LINE-1 Methylation Status Research Articles

Abstract 424: The transcriptomic and methylomic changes caused by subtoxic doses of 5-azacytidine and 5-aza-2’-deoxycytidine

Abstract Two demethylating agents, 5-azacytydine (Vidaza) and 5-Aza-2’-deoxycytidine (Decitabine), are used in the clinic to treat myelodysplastic syndrome (MDS) and some forms of leukemia. There are also several ongoing clinical trials using these demethylating agents to treat solid tumors. DNA incorporation of these drugs leads to the depletion of DNMTs (DNA methyltransferases) in the cell and passive DNA demethylation. The major difference between Decitabine and Vidaza is their respective level of incorporation into nucleic acid. Decitabine is only incorporated into DNA, while 70-90% of Vidaza incorporates into RNA and only 10-30% into DNA. Due to their mechanisms of action, the effects of these drugs on DNA are S-phase dependent. It has been previously demonstrated that various transcriptional changes are initiated by demethylation of DNA, but the majority of studies have focused on transcriptional activation of tumor suppressor genes. The objective of this study is to use genomics approaches to examine the effects of low doses of Vidaza and Decitabine on bladder cancer cells and normal immortalized urothelium. We hypothesize that Vidaza and Decitabine will have distinct biological effects on the methylome and transcriptome in these cells, including alternative splicing. We have generated DNA and RNA samples from normal (SV-HUC) and cancer bladder (T24 and SCaBer) cell lines treated with low concentrations (100nM, 200nM, and 1µM) of Vidaza and Decitabine. The cells were treated with the different concentration of drugs on day 2 and 4, and then harvested on day 5. Before utilizing these samples for genomic screening, we monitored the methylation status of three CpGs in the promoter region of LINE-1, a surrogate marker of pharmacologically induced DNA demethylation. Furthermore, we examined the mRNA expression of NY-ESO-1 cancer testis gene previously shown to be one of the reactivated genes by demethylating agents through a loss of promoter hypermethylation. T24 cells immediately showed a significant decrease of methylation of LINE-1 when treated with 100nM Decitibine, but there was barely any change in methylation status in the other cells lines at this low dose. Meanwhile, treatment with Vidaza did not change the methylation status of LINE-1 and mRNA expression of NY-ESO-1. However, these experiments were merely designed to monitor the general pharmacological effects of Vidaza and Decitabine. We are currently examining the effects of these drugs focusing on genomic regions (exonic and intronic) outside of gene promoters by utilizing deep-coverage paired-end RNA sequencing and 450K Illumina methylation array. The integration of these large-scale data will enable us to better understand pharmacologically induced demethylation in cancer and utilization of these agents in treatment of solid tumors including bladder cancer. Citation Format: Evelyn Smit, Nithya Krishnan, Jeffrey Conroy, Jianmin Wang, Song Lui, Anna Woloszynska-Read. The transcriptomic and methylomic changes caused by subtoxic doses of 5-azacytidine and 5-aza-2’-deoxycytidine. [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 424. doi:10.1158/1538-7445.AM2014-424

Abstract 408: The relationship between LINE-1 hypomethylation and fibrosis status in noncancerous liver tissues of hepatocellular carcinoma patients

Abstract Background: The DNA methylation alterations that occur in human cancers include global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation plays an important role in genomic instability, leading to cancer development. Since LINE-1 or the L1 retrotransposon constitutes a substantial portion of the human genome, LINE-1 methylation status reflects global DNA methylation. Our previous study has revealed that LINE-1 methylation level is a potential surrogate marker for epigenetic field defects induced by tobacco smoking in esophageal cancer patients. Given that liver fibrosis can be one phenomena of field cancerization theory in hepatocellular carcinoma (HCC), we examined the relationship between LINE-1 methylation level and fibrosis status in noncancerous liver tissues of HCC patients. Methods: Using histologically normal liver tissue samples and matched tumor tissue samples from 89 HCC cases, we measured LINE-1 methylation levels by pyrosequencing technology. Results: LINE-1 methylation of noncancerous liver tissues range was 35.8-97.3 of a 0-100 scale (mean: 78.1; median: 81.0; standard deviation: 11.9). HCC tissues showed significantly lower levels of LINE-1 methylation than matched normal tissue (P<0.01). LINE-1 methylation of noncancerous liver tissues was not associated with age, sex, Hepatitis B Virus infection, or Hepatitis C Virus infection. Interestingly, LINE-1 methylation level of noncancerous liver tissues with severe fibrosis (score 2-4) was significantly lower than that with mild fibrosis (score 0-1) (P=0.013). Conclusion: Liver fibrosis in noncancerous liver tissues of HCC patients was significantly associated with LINE-1 hypomethylation. This result supports the potential of LINE-1 methylation level as an indicator of epigenetic field for HCC cancerization. Citation Format: Yoshifumi Baba, Kazuto Harada, Keisuke Kosumi, Hiromitsu Hayashi, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. The relationship between LINE-1 hypomethylation and fibrosis status in noncancerous liver tissues of hepatocellular carcinoma patients. [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 408. doi:10.1158/1538-7445.AM2014-408

Methylation status of retinoic acid receptor beta2 promoter and global DNA in esophageal squamous cell carcinoma

Focal hypermethylation in promoter regions of tumor suppressor genes against the background of global hypomethylation is a landmark of carcinogenesis. This study aimed to investigate the methylation status of retinoic acid receptor beta2 (RARβ2) and long interspersed nuclear elements (LINE-1) in different stages of esophageal squamous cell carcinoma (ESCC). The tumor and adjacent normal esophageal tissues from 125 male ESCC patients who underwent primary surgery were analyzed for the methylation status of RARβ2 promoter and LINE-1 through methylation-specific polymerase chain reaction and pyrosequencing. RARβ2 hypermethylation was detected in 20% of the tumor samples, but not in the normal counterparts. The methylation frequency of LINE-1 was significantly lower in the tumor than in the normal parts (median: 67.7% vs. 80%, P < 0.0005). Ninety-eight patients (78.4%) had both RARβ2 hypermethylation and LINE-1 hypomethylation or either one. There was a trend toward higher risk of advanced T stage (P for trend = 0.05) or lymph node metastasis (P for trend = 0.02) when more adverse gene methylation profiles were present. Methylation status of RARβ2 and LINE-1 was related to the development and possibly the severity of ESCC.

Differences in LINE-1 Methylation Between Endometriotic Ovarian Cyst and Endometriosis-Associated Ovarian Cancer

Endometriosis in endometriosis-associated ovarian cancer (EAOC) refers to lesions that can derive from endometriotic ovarian cysts (ECs) that form in the ovarian endometrium with the potential to transform into full-blown ovarian cancer. Hypomethylation of long interspersed element-1 (LINE-1 or L1) is a common epigenomic event in several cancers and is strongly associated with ovarian cancer progression. To evaluated alterations in LINE-1 methylation between EC, ovarian endometrioid adenocarcinoma (OEA), EAOC, and ovarian clear cell carcinoma (OCC). First, LINE-1 methylation status in 19 normal endometrium, 29 EC, 35 OCC, and 22 OEA tissues from unrelated samples were compared. Then, specific areas of eutopic endometrium, contiguous endometriosis, and cancer arising from 16 EAOCs were collected by microdissection and analyzed for LINE-1 methylation status. The total LINE-1 methylation levels were significantly different among the endometrium, endometriosis, and ovarian cancer (P < 0.001). A stepwise decrease in LINE-1 methylation was observed in the following order: normal endometrium, EC, OEA, and OCC. Interestingly, endometriosis in EAOC of both OEA (P = 0.016) and OCC (P = 0.003) possessed a higher percentage of LINE-1 unmethylated loci than EC. Our data implicate that LINE-1 hypomethylation is an early molecular event involved in OEA and OCC malignant transformation. Precise measurements of LINE-1 methylation may help to distinguish EC and endometriosis in EAOC.

Abstract 678: Evaluation of oral bioavailability and intermittent dosing schedules for pharmacodynamic effects by SGI-110, a novel subcutaneous (SQ) second generation hypomethylating agent (HMA), in male cynomolgus monkeys .

Abstract Background: SGI-110 is a second generation hypomethylating agent consisting of a dinucleotide of decitabine and deoxyguanosine designed to release decitabine upon administration. SGI-110 is being evaluated in patients with relapsed/refractory MDS and AML. This study evaluated pharmacodynamic effects of three alternate weekly intermittent dosing schedules of Subcutaneous (SQ) SGI-110, and also the bioavailability of SGI-110 when given orally. Methods: Group A received a single 10 mg/kg dose of SGI-110 by oral gavage. Group B received SQ injections of 1.5 mg/kg on Days 1,2, 8,9 and 15,16; Group C received SQ injections on Days 1,4, 8,11, 15 and 18; group D received injections on Days 1,2,3 and 15,16,17. Pharmacokinetics of SGI-110 and decitabine were evaluated on Day 1 for all treatment groups. The pharmacodynamic effect of SGI-110 was assessed through Day 28 (Days 1, 4, 8, 11, 14, 21 and 28) by measuring LINE1 methylation status in PBMCs from monkeys in groups B, C and D. Results: Animals tolerated SGI-110 treatment well through the study. Relative oral bioavailability of decitabine appeared to be very low with only trace amounts of decitabine detected in plasma from Group A animals. A time-dependent LINE1 hypomethylating effect was observed in monkeys from Groups B, C and D relative to baseline pre-treatment values from Day 1. The maximal extent of hypomethylation varied between 11-14% within the groups and was achieved earlier in the cycle for Groups B and C (Day 11 and Day 14, respectively), whereas for Group D a second maximum peak effect was observed on Day 21. All groups trended towards baseline by Day 28 but Groups C and D still had significant hypomethylation (8-9%) present by the end of the study. On average, Group C and D seemed to achieve the maximum and most prolonged hypomethylation but only group C had a sustained effect. Conclusions: SGI-110 was well tolerated in cynomolgus monkeys for both single PO and repeat SQ dosing using intermittent alternate-weekly schedules. Oral bioavailability of SGI-110 was very low, suggesting very high first-pass contribution to SGI-110 and decitabine clearance upon oral administration. All SQ schedules achieved significant hypomethylating effect; the extent and the duration of effect were more favorable for Groups C and D with group C showing a more sustained effect over the 28-day cycle. We concluded that the twice weekly SQ regimen is a highly effective and convenient regimen to study in clinical trials as it may achieve a more sustained pharmacodynamic effect. Citation Format: Aram Oganesian, Pietro Taverna, Mohammad Azab. Evaluation of oral bioavailability and intermittent dosing schedules for pharmacodynamic effects by SGI-110, a novel subcutaneous (SQ) second generation hypomethylating agent (HMA), in male cynomolgus monkeys . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 678. doi:10.1158/1538-7445.AM2013-678

Changes in Gene Methylation Following Chemotherapy in Breast Cancer Cell Lines

Objective: Epigenetic modulation of gene expression by DNA promoter methylation may contribute to acquired resistance to chemotherapy in cancer cells. Decitabine (5-aza-2'- deoxycytidine), a demethylating agent, may act synergistically with standard chemotherapy regimens to activate epigenetically silenced genes. In the present in vitro study, it was investigated the effect of gene methylation level after treatment with decitabine and combination of decitabine with anthracycline-based therapeutics (5-fluorouracil plus epirubicine plus cyclophosphamide; FEC) on breast cancer cells (MCF-7 and MDA-MB-231). Methods: The effect of decitabine and its combination with FEC on different genes methylation level has been tested in MDA-MB-231 and MCF-7 human breast cancer cell lines. The effect of decitabine on the cell viability was assayed by MTT assay. Methylight real-time PCR and methylation specific PCR were carried out to determine the methylation status of certain genes: DAPK, TMS1, MGMT and the global methylation marker LINE-1. Results: The LINE-1 methylation status significantly decreased in both cell lines after treatment with the combination of decitabine with FEC. In MDA-MB-231 cells, methylation of the TMS1 and the MGMT gene promoter was significantly reduced by FEC plus decitabine while no effect was observed in MCF-7 cells. Conclusion: Anthracycline-based therapy regimens in combination with demethylating agents such as decitabine may affect chemotherapy outcome by modulation of apoptosis- relevant genes by methylation. More importantly, this modulation seems to be dependent on

DNA Methylation Pyrosequencing Assay Is Applicable for the Assessment of Epigenetic Active Environmental or Clinical Relevant Chemicals

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2'-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants.

FOLIC ACID PREVENTS LINE-1 HYPOMETHYLATION IN RATS WITH HYPERHOMOCYSTEINEMIA

ObjectivesLINE-1 (Long Interspersed Nuclear Elements-1) hypomethylation has been associated with risk of cardiovascular disease. Increased plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. However, it is still...

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

ObjectiveEarly-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.DesignWe analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.ResultsMean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).ConclusionsLINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.

LINE-1 methylation status and its association with tetralogy of fallot in infants

BackgroundMethylation levels of long interspersed nucleotide elements (LINE-1) are representative of genome-wide methylation status and play an important role in maintaining genomic stability and gene expression. To derive insight into the association between genome-wide methylation status and tetralogy of fallot (TOF), we compared the methylation status of LINE-1 element between TOF patients and controls. The methylation of the NKX 2–5, HAND 1, and TBX 20 promoter regions was also evaluated.MethodsGenomic DNA from right ventricular tissue samples was obtained from 32 patients with TOF and 15 control subjects. Sequenom MassARRAY platform was performed to examine the methylation levels of LINE-1, NKX2-5, HAND1 and TBX20. Mann–Whitney U test was used to compare differences in methylation levels between two groups.ResultsThe methylation level of LINE-1 was significantly lower in patients with TOF, with a median of 57.95% (interquartile range [IQR]: 56.10%–60.04%), as opposed to 59.70% in controls (IQR: 59.00%–61.30%; P = 0.0021). The highest LINE-1 methylation level was 61.3%. The risk of TOF increased in subjects with the lowest methylation levels (less than or equal to 59.0%; OR = 14.7, 95% CI: 1.8–117.7, P = 0.014) and in those with medium methylation levels (59.0%–61.3%; OR = 2.0, 95% CI: 0.3–14.2, P = 0.65). An ROC curve analysis showed a relatively high accuracy of using the LINE-1 methylation level in predicting the presence of TOF (AUC = 0.78, 95% CI: 0.65–0.91; P = 0.002). The association of the LINE-1 methylation level with TOF was only observed in males (P = 0.006) and not in females (P = 0.25). Neither age nor gender was found to be associated with the LINE-1 methylation level in patients or controls. Higher methylation levels of NKX2-5 and HAND1 and lower methylation levels of TBX20 were also observed in patients with TOF than in controls. No association was found between the methylation levels of NKX2-5, HAND1 and TBX 20 with the LINE-1 methylation level.ConclusionsLower LINE-1 methylation levels are associated with increased risk of TOF and may provide important clues for the development of TOF.

Abstract 2665: Oxaliplatin as an alternative for the treatment of high grade prostate cancers. Identification of serine hydroxymethyl transferases (SHMT) as selective biomarkers of oxaliplatin sensitivity via the modulation of DNA methylation.

Abstract Prostate cancer (PCa) is one of the leading causes of death from cancer in men. High Gleason grade PCa are characterized by aggressive tumors with poorly differentiated cells and a high metastatic potential. They are often refractory to chemical castration but still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low pointing towards the need for new alternative therapies. Using an in silico approach based on a 86 genes signature which could distinguish between low-grade and high-grade tumors, we identified 9 genes (PCCB, SHMT2, DPM1, RHOT2, RPL13, CD59, EIF4AI, CDKN2C, JUN) for which expression levels across the NCI-60 cell lines panel was significantly correlated (p&amp;lt; 0.05) to oxaliplatin but not to cisplatin sensitivity. Among them, SHMT2 which is overexpressed in high grade PCa, encodes the mitochondrial isoform of serine hydroxymethyl transferase, which converts glycine to serine, which is then exported to the cytoplasm to generate the one-carbon units that are required for the biosynthesis of purines. SiRNA-mediated donwregulation of SHMT2 in DU145 or LNCaP prostate tumor cell lines rendered cells resistant to oxaliplatin but not to cisplatin. CHO 51-11 cells which express a catalytically inactive SHMT2 were also resistant to oxaliplatin but not to cisplatin as compared to CHO K1 control cells. The selective resistance to oxaliplatin was not due to the oxalate moiety since addition of oxalate to cisplatin could not restore the level of sensitivity of CHO cells to oxaliplatin. Selectivity could however be attributed to the diamino cyclohexane (DACH) moiety of oxaliplatin since CHO 51-11 cells were also resistant to dichloro(1,2-diaminocyclohexane)-platinum(II). Resistance to oxaliplatin could also be achieved by SHMT1 downregulation (the cytoplasmic form of SHMT) or by an alteration of the pool of glycine and serine. SHMTs can indirectly regulate the methylation status of DNA, and we showed that siRNA-mediated downregulation of SHMTs in hypomethylated LNCaP cells increased the global level of DNA methylation, as measured by 4 CpGs methylation status of LINE-1. Interestingly, increased level of global methylation status was associated with increased resistance to oxaliplatin. Altogether, our results identified oxaliplatin as a potential alternative for the treatment of high grade prostate cancers and SHMT2 (and SHMT1) as selective markers of oxaliplatin sensitivity which involves, at least in part, the regulation of global levels of DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2665. doi:1538-7445.AM2012-2665

ALU and LINE‐1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications

Focal CpG island hypermethylation and diffuse genomic hypomethylation signify the changes in the DNA methylation status in cancer cells. ALU and LINE-1 repetitive DNA elements comprise ~28% of the human genome. PCR-based measurements of these repetitive DNA elements can be used as a surrogate marker of the genomewide methylation content. Our study aimed to identify the timing of ALU and LINE-1 hypomethylations during multistep gastric carcinogenesis and their prognostic implications in gastric cancer (GC). In our study, we analyzed the methylation statuses of ALU and LINE-1 in 249 cases of gastric biopsy samples and another independent set of 198 cases of advanced GC by pyrosequencing. Regardless of the Helicobacter pylori infection status, a significant decrease in the ALU methylation levels was noted during the transitions from chronic gastritis to intestinal metaplasia and from gastric adenoma to GC. LINE-1 methylation decreased during the transition from intestinal metaplasia to gastric adenoma and no further decrease occurred during the transition from gastric adenoma to GC. A low LINE-1 methylation status was strongly associated with poor prognosis in GC. A multivariate analysis revealed that LINE-1 methylation status was an independent prognostic factor. Our findings suggest that ALU and LINE-1 hypomethylations are early events during multistep gastric carcinogenesis. Furthermore, the LINE-1 methylation status can be used as a molecular biomarker to define a subset of GC patients with poor prognosis.

A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation

BackgroundThe methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation.FindingsUsing high resolution melt (HRM) curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986) with a high amplification efficiency (90-105%), capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%)--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach.ConclusionsIn summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies.

Abstract 92: Effects of p16 methylation on the efficacy of cetuximab in metastatic colorectal cancer

Abstract Background: Promoter DNA methylation is an important epigenetic factor of gene inactivation in human carcinogenesis and gene silencing of tumor suppressor genes in colorectal cancer. Global DNA methylation, reflected by the status of LINE-1 methylation, plays an important role in genomic instability and carcinogenesis. We examined the role of p16 methylation and LINE-1 methylation in metastatic colorectal cancer patients treated with cetuximab chemotherapy. Methods: The study was performed using fresh frozen specimens from 42 metastatic colorectal cancer patients treated with cetuximab. Samples were analyzed by bisulfied-based approaches to study the methylation status of p16 and LINE-1 by pyrosequecing. KRAS mutation was determined by pyrosequencing. Results: Methylation of p16 was associated with decreased progression-free survival in all patients (n=42, 4.9 months in p16 methylated patients vs 15.7 months in p16 unmethylated patients, p=0.01). When proximal location tumors were analyzed separately, methylation of p16 was associated with decreased progression-free survival (n=11, 4 months vs 15.7 months, p&amp;lt;0.01), and decreased overall survival (14.5 months vs 35 months, p&amp;lt;0.01). Among KRAS wild type tumors (n=35), methylation of p16 demonstrated a tendency toward decreased progression-free survival (6.3 months in methylated patients vs 15.7 months in unmethylated patients, p=0.07). In 3rd line erbitux treatment patients (n=13), the median PFS was 4.0 months for p16 methylated patients and 9.6 months for unmethylated patients (p=0.25). In the response rate analysis, compared with KRAS wild type tumors, KRAS mutant tumors had a significantly lower response rate (0 % vs 45.7%, p=0.033). Compared with P16-methylated tumors, p16 unmethylated tumors demonstrated a tendency toward better response rate (10% vs 46.9%, p=0.061). LINE-1 methylation was not associated with either progression-free survival (p=0.53) or response rate (p = 0.53). Conclusions: 16 methylation may be a factor other than KRAS mutation associated with resistance to anti-EGFR monoclonal antibodies. Because we analyzed heterogeneous population and our sample size was too small, we suggest that further studies are necessary to determine the predictive role of DNA methylation markers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 92. doi:10.1158/1538-7445.AM2011-92

LINE-1 Hypomethylation During Primary Colon Cancer Progression

BackgroundMethylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.Methodology/Principal FindingsDNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).Conclusion/SignificanceBy using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.

Correlation between hypomethylation of long interspersed element-1 and clinicopathologic features of gastric cancer

Objective To elucidate the correlation of methylation of long interspersed nucleotide element-1 (Line-1) with clinicalopathological features of gastric cancer.Methods Gastric cancer tissues and paracancerous tissues were collected from 30 patients and methylation status of Line-1 was detected in caner and corresponding normal tissues by using Pyrosequencing.Results The methylation level of Line-1in normal gastric and gastric cancer tissues was 75.1% and 65.8% respectively (P＜0.05).Among 30cancer tissues,methylation status of Line-1 was closely related to tumor infiltration (T1/T2:68.9%,T3/T4:61.3% ),lymph nodes metastasis ( NO:70.0%,N1:69.9%,N2:61.7%,N3:57.6% ) and distant metastasis ( MO:67.9%,M1:59.1% ) ( P ＜0.05) and declined with tumor progression,but wasn't associated with age,gender,location and differentiation ( P＞ 0.05 ).Conclusion Hypomethylation of Line-1 was remarkably related to infiltration and metastasis of gastric cancer and may contribute to the tumor progression. Key words: Gastric carcinoma; Line-1; Methylation

DNA methylation predicts recurrence from resected stage III proximal colon cancer

In colorectal cancer (CRC), DNA methylation anomalies define distinct subgroups termed CpG island methylator phenotype 1 (CIMP1), CIMP2, and CIMP-negative. The role of this classification in predicting recurrence and disease-free survival (DFS) in resected stage III CRC was evaluated. Sporadic cancers from 161 patients were analyzed. Bisulfite pyrosequencing was used to examine the methylation of 2 global DNA methylation markers (LINE-1, Alu) and 9 loci (MINT1, MINT2, MINT31, P16, hMLH1, P14, SFRP1, SFRP2, and WNT5A). Mutations in BRAF and KRAS were assayed. Gene hypermethylation clustered in discrete groups of patients, indicating the presence of CIMP. K-means clustering analysis identified 3 discrete subgroups: CIMP1 (n = 22, 13.7%), associated with proximal location and BRAF mutations; CIMP2 (n = 40, 24.8%), associated with KRAS mutations; and CIMP-negative (n = 99, 61.5%), associated with distal location. In proximal CRC, CIMP1 was correlated with a higher recurrence rate (53% for CIMP1, 18% for CIMP2, and 26% for CIMP-negative) and a worse DFS (P = .015). Also in proximal CRC, LINE-1 methylation was lower in patients whose cancer recurred compared with those whose cancer did not recur (P = .049). In multivariate analysis, CIMP1 and low LINE1 methylation were independent prognostic factors for DFS in proximal CRC (P = .008 for classification by K-means clustering analysis; P = .040 for LINE-1 methylation status). DNA methylation is a useful biomarker of recurrence in resected stage III proximal but not distal CRC. However, as the number of CIMP1 cases was small in distal CRC, further study is required to validate our findings.

Abstract 1896: Selenium and isothiocyanates increases TrxR1 and GI-GPx expression but DNA methylation of LINE-1 remains unchanged in Caco-2 cells

Abstract Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression through epigenetic events to modify cancer susceptibility. Evidence from epidemiological, in vivo and in vitro studies have suggested that the chemopreventive effect of cruciferous vegetables is attributed to the glucosinolate break-down products, isothiocyanates (ITCs). Similarly, selenium (Se) is an integral part of many cellular antioxidant enzymes and like isothiocyanates has been inversely associated with the incidence of cancer as a result of influencing multiple pathways in the development of cancer. However, their efficacy might depend on the chemical form in which they are administered. We therefore examined the synergistic effect of two forms of selenium, selenium-methylselenocysteine (Se-MSC) and selenite (200nM) with ITCs (6µM) such as iberin and sulforaphane on the expression of antioxidant selenoenzymes such as thioredoxin-reductase (TrxR1) and gastrointestinal glutathione peroxidise (GI-GPx) in an in vitro model using Caco-2 cells. Our preliminary results suggest that the simultaneous addition of Se-MSC and iberin significantly induced TrxR1 and GI-GPx mRNA and protein expression more than either compound alone by ∼6 fold (p&amp;lt;0.001). In addition to the antioxidant protection provided by these selenoproteins, DNA methylation is essential for normal development, but aberrant DNA methylation patterns are a hallmark of most cancers and include global DNA hypomethylation, region-specific hypermethylation and increased DNA methyltransferases (DNMTs) activity. We therefore also examined the impact of Se and ITCs on DNA methylation, specifically examining factors modulating DNMT1, 3A, 3B expression and methylation of the LINE-1 retrotransposon, a surrogate marker of global DNA methylation. Caco-2 cells were treated with Se-MSC and selenite using 0.2, 0.5, 1, 2 and 5µM, in addition to the ITCs (6µM) after 5 and 10 days of treatment. Our initial data suggests that methylation of LINE-1 in the control group was indeed low (55%) and treatment of cells with DNMT inhibitor 5-aza-2 deoxycytidine resulted in a 45% reduction in LINE-1 methylation as expected. Unexpectedly, none of the food compounds or dose used in this study modified significantly the methylation status of LINE-1. Interestingly, although the methylation levels of this marker remain unchanged, the mRNA expression of the DNMTs showed the tendency to decrease, following the same pattern in the different treatments and time courses albeit with small differences, which suggests that both de novo and maintenance DNMTs work in concert to create and propagate methylation patterns. However, only DNMT3B showed a significant reduction of 35 and 38% after 10 days of treatment with Se-MSC (500nM) (p=0.04) and selenite (1µM) (p=0.035) respectively. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1896.

Abstract 156: Implications of LINE1 methylation for bladder cancer risk in women

Abstract Bladder cancer is the ninth most incident form of cancer in the United States and is an exposure-related disease which contributes significantly to morbidity and health care costs. Of concern is the fact that incidence rates have been stable in men in the last twenty years but have been increasing in women at a rate of 0.2% a year. Although tobacco use and occupational exposures have been linked to bladder cancer, much of the etiology of this disease remains unexplained. Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of this disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer. LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case control study of bladder cancer in New Hampshire. Cases included white, 25-74 year olds, with a histopathologically confirmed diagnosis of bladder cancer while controls were selected from records of the Department of Transportation and the Medicare Program. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation and for a number of different risk factors for bladder cancer including arsenic. Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer (95% CI, 1.12-2.90) in models controlling for gender, age and smoking, with the association being stronger in women than in men (ORs = 2.48, 95% CI 1.19-5.17 in women and 1.47, 95% CI 0.79-2.74 in men). Amongst non-invasive cancers, being in the lowest LINE1 methylation decile conferred an almost two fold risk of bladder cancer (OR 1.94, 95% CI, 1.17-3.22) while the risk was non-significant amongst invasive cancers after adjusting for gender, age and smoking. Amongst controls, women were more likely to have lower LINE1 methylation than men (p-value 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (p-value 0.04). This investigation has identified a novel epigenetic marker of bladder cancer which has the potential for utility as a screening strategy for this disease, and which may aid in providing a more comprehensive understanding of the etiology of bladder cancer. In addition, this study shows an increased risk of LINE1 hypomethylation with high levels of arsenic, raising important questions about the nature of the causal pathway for alterations in DNA repeat methylation to contribute to cancer risk. These results also suggest that women demonstrate lower levels of LINE1 methylation, which may also aid in explaining the disparate incidence of this disease in men and women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 156.

Implications of LINE1 Methylation for Bladder Cancer Risk in Women

Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer. LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case-control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation. Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer [95% confidence interval (95% CI), 1.12-2.90] in models controlling for gender, age, and smoking, and the association was stronger in women than in men (odds ratio, 2.48; 95% CI, 1.19-5.17 in women; and odds ratio, 1.47; 95% CI, 0.79-2.74 in men). Among controls, women were more likely to have lower LINE1 methylation than men (P = 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (P = 0.04). LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially among women.