Long Interspersed Nucleotide Element-1 Hypomethylation Research Articles

Correlation of LINE-1 Hypomethylation With Size and Pathologic Extent of Dysplasia in Colorectal Tubular Adenomas.

INTRODUCTION:Conventional adenomas (tubular adenoma [TA] or tubulovillous adenoma) and sessile serrated lesions (SSLs) are neoplastic precancerous lesions frequently detected in patients undergoing average risk screening colonoscopy and polyp surveillance. Metachronous risk stratification of adenomas is currently limited to histologic features and size of polyps. We report long interspersed nucleotide element-1 (LINE-1) methylation levels in SSL in comparison to TA and the impact of TA size and presence of high-grade dysplasia (HGD) on LINE-1 methylation.METHODS:LINE-1 methylation was assessed by pyrosequencing of bisulfite-converted DNA. We compared LINE-1 methylation between TA and SSL, among varying sizes of TA, and between TA with HGD and low-grade dysplasia (LGD).RESULTS:LINE-1 methylation declined with increasing polyp size in TA when comparing those <5 mm (72.31 ± 6.11), 5 to <10 mm (67.50 ± 7.00), and ≥10 mm (66.75 ± 11.89). There were lower LINE-1 methylation levels in TA with LGD (n = 119) compared with SSLs (n = 29) (69.11 ± 8.62 vs 81.41 ± 2.43, P < 0.001). TA containing HGD (n = 26) had lower LINE-1 methylation levels than those with LGD (n = 119) (59.86 ± 7.93 vs 69.11 ± 8.62, P < 0.001).DISCUSSION:HGD and increasing size of TA/tubulovillous adenoma were associated with lower LINE-1 methylation. This supports a hypothesis that LINE-1 hypomethylation in TAs indicates advancement along the CRC tumorigenesis pathway. Lower LINE-1 methylation and greater variance of global DNA methylation was seen in TA compared with SSL. LINE-1 methylation in adenomas correlates with polyp size and degree of dysplasia and deserves further study as a predictor of metachronous colorectal cancer risk.

Tumor Long Interspersed Nucleotide Element-1 (LINE-1) Hypomethylation in Relation to Age of Colorectal Cancer Diagnosis and Prognosis.

Simple SummaryThe rising incidence of early-onset cancers diagnosed before 50 years in many body sites (including the colorectum) is a growing concern. Despite the many studies of early-onset colorectal cancer, the characteristics of colorectal cancer diagnosed at age 50 or slightly after (close to age 50) have not been adequately examined. Although epigenetic alterations are considered to play a role in early-onset colorectal cancer, the association of LINE-1 hypomethylation (that reflects global DNA hypomethylation) with the age of colorectal cancer diagnosis has not been thoroughly clarified. Using a database of 1356 colorectal cancers, we found that tumor LINE-1 hypomethylation was increasingly more common with decreasing age of colorectal cancer diagnosis and was associated with higher colorectal cancer-specific mortality. Our findings support the “age continuum” model that has substantial implications in research on cancers in not only the colorectum but also many other body sites.Evidence indicates the pathogenic role of epigenetic alterations in early-onset colorectal cancers diagnosed before age 50. However, features of colorectal cancers diagnosed at age 50–54 (hereafter referred to as “intermediate-onset”) remain less known. We hypothesized that tumor long interspersed nucleotide element-1 (LINE-1) hypomethylation might be increasingly more common with decreasing age of colorectal cancer diagnosis. In 1356 colorectal cancers, including 28 early-onset and 66 intermediate-onset cases, the tumor LINE-1 methylation level measured by bisulfite-PCR-pyrosequencing (scaled 0 to 100) showed a mean of 63.6 (standard deviation (SD) 10.1). The mean tumor LINE-1 methylation level decreased with decreasing age (mean 64.7 (SD 10.4) in age ≥70, 62.8 (SD 9.4) in age 55–69, 61.0 (SD 10.2) in age 50–54, and 58.9 (SD 12.0) in age <50; p < 0.0001). In linear regression analysis, the multivariable-adjusted β coefficient (95% confidence interval (CI)) (vs. age ≥70) was −1.38 (−2.47 to −0.30) for age 55–69, −2.82 (−5.29 to −0.34) for age 50–54, and −4.54 (−8.24 to −0.85) for age <50 (Ptrend = 0.0003). Multivariable-adjusted hazard ratios (95% CI) for LINE-1 methylation levels of ≤45, 45–55, and 55–65 (vs. >65) were 2.33 (1.49–3.64), 1.39 (1.05–1.85), and 1.29 (1.02–1.63), respectively (Ptrend = 0.0005). In conclusion, tumor LINE-1 hypomethylation is increasingly more common with decreasing age of colorectal cancer diagnosis, suggesting a role of global DNA hypomethylation in colorectal cancer arising in younger adults.

Impact of Tumor LINE-1 Methylation Level and Neoadjuvant Treatment and Its Association with Colorectal Cancer Survival.

Recent studies suggest that long-interspersed nucleotide element-1 (LINE-1) hypomethylation is commonly found in colorectal cancer (CRC), and is associated with worse prognosis. However, the utility of LINE-1 methylation on the prognosis of CRC is still controversial, and may be due to the fact that some clinical and pathological features may affect LINE-1 methylation. Thus, the aim of this study was to assess the prognostic value of tumor LINE-1 methylation in CRC, through their association with the CRC clinical and pathological characteristics. Survival of sixty-seven CRC patients was evaluated according to the median of tumor LINE-1 methylation, as well as pathological and oncological variables. We also studied the association between LINE-1 methylation and pathological features, and finally, we assessed the overall and disease-free survival of LINE1 methylation, stratified by neoadjuvant treatment and further checked by multivariate Cox regression to assess the statistical interactions. LINE-1 was hypomethylated in the CRC tumor with respect to the tumor adjacent-free area (p < 0.05), without association with any other clinical and oncological features, nor with overall and disease-free survival rates for CRC. Relevantly, in neoadjuvant treatment, LINE-1 methylation was associated with survival rates. Thus, disease-free and overall survival rates of treated CRC patients were worse in the hypomethylated LINE-1 tumors than those with normal LINE-1 methylation (p = 0.004 and 0.0049, respectively). Indeed, LINE-1 was hypermethylated more in the treated patients than in the non-treated patients (p < 0.05). The present study showed that tumor LINE-1 hypomethylation was associated with worse survival rates in only treated patients. Our data suggest an interactive effect of neoadjuvant treatment and tumor LINE-1 methylation, which could be a specific-tissue biomarker to predict survival of the treated patients, and help to personalize treatment in CRC.

Long interspersed nuclear element 1 hypomethylation has novel prognostic value and potential utility in liquid biopsy for oral cavity cancer

BackgroundNew biomarkers are urgently needed to improve personalized treatment approaches for head and neck squamous cell carcinoma (HNSCC). Global DNA hypomethylation has wide-ranging functions in multistep carcinogenesis, and the hypomethylation of long interspersed nucleotide element-1 (LINE-1) is related to increased retrotransposon activity and induced genome instability. However, little information is available regarding LINE-1 hypomethylation and its prognostic implications in HNSCC.MethodsIn this study, we analyzed LINE-1 hypomethylation levels in a well-characterized dataset of 317 primary HNSCC tissues and 225 matched pairs of normal mucosa tissues, along with five oral cavity cancer (OCC) circulating tumor DNA (ctDNA) samples using quantitative real-time methylation and unmethylation PCR. The analysis was performed according to various clinical characteristics and prognostic implications.ResultsThe results demonstrated that LINE-1 hypomethylation levels were significantly higher in the HNSCC tissues than in corresponding normal tissues from the same individuals (P < 0.001). Univariate analysis revealed that high levels of LINE-1 hypomethylation were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038), whereas multivariate analysis demonstrated that they were significant independent prognostic factor for DFS (hazard ratio: 2.10, 95% confidence interval: 1.02–4.36; P = 0.045). Moreover, samples with high LINE-1 hypomethylation levels exhibited the greatest decrease in 5-hydroxymethylcytosine (5-hmC) levels and increase in tumor-suppressor gene methylation index (P = 0.006 and P < 0.001, respectively). Further, ctDNA studies also showed that LINE-1 hypomethylation had high predictive ability in OCC.ConclusionsLINE-1 hypomethylation is associated with a higher risk of early OCC relapse, and is hence, a potential predictive biomarker for OCC. Furthermore, 5-hmC levels also exhibited predictive potential in OCC, based on their inverse correlation with LINE-1 hypomethylation levels. LINE-1 hypomethylation analysis, therefore, has applications in determining patient prognosis and real-time surveillance of disease recurrence, and could serve as an alternative method for OCC screening.

Low Maternal Dietary Folate Alters Retrotranspose by Methylation Regulation in Intrauterine Growth Retardation (IUGR) Fetuses in a Mouse Model.

BackgroundMaternal folate deficiency-mediated metabolic disruption is considered to be associated with the risk of intrauterine growth retardation (IUGR), but the exact mechanism remains unclear. The retrotransposon long interspersed nucleotide element-1 (LINE-1), which can induce birth defects via RNA intermediates, plays crucial roles during embryonic development. We investigated potential relationships between maternal folate and DNA methylation, and possible roles of LINE-1 in IUGR.Material/MethodsThe IUGR model was established by feeding female mice 1 of 3 diets – control diet (CD), folate-deficient diet for 2 weeks (FD2w), and folate-deficient diet for 4 weeks (FD4w) – prior to mating. Maternal serum folate, 5-methyltetrahydrofolate (5-MeTHF), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) concentrations and global DNA methylation were assessed by LC/MS/MS method. LINE-1 methylation levels in fetuses were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LINE-1 expression levels were validated by real-time PCR.ResultsMaternal folate deficiency caused plasma folate and 5-MeTHF levels to decrease and SAH level to increase in the FD4w group. Compared with the CD group, methylation levels of genomic DNA and LINE-1 decreased significantly in placenta and fetal tissues from the FD4w group. Expression of LINE-1 open reading frame 1 (ORF1) protein was elevated in fetal liver tissues. Furthermore, a strong correlation was found between methylation and disrupted one-carbon metabolism, implying that dietary folate plays important roles during embryogenesis.ConclusionsMaternal dietary folate deficiency impaired one-carbon metabolism, leading to global DNA and LINE-1 hypomethylation, and then increased retrotransposition in fetuses, which can lead to IUGR.

Tumor Long-interspersed Nucleotide Element-1 Methylation Level and Immune Response to Esophageal Cancer.

To examine the relationship between tumor long-interspersed nucleotide element-1 (LINE-1) methylation level and immune response to esophageal cancer. Evidence points to a correlation between the abundance of immune cells and a favorable prognosis in esophageal cancer patients. Accumulating evidence indicates a critical role of tumor LINE-1 hypomethylation in the aggressive behavior of esophageal cancer, which in turn leads to an unfavorable prognosis. Utilizing a nonbiased database of 292 resected esophageal cancers, we measured tumor LINE-1 methylation level by pyrosequencing assay, and examined the relationship between LINE-1 methylation and the density of T cells (CD8 and FOXP3) and the lymphocytic reaction patterns (follicle lymphocytic reaction, peritumoral lymphocytic reaction, stromal lymphocytic reaction, and tumor-infiltrating lymphocytes) in esophageal carcinoma tissue. LINE-1 hypomethylation was associated with male gender and advanced stage cancer (P = 0.03 and P = 0.048, respectively). Tumor LINE-1 methylation level was significantly positively associated with peritumoral lymphocytic reaction (P = 0.004), but not with others. Compared with LINE-1 hypermethylation group, LINE-1 hypomethylation group showed much lower level of peritumoral lymphocytic reaction (univariable odds ratio 0.32, 95% confidence interval 0.16-0.64, P = 0.002). In multivariable model to control for potential confounders including disease stage, the similar finding was observed (multivariable odds ratio 0.31, 95% confidence interval 0.14-0.66, P = 0.004). Tumor LINE-1 hypomethylation level is associated with a diminished peritumoral lymphocytic reaction, providing impetus for further investigations on potential interactive roles of tumor LINE-1 hypomethylation and host immunity in esophageal cancer development.

Soy Isoflavone Supplementation Increases Long Interspersed Nucleotide Element-1 (LINE-1) Methylation in Head and Neck Squamous Cell Carcinoma

Aim: Soy isoflavones have been suggested as epigenetic modulating agents with effects that could be important in carcinogenesis. Hypomethylation of LINE-1 has been associated with head and neck squamous cell carcinoma (HNSCC) development from oral premalignant lesions and with poor prognosis. To determine if neoadjuvant soy isoflavone supplementation could modulate LINE-1 methylation in HNSCC, we undertook a clinical trial.Methods: Thirty-nine patients received 2–3 weeks of soy isoflavone supplements (300 mg/day) orally prior to surgery. Methylation of LINE-1, and 6 other genes was measured by pyrosequencing in biopsy, resection, and whole blood (WB) specimens. Changes in methylation were tested using paired t tests and ANOVA. Median follow up was 45 months.Results: LINE-1 methylation increased significantly after soy isoflavone (P < 0.005). Amount of change correlated positively with days of isoflavone taken (P = 0.04). Similar changes were not seen in corresponding WB samples. No significant changes in tumor or blood methylation levels were seen in the other candidate genes.Conclusion: This is the first demonstration of in vivo increases in tissue-specific global methylation associated with soy isoflavone intake in patients with HNSCC. Prior associations of LINE-1 hypomethylation with genetic instability, carcinogenesis, and prognosis suggest that soy isoflavones maybe potential chemopreventive agents in HNSCC.

LINE-1 hypomethylation in colon cancer.

588 Background: Global hypomethylation of the genome is one of the causes of chromosomal instability and aneuploidy and is associated with an increased risk of colorectal cancer (CRC). The long interspersed nucleotide element-1 (LINE-1) is the main part of the mobile repeated elements of the genome, and the level of its methylation is concordant with genome-wide methylation. The aim of the study was to assess the methylation of LINE-1, as well as methylation of the promoter of six cancer-associated genes adjusting for the clinical and pathological characteristics in colorectal cancer (CRC). Methods: Using pyrosequencing (PyroMark Q24 analysis system, Qiagen, Germany) DNA methylation levels of LINE-1 and 6 genes ( APC, CDH13, MLH1, MGMT, P16 and RASSF1A) were measured in tumoral and normal tissues of 50 patients with CRC. Results: The level of LINE-1 methylation was 66.8 ± 0.89% in tumors, 72.5 ± 0.45% in normal tissues ( p = 0.0013). The differences in LINE-1 methylation level were not associated with age, differentiation grade, tumor localization, and the presence of activating mutation within the KRAS gene. At the same time, the MGMT methylation was positively associated with age ( p = 0.04), and the hypermethylation of the CDH13 gene was more common in tumors of the proximal colon ( p = 0.004). Methylation of the CDH13 promoter negatively correlated with methylation level of LINE1 ( r = -0.427, p &lt; 0.05). Hypomethylation of LINE-1 was associated with progression of TNM-stages of the tumors ( r = +0.606, p &lt; 0.001). The cancer-associated genes were hypermethylated in 70% of CRC cases when compared with normal epithelium, and hypomethylation of LINE-1 was observed in 90% of CRC cases. Conclusions: The present study demonstrated the association of LINE-1 methylation with the TNM tumor stage, which may be used as a biomarker for early diagnosis and prognosis of sporadic CRC, together with assessment of hypermethylation of tumor suppressors.

Prognostic significance of LINE-1 hypomethylation in oropharyngeal squamous cell carcinoma

BackgroundInclusion of new biomarkers to improve a personalized treatment approach for oropharyngeal squamous cell carcinoma (OPSCC) is urgently needed. Hypomethylation of the Long interspersed nucleotide element-1 (LINE-1) repetitive elements, a widely accepted surrogate of overall genomic DNA methylation content, was found to be associated with a poor prognosis in several cancers. At present, no studies have investigated the influence of LINE-1 methylation levels on OPSCC relapse. The main goal of this study was the evaluation of the prognostic value of LINE-1 methylation status in predicting early tumor relapse in locally advanced OPSCC.MethodsWe retrospectively reviewed a cohort of 77 patients with stage III–IVB OPSCC. Methylation of LINE-1 repetitive sequences was evaluated by real-time quantitative methylation-specific PCR in formalin-fixed paraffin-embedded tissues. The prognostic relevance of LINE-1 methylation was assessed by comparing patients who relapsed within 2 years from the end of treatment (cases) with those who did not (controls). Results were validated in an independent cohort of 33 patients with OPSCC.ResultsWith respect to early OPSCC relapse, the mean LINE-1 methylation level was significantly lower in relapsed cases than in control group (p < 0.01). Interestingly, LINE-1 methylation was lower in relapsed cases than in controls in both HPV16-negative and HPV16-positive OPSCC patients, even if statistical significance was reached only for the former group (p = 0.01). LINE-1 methylation levels were also significantly reduced in relapsed cases with respect to the controls in OPSCC current smokers (p = 0.02). Consistently, in HPV16-negative current smokers, OPSCC relapse was significantly associated with decreased levels of LINE-1 methylation (p = 0.02). Using logistic regression model, we found that patients with hypomethylated LINE-1 were associated with a 3.5 higher risk of early relapse than hypermethylated ones (OR = 3.51; 95% CI 1.03–12.00). Adjustment for potential confounders did not substantially change the risk magnitude. Results from the validation cohort confirmed the lower LINE-1 methylation in patients who early relapsed compared to relapse-free patients.ConclusionsLINE-1 hypomethylation is associated with higher risk of early relapse in stage III–IVB OPSCC. Further validation in a prospective study is needed for its application in daily clinical practice.

LINE-1 methylation level and prognosis in pancreas cancer: pyrosequencing technology and literature review

Global DNA hypomethylation plays an important role in genomic instability and carcinogenesis. The long interspersed nucleotide element-1 (LINE-1) methylation level is a good surrogate marker of the global DNA methylation level. Previously, we demonstrated a strong relationship between LINE-1 hypomethylation and poor prognosis in certain cancers. However, the relationship between the LINE-1 methylation level and the clinical outcome of pancreatic cancer (PC) remains unclear. We used a pyrosequencing assay to measure LINE-1 methylation levels in 126 samples of resected PC and evaluated the prognostic value of the LINE-1 methylation level. LINE-1 methylation levels were significantly lower in PC tissues than in matched noncancerous pancreatic tissues (p=0.039, n=36). The tumoral LINE-1 methylation range was 41.3-92.8 (n=126, mean 77.7, median 78.5, standard deviation 5.7). The LINE-1 methylation level was unrelated to clinical and pathological features. Moreover, LINE-1 hypomethylation was not significantly associated with overall survival, cancer specific survival, or disease-free survival (log-rank p=0.30, p=0.18 and p=0.50, respectively). The LINE-1 methylation level appears not to be associated with poor prognosis in PC. The effect of the LINE-1 methylation level on the survival of PC patients needs to be confirmed in a larger-cohort study.

The role of LINE-1 methylation in predicting survival among colorectal cancer patients: a meta-analysis.

The prognostic value of long interspersed nucleotide element-1 (LINE-1) methylation in patients with colorectal cancer (CRC) remains uncertain. We have therefore performed a meta-analysis to elucidate this issue. The PubMed and Web of Science databases were searched for studies published up to 30 June 2016 which reported on an association between LINE-1 methylation and overall survival (OS), disease-free survival (DFS), or cancer-specific survival (CSS) among CRC patients. The reference lists of the identified studies were also analyzed to identify additional eligible studies. Hazard ratios (HRs) with 95% confidence intervals (CIs) were pooled using the fixed-effects or the random-effects model. Stratification analysis and meta-regression analysis were performed to detect the source of heterogeneity. Analyses of sensitivity and publication bias were also carried out. Thirteen independent studies involving 3620 CRC patients were recruited to the meta-analysis. LINE-1 hypomethylation was found to be significantly associated with shorter OS (HR 2.92, 95% CI 2.20-3.88, p<0.001) and DFS (HR 2.18, 95% CI 1.46-3.27, p<0.001), as well as unfavorable CSS (HR 1.96, 95% CI 1.35-2.85, p<0.001). No heterogeneity was found among the studies evaluating the associations between LINE-1 hypomethylation and OS or DFS, with the exception being CSS. Moreover, meta-regression analysis suggested that one of the contributors to between-study heterogeneity on the association between LINE-1 methylation and CSS was statistical methodology. The subgroup analysis suggested that the association in studies using the Cox model statistical method (HR 2.76, 95% CI 1.90-4.01, p<0.001) was stronger than that in studies using the Log-rank test (HR 1.41, 95% CI 1.07-1.87, p=0.015). The results of this meta-analysis suggest that LINE-1 methylation is significantly associated with the survival of CRC patients and that it could be a predictive factor for CRC prognosis.

Prognostic and predictive significance of long interspersed nucleotide element-1 methylation in advanced-stage colorectal cancer.

BackgroundHypomethylation of Long Interspersed Nucleotide Element-1 (LINE-1) is associated with worse prognosis in colorectal cancer (CRC). However, little is known about the relevance of this marker for the prognosis and response to chemotherapy of metastatic and recurrent (advanced-stage) CRC. Our aim was therefore to investigate whether tumor LINE-1 hypomethylation correlates with patient survival and with response to 5-fluorouracil (5-FU)/ oxaliplatin (FOLFOX) chemotherapy in advanced-stage CRC.MethodsThe study included 40 CRC patients who developed metastasis or local recurrence after surgery and subsequently underwent FOLFOX therapy. Progression-free and overall survival were estimated using the Kaplan-Meier method. LINE-1 methylation levels in formalin-fixed and paraffin-embedded primary tumor tissues were measured by MethyLight assay and correlated with patient survival. In vitro analyses were also conducted with human colon cancer cell lines having different LINE-1 methylation levels to examine the effects of 5-FU and oxaliplatin on LINE-1 activity and DNA double-strand-breaks.ResultsPatients with LINE-1 hypomethylation showed significantly worse progression-free (median: 6.6 vs 9.4 months; P = 0.02) and overall (median: 16.6 vs 23.2 months; P = 0.01) survival following chemotherapy compared to patients with high methylation. LINE-1 hypomethylation was an independent factor for poor prognosis (P = 0.018) and was associated with a trend for non-response to FOLFOX chemotherapy. In vitro analysis showed that oxaliplatin increased the LINE-1 score in LINE-1-expressing (hypomethylated) cancer cells, thereby enhancing and prolonging the effect of 5-FU against these cells. This finding supports the observed correlation between tumor LINE-1 methylation and response to chemotherapy in CRC patients.ConclusionsTumor LINE-1 hypomethylation is an independent marker of poor prognosis in advanced-stage CRC and may also predict non-response to combination FOLFOX chemotherapy. Prospective studies are needed to optimize the measurement of tumor LINE-1 methylation and to confirm its clinical impact, particularly as a predictive marker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2984-8) contains supplementary material, which is available to authorized users.

Abstract 2776: The relationship between UHRF1 overexpression and LINE-1 hypomethylation in esophageal squamous cell carcinoma

Abstract Background: Global DNA hypomethylation contributes to oncogenesis through multiple mechanism. The level of long interspersed nucleotide element-1 (LINE-1) methylation is regarded as a surrogate marker of global DNA methylation, and is drawing interest as a good indicator for predicting cancer prognosis. However, the mechanism by which LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully explored. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays an essential role in DNA methylation. Previous studies have demonstrated that UHRF1 overexpression might be a mechanism of underlying DNA hypomethylation in hepatocellular carcinoma, and contribute to poor prognosis. Nonetheless, the epigenetic role of UHRF1 in esophageal squamous cell carcinoma (ESCC) remains unclear. Methods: Using 16 frozen tissues (matched pairs of cancer and normal epithelium), and 160 curatively resected esophageal cancer specimens, we analyzed the relationship between UHRF1 expression, LINE-1 methylation levels and patient outcome. We measured UHRF1 expression by real-time PCR and immunohistochemistry, and LINE-1 methylation levels by bisulfite- pyrosequencing technology. In addition, to investigate the relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) using UHRF1 overexpression vector and siRNA for UHRF1 with ESCC tissues and ESCC cell lines. Results: In ESCC tissues, UHRF1 expression were significantly associated with LINE-1 methylation levels, and UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients. Furthermore, UHRF1 vector-mediated overexpression caused LINE-1 (global DNA) hypomethylation, and conversely, UHRF1 knockdown using siRNA increased DNA methylation in ESCC cell lines. Conclusion: These results may suggest that UHRF1 is an epigenetic key regulator of DNA methylation, moreover, can be applicable to attractive cancer therapies. Citation Format: Kenichi Nakamura, Yoshifumi Baba, Keisuke Kosumi, Kazuto Harada, Hironobu Shigaki, Keisuke Miyake, Junji Kurashige, Takatsugu Ishimoto, Masaaki Iwatsuki, Naoya Yoshida, Hideo Baba. The relationship between UHRF1 overexpression and LINE-1 hypomethylation in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2776.

Abstract 2782: The effect of LINE-1 methylation level on survival in pancreatic cancer

Abstract Background: Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1 (LINE-1) is a good indicator of the global DNA methylation level. In previous studies, we demonstrated that LINE-1 hypomethylation was strongly associated with a poor prognosis in some types of cancers including esophageal cancer, gastric cancer, and hepatocellular carcinoma, supporting its potential role as a prognostic marker. However, the relationship between LINE-1 hypomethylation and clinical outcome in pancreatic cancer remains unclear. Methods: LINE-1 methylation level in 126 samples resected pancreatic cancers was measured by pyrosequencing assay, and the prognostic value of LINE-1 methylation level in pancreatic cancer was examined. Results: Pancreatic cancers showed significantly lower LINE-1 methylation levels compared to matched non tumorous parenchyma (P = 0.039; N = 36). Tumoral LINE-1 methylation range was 41.3-92.8 (N = 126; mean: 77.7; median: 78.5; standard deviation: 5.7). However, LINE-1 hypomethylation was not significantly associated with overall survival, cancer specific survival, disease-free survival [log-rank p = 0.30, p = 0.18 and p = 0.50, respectively]. Conclusion: The present result suggested that LINE-1 hypomethylation in pancreatic cancer is not associated with poor prognosis. In this study, our cohort was small, therefore further accumulation of data may be needed in order to confirm the effect of LINE-1 methylation level on survival in pancreatic cancer. Citation Format: Kensuke Yamamura, Yoshifumi Baba, Lei Zhou, Xiaobo Zhang, Keisuke Kosumi, Katunobu Taki, Kazuto Harada, Takatsugu Ishimoto, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Tan Xiaodong, Hideo Baba. The effect of LINE-1 methylation level on survival in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2782.

Tumor LINE-1 methylation level and colorectal cancer location in relation to patient survival.

Colorectal tumors arise with genomic and epigenomic alterations through interactions between neoplastic cells, immune cells, and microbiota that vary along the proximal to distal axis of colorectum. Long interspersed nucleotide element-1 (LINE-1) hypomethylation in colorectal cancer has been associated with worse clinical outcome. Utilizing 1,317 colon and rectal carcinoma cases in two U.S.-nationwide prospective cohort studies, we examined patient survival according to LINE-1 methylation level stratified by tumor location. Cox proportional hazards model was used to assess a statistical interaction between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, and KRAS, BRAF, and PIK3CA mutations. A statistically significant interaction was found between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis (Pinteraction = 0.011). The association of LINE-1 hypomethylation with higher colorectal cancer-specific mortality was stronger in proximal colon cancers (multivariable hazard ratio [HR], 1.66; 95% confidence interval [CI], 1.21 to 2.28) than in distal colon cancers (multivariable HR, 1.18; 95% CI, 0.81 to 1.72) or rectal cancers (multivariable HR, 0.87; 95% CI, 0.57 to 1.34). Our data suggest the interactive effect of LINE-1 methylation level and colorectal cancer location on clinical outcome.

APOBEC3B is an enzymatic source of molecular alterations in esophageal squamous cell carcinoma.

APOBEC3B belongs to the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family of enzymes and induces C to T transitions of target DNA by cytidine deamination. Recently, several mutations in various cancers have been linked to APOBEC3B, suggesting a crucial role for this protein in carcinogenesis and cancer development. However, the significance of APOBEC3B in esophageal squamous cell carcinoma (ESCC) remains uncertain. In addition, the APOBEC3B immunoreactivity in cancer tissues is uncertain. Recently, we have demonstrated that PIK3CA mutation and the methylation level of long interspersed nucleotide element 1 (LINE-1) (a surrogate marker of global DNA methylation level) are prognostic markers and have crucial role on malignancy in ESCC patients. This study aims to clarify the impact of APOBEC3B on the clinical, pathological, and molecular features of ESCC. We evaluated APOBEC3B expression in ESCC and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathological features, and the molecular features of ESCC (PIK3CA mutation, p53 expression, and LINE-1 methylation level). The immunoreactivity and mRNA level of APOBEC3B were significantly higher in cancer tissues than in noncancerous esophageal mucosae (P=0.050). APOBEC3B expression was significantly correlated with PIK3CA mutation (P=0.013), particularly with C to T transitions of PIK3CA (P=0.041). Moreover, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethylation (P=0.027). Given the crucial roles of PIK3CA mutation and LINE-1 methylation levels, our findings might provide new insights into the biological mechanisms of ESCC tumorigenesis and progression.

Aberrant DNA methylation profiles of inherited and sporadic colorectal cancer

BackgroundAberrant DNA methylation has been widely investigated in sporadic colorectal carcinomas (CRCs), and extensive work has been performed to characterize different methylation profiles of CRC. Less information is available about the role of epigenetics in hereditary CRC and about the possible clinical use of epigenetic biomarkers in CRC, regardless of the etiopathogenesis. Long interspersed nucleotide element 1 (LINE-1) hypomethylation and gene-specific hypermethylation of 38 promoters were analyzed in multicenter series of 220 CRCs including 71 Lynch (Lynch colorectal cancer with microsatellite instability (LS-MSI)), 23 CRCs of patients under 40 years in which the main inherited CRC syndromes had been excluded (early-onset colorectal cancer with microsatellite stability (EO-MSS)), and 126 sporadic CRCs, comprising 28 cases with microsatellite instability (S-MSI) and 98 that were microsatellite stable (S-MSS). All tumor methylation patterns were integrated with clinico-pathological and genetic characteristics, namely chromosomal instability (CIN), TP53 loss, BRAF, and KRAS mutations.ResultsLS-MSI mainly showed absence of extensive DNA hypo- and hypermethylation. LINE-1 hypomethylation was observed in a subset of LS-MSI that were associated with the worse prognosis. Genetically, they commonly displayed G:A transition in the KRAS gene and absence of a CIN phenotype and of TP53 loss. S-MSI exhibited a specific epigenetic profile showing low rates of LINE-1 hypomethylation and extensive gene hypermethylation. S-MSI were mainly characterized by MLH1 methylation, BRAF mutation, and absence of a CIN phenotype and of TP53 loss. By contrast, S-MSS showed a high frequency of LINE-1 hypomethylation and of CIN, and they were associated with a worse prognosis. EO-MSS were a genetically and epigenetically heterogeneous group of CRCs. Like LS-MSI, some EO-MSS displayed low rates of DNA hypo- or hypermethylation and frequent G:A transitions in the KRAS gene, suggesting that a genetic syndrome might still be unrevealed in these patients. By contrast, some EO-MSS showed similar features to those observed in S-MSS, such as LINE-1 hypomethylation, CIN, and TP53 deletion. In all four classes, hypermethylation of ESR1, GATA5, and WT1 was very common.ConclusionsAberrant DNA methylation analysis allows the identification of different subsets of CRCs. This study confirms the potential utility of methylation tests for early detection of CRC and suggests that LINE-1 hypomethylation may be a useful prognostic marker in both sporadic and inherited CRCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0165-2) contains supplementary material, which is available to authorized users.

Abstract 4829: The clinical significance of APOBEC3B in esophageal squamous cell carcinoma

Abstract Backgrounds: APOBEC3B is one of the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family, and APOBEC3B can induce transitions into target DNA through cytidine deamination. To date, APOBEC attracts attention due to its functioning as part of the innate immune system by protecting against retrovirus and retrotransposon propagation. And, recent studies revealed that APOBEC3B might contribute to several mutations in various types of cancers, including cervical, breast, head and neck, lung, bladder cancers, and APOBEC3B may have a crucial role in carcinogenesis and cancer development. Esophageal squamous cell carcinoma (ESCC) reportedly has several molecular features, and we recently reported that long interspersed nucleotide element 1 (LINE-1) hypomethylation might contribute to poor prognosis and PIK3CA mutation to good prognosis in ESCC. However, there is no report which investigates the relationship between APOBEC3B expression, clinical and pathological features in ESCC. Methods: We compared the expression of APOBEC3B mRNA between cancer tissues and noncancerous esophageal mucosae by using GEO profiles and ESCC frozen tissues. In addition, we investigated the relationships between the APOBEC3B immunohistochemical expression levels, clinical and pathological features, and molecular features including p53 expression, global DNA methylation level and PIK3CA mutation. We quantified LINE-1 retrotransposons, of which a substantial portion of the human genome consists, to assess global DNA methylation. Results: APOBEC3B expression at mRNA level was significantly higher in cancer tissues than in noncancerous esophageal mucosae (P = 0.050). In the immunohistochemical study, the APOBEC3B expression of ESCC was stronger than that of noncancerous esophageal mucosa. We found no significant correlation of APOBEC3B expression with tumor size, T stage, N stage and pStage (P = 0.74, P = 0.32, P = 0.19, P = 0.23, respectively). There were no correlation between APOBEC3B expression and prognosis. There was no correlation between APOBEC3B expression and p53 expression (P = 0.23), but, interestingly, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethyaltion and PIK3CA mutation (P = 0.029, P = 0.020). Conclusions: APOBEC3B high expression had significant relationships with LINE-1 hypomethylation and PIK3CA mutation, supporting that APOBEC3B might contribute to malignant behavior through the molecular features in ESCC. Citation Format: Keisuke Kosumi, Yoshifumi Baba, Takatsugu Ishimoto, Kenichi Nakamura, Kazuto Harada, Mayuko Ohuchi, Yuki Kiyozumi, Daisuke Izumi, Ryuma Tokunaga, Junji Kurashige, Ryuichi Karashima, Yukiharu Hiyoshi, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. The clinical significance of APOBEC3B in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4829. doi:10.1158/1538-7445.AM2015-4829

Tissue-Specific Methylation of Long Interspersed Nucleotide Element-1 of Homo Sapiens (L1Hs) During Human Embryogenesis and Roles in Neural Tube Defects.

Epigenetic regulation of long interspersed nucleotide element-1 (LINE-1) retrotransposition events plays crucial roles during early development. Previously we showed that LINE-1 hypomethylation in neuronal tissues is associated with pathogenesis of neural tube defect (NTD). Herein, we further evaluated LINE-1 Homo sapiens (L1Hs) methylation in tissues derived from three germ layers of stillborn NTD fetuses, to define patterns of tissue specific methylation and site-specific hypomethylation at CpG sites within an L1Hs promoter region. Stable, tissue-specific L1Hs methylation patterns throughout three germ layer lineages of the fetus, placenta, and maternal peripheral blood were observed. Samples from maternal peripheral blood exhibited the highest level of L1Hs methylation (64.95%) and that from placenta showed the lowest (26.82%). Between samples from NTDs and controls, decrease in L1Hs methylation was only significant in NTD-affected brain tissue at 7.35%, especially in females (8.98%). L1Hs hypomethylation in NTDs was also associated with a significant increase in expression level of an L1Hs-encoded transcript in females (r = -0.846, p = 0.004). This could be due to genomic DNA instability and alternation in chromatins accessibility resulted from abnormal L1Hs hypomethylation, as showed in this study with HCT-15 cells treated with methylation inhibitor 5-Aza.

Abstract 1357: Clinical, pathological and prognostic value of LINE-1 methylation in hepatocellular carcinoma

Abstract Objectives:Cancer cells exhibit 2 types of deoxyribonucleic acid (DNA) methylation alterations: genome-wide DNA hypomethylation and site-specific CpG island promoter hypermethylation. Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. As long interspersed nucleotide element-1 (LINE-1) retrotransposon constitutes a substantial portion (approximately 17%) of the humangenome, the level of LINE-1 methylation is regarded to be a surrogate marker of genome-wide DNA methylation. Importantly, LINE-1 hypomethylation is associated with poor prognosis in several type of cancers, suggesting the potencial role of the LINE-1 methylation level as a useful marker for predicting cancer prognosis. However, clinical, pathological and prognostic significance of LINE-1 hypomethylaiton in hepatocellular carcinoma (HCC) remains unclear. Methods: Using 160 HCC resected specimens, we quantified the LINE-1 methylation by utilizing the bisulfite pyrosequencing technology. This assay amplifies a region of LINE-1 element (position 305-331 in accession no. X58075), which includes 4 CpG cites. The average of the relative amounts of C in the 4 CpG sites was used as the overall LINE-1 methylation level.Results: We first examined the LINE-1 methylation level in 85 HCC tissues and matched normal hepatic parenchyma samples. The cancer tissues exhibited significantly lower levels of LINE-1 methylation (median, 64.5 ; range, 99.1-21.5) than the matched normal hepatic parenchyma (median, 81.0 ; range, 97.2-35.8) (P &amp;lt; 0.0001 by the paired t test). The LINE-1 methylation level of HCC was not associated with any of theclinical, epidemiological, or pathological characteristics, including sex, age, AFP, PIVKA-II, infection of hepatitis B virus and hepatitis C virus, ICG-R15, stage of fibrosis, differentiation, tumor size, tumor type, vascular invasion and stage (all P &amp;gt; 0.09). In the Kaplan-Meier analysis, LINE-1 hypomethylators (methylation level &amp;lt; 74.4) experienced significantly shorter disease-free survival than those with hypermethylators (log-rank P=0.029). Over-all survival is not associated with LINE-1 methylation ( log-rank P=0.16). Conclusions: Genome-wide DNA hypomethylation, as measured in LINE-1, is associated with disease-free survival among patients with HCC, suggesting that LINE-1 hypomethylation may be a biomarker that can be used to identify patients who will experience an inferior outcome. Citation Format: kazuto harada, Yoshirumi Baba, Akira Chikamoto, Takatsugu Ishimoto, Keisuke Kosumi, Asuka Murata, Naoya Yoshida, Toru Beppu, Hideo Baba. Clinical, pathological and prognostic value of LINE-1 methylation in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2014-1357