-1 compared with patients with solitary CRC and controls. However, and in line with our results, there were no differences between the two latter groups. Another study has shown that normal colorectal mucosa samples from different risk groups displayed differences in LINE-1 methylation that mirrored differences between their respective tumor specimens. A more recent study has shown that adjacent mucosa from individuals with multiple/large serrated polyps display significantly lower LINE-1 methylation levels compared with individuals without such polyps. We tested the hypothesis that LINE-1 methylation in normal mucosa could be used as a biomarker for risk of sporadic CRC, focusing on patients with conventional adenomas. By analyzing a prospective cohort of patients with CC and controls, we found that our data suggest that methylation status of LINE-1 in normal mucosa is not a suitable biomarker to predict CC risk in patients with sporadic cancer. However, the potential field defect for LINE-1 methylation in patients with serrated polyps needs further assessment. In addition, our data show different methylation levels in normal mucosa between the descending colon and the rectum, suggesting different methylation profiling depending on the colonic segment.
Our results shed some light on the role of genome-wide methylation in rectal ACF and argue against its utility as a biomarkers for assessing CC risk.
MP and FB share senior authorship.
Aberrant crypt foci
Long interspersed element-1
Normal descending mucosa
Normal rectal mucosa
This work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS) (Proyectos de Evaluación de Tecnologías Sanitarias PS09/00669); the Banc de Tumors-Biobanc Hospital Clinic-IDIBAPS and Xarxa de Bancs de Tumors de Catalunya (XBTC). The Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD) is funded by Instituto de Salud Carlos III. ML-C was a Research Fellow from the FIS (Rio Hortega contract). IQ is funded by the Ministerio de Educación (FPU2012 program). FB is funded by Instituto de Salud Carlos III (PI10/00384 and PI13/00719). MP is funded by Instituto de Salud Carlos III (PI12/01481); “Cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER). Unión Europea. Una manera de hacer Europa”. JC was awarded a grant from the Asociación Española Contra el Cáncer, and is supported by Instituto de Salud Carlos III (CP13/00160).
- Takayama T, Katsuki S, Takahashi Y, Ohi M, Nojiri S, Sakamaki S, Kato J, Kogawa K, Miyake H, Niitsu Y: Aberrant crypt foci of the colon as precursors of adenoma and cancer. N Engl J Med. 1998, 339: 1277-1284. 10.1056/NEJM199810293391803.View ArticlePubMedGoogle Scholar
- McLellan EA, Medline A, Bird RP: Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon. Carcinogenesis. 1991, 12: 2093-2098. 10.1093/carcin/12.11.2093.View ArticlePubMedGoogle Scholar
- Bird RP, Good CK: The significance of aberrant crypt foci in understanding the pathogenesis of colon cancer. Toxicol Lett. 2000, 112–113: 395-402.View ArticlePubMedGoogle Scholar
- Yokota T, Sugano K, Kondo H, Saito D, Sugihara K, Fukayama N, Ohkura H, Ochiai A, Yoshida S: Detection of aberrant crypt foci by magnifying colonoscopy. Gastrointest Endosc. 1997, 46: 61-65. 10.1016/S0016-5107(97)70212-8.View ArticlePubMedGoogle Scholar
- Gupta S, Ashfaq R, Kapur P, Afonso BB, Nguyen TP, Ansari F, Boland CR, Goel A, Rockey DC: Microsatellite instability among individuals of Hispanic origin with colorectal cancer. Cancer. 2011, 116: 4965-4972.View ArticleGoogle Scholar
- Carr NJ SL, Neiderau C: Aberrrant crypt foci. Pathology and genetics: Tumors of the digestive system. WHO Classification of Tumours Series. 2000, Lyon: IARC PressGoogle Scholar
- Adler DG, Gostout CJ, Sorbi D, Burgart LJ, Wang L, Harmsen WS: Endoscopic identification and quantification of aberrant crypt foci in the human colon. Gastrointest Endosc. 2002, 56: 657-662. 10.1016/S0016-5107(02)70113-2.View ArticlePubMedGoogle Scholar
- Pretlow TP, Pretlow TG: Mutant KRAS in aberrant crypt foci (ACF): initiation of colorectal cancer?. Biochim Biophys Acta. 2005, 1756: 83-96.PubMedGoogle Scholar
- Suehiro Y, Hinoda Y: Genetic and epigenetic changes in aberrant crypt foci and serrated polyps. Cancer Sci. 2008, 99: 1071-1076. 10.1111/j.1349-7006.2008.00784.x.View ArticlePubMedGoogle Scholar
- Mutch MG, Schoen RE, Fleshman JW, Rall CJ, Dry S, Seligson D, Charabaty A, Chia D, Umar A, Viner J, Hawk E, Pinsky PF: A multicenter study of prevalence and risk factors for aberrant crypt foci. Clin Gastroenterol Hepatol. 2009, 7: 568-574. 10.1016/j.cgh.2009.01.016.View ArticlePubMedGoogle Scholar
- Speek M: Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes. Mol Cell Biol. 2001, 21: 1973-1985. 10.1128/MCB.21.6.1973-1985.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Kazazian HH: Mobile elements: drivers of genome evolution. Science. 2004, 303: 1626-1632. 10.1126/science.1089670.View ArticlePubMedGoogle Scholar
- Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A: Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Oncogene. 2008, 27: 404-408. 10.1038/sj.onc.1210631.View ArticlePubMedGoogle Scholar
- Pavicic W, Joensuu EI, Nieminen T, Peltomaki P: LINE-1 hypomethylation in familial and sporadic cancer. J Mol Med (Berl). 2012, 90: 827-835. 10.1007/s00109-011-0854-z.View ArticleGoogle Scholar
- Sunami E, de Maat M, Vu A, Turner RR, Hoon DS: LINE-1 hypomethylation during primary colon cancer progression. PLoS One. 2011, 6: e18884-10.1371/journal.pone.0018884.PubMed CentralView ArticlePubMedGoogle Scholar
- Ibrahim AE, Arends MJ, Silva AL, Wyllie AH, Greger L, Ito Y, Vowler SL, Huang TH, Tavare S, Murrell A, Brenton JD: Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut. 2011, 60: 499-508. 10.1136/gut.2010.223602.View ArticlePubMedGoogle Scholar
- Kamiyama H, Suzuki K, Maeda T, Koizumi K, Miyaki Y, Okada S, Kawamura YJ, Samuelsson JK, Alonso S, Konishi F, Perucho M: DNA demethylation in normal colon tissue predicts predisposition to multiple cancers. Oncogene. 2012, 31: 5029-5037. 10.1038/onc.2011.652.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamada A, Minamiguchi S, Sakai Y, Horimatsu T, Muto M, Chiba T, Boland CR, Goel A: Colorectal advanced neoplasms occur through dual carcinogenesis pathways in individuals with coexisting serrated polyps. PLoS One. 2014, 9: e98059-10.1371/journal.pone.0098059.PubMed CentralView ArticlePubMedGoogle Scholar
- Antelo M, Balaguer F, Shia J, Shen Y, Hur K, Moreira L, Cuatrecasas M, Bujanda L, Giraldez MD, Takahashi M, Cabanne A, Barugel ME, Arnold M, Roca EL, Andreu M, Castellvi-Bel S, Llor X, Jover R, Castells A, Boland CR, Goel A: A high degree of LINE-1 hypomethylation is a unique feature of early-onset colorectal cancer. PLoS One. 2012, 7: e45357-10.1371/journal.pone.0045357.PubMed CentralView ArticlePubMedGoogle Scholar
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