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LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis.

Shigaki H, Baba Y, Watanabe M, Murata A, Iwagami S, Miyake K, Ishimoto T, Iwatsuki M, Baba H - Gastric Cancer (2012)

Bottom Line: Tumoral LINE-1 methylation range was 11.6-97.5 on a 0-100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9).LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09-3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02-3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04-4.04, p = 0.036].No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto, 860-8556, Japan.

ABSTRACT

Background: Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, LINE-1 methylation level is attracting interest as a predictive marker for patient prognosis. However, the prognostic significance of LINE-1 hypomethylation in gastric cancer remains unclear.

Methods: Using 203 resected gastric cancer specimens, we quantified LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical and pathological variables.

Results: Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p < 0.0001; n = 74). Tumoral LINE-1 methylation range was 11.6-97.5 on a 0-100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9). LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09-3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02-3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04-4.04, p = 0.036]. No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25).

Conclusions: LINE-1 hypomethylation in gastric cancer is associated with shorter survival, suggesting that it has potential for use as a prognostic biomarker.

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Related in: MedlinePlus

Pyrosequencing assay used to measure the long interspersed nucleotide element-1, L1 (LINE-1) methylation level. a A LINE-1 hypermethylated tumor (methylation level, 78 %). b A LINE-1 hypomethylated tumor (methylation level, 39 %). The percent (%) (blue) is the proportion of C at each CpG site after bisulfite conversion, and the methylation level of each CpG site was estimated by the proportion of C (%). The overall LINE-1 methylation level was calculated as the average of the proportions of C (%) at the 4 CpG sites. The first, third, and fourth CpG sites follow mononucleotide T repeats, resulting in higher T peaks than the second CpG site, and the proportion of C (%) has been adjusted accordingly. Arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion
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Fig1: Pyrosequencing assay used to measure the long interspersed nucleotide element-1, L1 (LINE-1) methylation level. a A LINE-1 hypermethylated tumor (methylation level, 78 %). b A LINE-1 hypomethylated tumor (methylation level, 39 %). The percent (%) (blue) is the proportion of C at each CpG site after bisulfite conversion, and the methylation level of each CpG site was estimated by the proportion of C (%). The overall LINE-1 methylation level was calculated as the average of the proportions of C (%) at the 4 CpG sites. The first, third, and fourth CpG sites follow mononucleotide T repeats, resulting in higher T peaks than the second CpG site, and the proportion of C (%) has been adjusted accordingly. Arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion

Mentions: PCR and subsequent pyrosequencing for LINE-1 were performed as previously described by Ogino et al., using the PyroMark kit (Qiagen) [14, 19, 20]. This assay amplifies a region of LINE-1 element (position 305–331 in accession no. X58075), which includes four CpG cites. The PCR conditions were 45 cycles of 95 °C for 20 s, 50 °C for 20 s, and 72 °C for 20 s, followed by 72 °C for 5 min. The biotinylated PCR product was purified and made single-stranded to act as a template in a pyrosequencing reaction, using the Pyrosequencing Vacuum Prep Tool (Qiagen). Pyrosequencing reactions were performed in the PyroMark Q24 System (Qiagen). The nucleotide dispensation order was ACT CAG TGT GTC AGT CAG TTA GTC TG. The non-CpG cytosine in LINE-1 repetitive sequences has been documented to be rarely methylated. Thus, complete conversion of cytosine at a non-CpG site ensured successful bisulfite conversion. The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as the percentage (i.e., 0–100). The average of the relative amounts of C in the 4 CpG sites was used as the overall LINE-1 methylation level in a given tumor (Fig. 1). In published literature, we have validated our LINE-1 methylation pyrosequencing assay; we have performed bisulfite conversion on five different DNA specimen aliquots and repeated PCR-pyrosequencing five times using four macro-dissected cancers. Bisulfite-to-bisulfite (between-bisulfite treatment) standard deviation (SD) ranged from 1.4 to 2.9 (median, 2.3), and run-to-run (between-PCR pyrosequencing run) SD ranged from 0.6 to 3.3 (median, 1.2) [21]. In this study, we used “LINE-1 methylation level” for LINE-1 methylation as a continuous variable and “LINE-1 hypomethylation” for LINE-1 methylation as a categorical variable (i.e., hypomethylation vs. hypermethylation).Fig. 1


LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis.

Shigaki H, Baba Y, Watanabe M, Murata A, Iwagami S, Miyake K, Ishimoto T, Iwatsuki M, Baba H - Gastric Cancer (2012)

Pyrosequencing assay used to measure the long interspersed nucleotide element-1, L1 (LINE-1) methylation level. a A LINE-1 hypermethylated tumor (methylation level, 78 %). b A LINE-1 hypomethylated tumor (methylation level, 39 %). The percent (%) (blue) is the proportion of C at each CpG site after bisulfite conversion, and the methylation level of each CpG site was estimated by the proportion of C (%). The overall LINE-1 methylation level was calculated as the average of the proportions of C (%) at the 4 CpG sites. The first, third, and fourth CpG sites follow mononucleotide T repeats, resulting in higher T peaks than the second CpG site, and the proportion of C (%) has been adjusted accordingly. Arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion
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Related In: Results  -  Collection

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Fig1: Pyrosequencing assay used to measure the long interspersed nucleotide element-1, L1 (LINE-1) methylation level. a A LINE-1 hypermethylated tumor (methylation level, 78 %). b A LINE-1 hypomethylated tumor (methylation level, 39 %). The percent (%) (blue) is the proportion of C at each CpG site after bisulfite conversion, and the methylation level of each CpG site was estimated by the proportion of C (%). The overall LINE-1 methylation level was calculated as the average of the proportions of C (%) at the 4 CpG sites. The first, third, and fourth CpG sites follow mononucleotide T repeats, resulting in higher T peaks than the second CpG site, and the proportion of C (%) has been adjusted accordingly. Arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion
Mentions: PCR and subsequent pyrosequencing for LINE-1 were performed as previously described by Ogino et al., using the PyroMark kit (Qiagen) [14, 19, 20]. This assay amplifies a region of LINE-1 element (position 305–331 in accession no. X58075), which includes four CpG cites. The PCR conditions were 45 cycles of 95 °C for 20 s, 50 °C for 20 s, and 72 °C for 20 s, followed by 72 °C for 5 min. The biotinylated PCR product was purified and made single-stranded to act as a template in a pyrosequencing reaction, using the Pyrosequencing Vacuum Prep Tool (Qiagen). Pyrosequencing reactions were performed in the PyroMark Q24 System (Qiagen). The nucleotide dispensation order was ACT CAG TGT GTC AGT CAG TTA GTC TG. The non-CpG cytosine in LINE-1 repetitive sequences has been documented to be rarely methylated. Thus, complete conversion of cytosine at a non-CpG site ensured successful bisulfite conversion. The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as the percentage (i.e., 0–100). The average of the relative amounts of C in the 4 CpG sites was used as the overall LINE-1 methylation level in a given tumor (Fig. 1). In published literature, we have validated our LINE-1 methylation pyrosequencing assay; we have performed bisulfite conversion on five different DNA specimen aliquots and repeated PCR-pyrosequencing five times using four macro-dissected cancers. Bisulfite-to-bisulfite (between-bisulfite treatment) standard deviation (SD) ranged from 1.4 to 2.9 (median, 2.3), and run-to-run (between-PCR pyrosequencing run) SD ranged from 0.6 to 3.3 (median, 1.2) [21]. In this study, we used “LINE-1 methylation level” for LINE-1 methylation as a continuous variable and “LINE-1 hypomethylation” for LINE-1 methylation as a categorical variable (i.e., hypomethylation vs. hypermethylation).Fig. 1

Bottom Line: Tumoral LINE-1 methylation range was 11.6-97.5 on a 0-100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9).LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09-3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02-3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04-4.04, p = 0.036].No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Kumamoto, Kumamoto, 860-8556, Japan.

ABSTRACT

Background: Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, LINE-1 methylation level is attracting interest as a predictive marker for patient prognosis. However, the prognostic significance of LINE-1 hypomethylation in gastric cancer remains unclear.

Methods: Using 203 resected gastric cancer specimens, we quantified LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical and pathological variables.

Results: Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p < 0.0001; n = 74). Tumoral LINE-1 methylation range was 11.6-97.5 on a 0-100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9). LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09-3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02-3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04-4.04, p = 0.036]. No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25).

Conclusions: LINE-1 hypomethylation in gastric cancer is associated with shorter survival, suggesting that it has potential for use as a prognostic biomarker.

Show MeSH
Related in: MedlinePlus