Dajun Deng

Epigenetic Alterations as Cancer Diagnostic, Prognostic, and Predictive Biomarkers

Alterations of DNA methylation and transcription of microRNAs (miRNAs) are very stable phenomena in tissues and body fluids and suitable for sensitive detection. These advantages enable us to translate some important discoveries on epigenetic oncology into biomarkers for control of cancer. A few promising epigenetic biomarkers are emerging. Clinical trials using methylated CpG islands of p16, Septin9, and MGMT as biomarkers are carried out for predication of cancer development, diagnosis, and chemosensitivity. Circulating miRNAs are promising biomarkers, too. Breakthroughs in the past decade imply that epigenetic biomarkers may be useful in reducing the burden of cancer.

Methylation of p16 CpG Islands Associated with Malignant Transformation of Gastric Dysplasia in a Population-Based Study

Purpose: Inactivation of p16 by aberrant methylation of CpG islands is a frequent event in carcinomas and precancerous lesions of various organs, including the stomach. The aim of this study is to investigate the relationship between p16 methylation and malignant transformation of human gastric dysplasia (DYS) based on follow-up endoscopic screening in a high-risk population. Experimental Design: Genomic DNA samples were extracted from paraffin blocks of gastric mucosal biopsies that were histopathologically diagnosed as low-grade DYS from patients who developed gastric carcinomas [GCs (n = 21)] and those that did not do so (n = 21) during 5 years of follow-up. The methylation status of p16 CpG islands of each sample was detected by methylation-specific PCR, denatured high-performance liquid chromatography, and sequencing. Results: Aberrant p16 methylation was observed in 5 of 21 samples of DYS that progressed to GC but in 0 of 21 samples that did not progress to GC (P = 0.048, two-sided). Sequencing results confirmed that all CpG sites were methylated in the analyzed sequence from these five p16-methylated cases. Furthermore, p16 methylation was also observed in the five subsequent GCs. Unmethylated p16 CpG islands were detected in all of the samples without p16 methylation. Conclusions: Our findings suggest p16 methylation is correlated with the malignant transformation of gastric DYS, and p16 methylation might be a useful biomarker for prediction of malignant potential of gastric DYS.

Methylation of p16 CpG Island Associated with Malignant Progression of Oral Epithelial Dysplasia: A Prospective Cohort Study

Purpose: Inactivation of p16 gene by CpG methylation is a frequent event in oral epithelial dysplasia. To investigate the predictive value of p16 methylation on malignant potential in oral epithelial dysplasia, we carried out the prospective cohort study. Experimental Design: One hundred one patients with histologically confirmed mild or moderate oral epithelial dysplasia were included in the present cohort study. p16 Methylation status of the oral epithelial dysplasia lesions from 93 cases was obtained by methylation-specific PCR. Progression of the oral epithelial dysplasia lesions was examined in 78 cases histologically during a 45.8 months follow-up period. The association between p16 methylation and progression of oral epithelial dysplasia was analyzed. Results: Of the 93 enrolled cases, 15 cases were lost during the follow-up because of changes of contact information, with a compliance of 83.9%. p16 Methylation was detectable in oral epithelial dysplasia lesions from 32 (41.0%) of 78 enrolled patients. Oral epithelial dysplasia–related squamous cell carcinomas were observed in 22 patients (28.2%) during the follow-up. Rate of progression to oral cancer in patients with the p16-methylated oral epithelial dysplasia was significantly higher than that with the p16-unmethylated oral epithelial dysplasia (43.8% versus 17.4%; adjusted odds ratio, 3.7; P = 0.013), especially for patients at the baseline age of ≥60 years (adjusted odds ratio, 12.0; P = 0.003) and patients with moderate oral epithelial dysplasia (adjusted odds ratio, 15.6; P = 0.022). The overall sensitivity and specificity of prediction of malignant transformation of oral epithelial dysplasia by p16 methylation were 63.6% and 67.9%, respectively. Conclusion: p16 Methylation was correlated with malignant transformation of oral epithelial dysplasia and is a potential biomarker for prediction of prognosis of mild or moderate oral epithelial dysplasia. (Clin Cancer Res 2009;15(16):5178–83)

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: A population-based study

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population‐based study was conducted in Linqu County, a high‐risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation‐specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C‐urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p < 0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94–30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60–33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44–8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90–7.10), and dysplasia (OR, 2.48; 95%CI: 1.02–5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02–16.13 times higher in subjects with mild H. pylori infection, and 2.69–38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island.

Methylation of CpG islands of p16 associated with progression of primary gastric carcinomas.

Inactivation of p16 by methylation of CpG islands is a frequent early event in gastric carcinogenesis. The positive relationship between p16 methylation and the clinical characteristics of gastric carcinomas (GC) has not been reported to date. In the present study, a DHPLC assay to quantify p16 methylation was established (detection limit by fluorescence detector: 1:255 (Methlyated vs Unmethylated)). The proportion of methylated p16 in the representative samples was confirmed and standardized by clone sequencing. Then, the DHPLC and two regular methylation-specific PCR (MSP) assays were used to detect p16 methylation in 82 paired, resected GCs and their adjacent normal tissues. Results showed that the average proportion of methylated p16 in GCs was significantly higher than that in their adjacent samples (12.90 vs 0.63%; t-test P=0.005). A much higher proportion of methylated p16 was detected in GCs with metastases (local or distant) than without metastases (14.76 vs 2.61%; t-test P=0.014). A proportional relationship was observed between clinical stages and positive rates of p16 methylation in GCs and/or adjacent tissues: 27.3, 37.5, and 58.8% (by DHPLC) for stage-I, -II, -III–IV of GCs, respectively (two-sided Fishers exact test P=0.016). To confirm the data obtained by DHPLC, two MSP primer sets (p16-M and p16-M2) were also used to analyze p16 methylation in the same set of samples simultaneously. Data of MSP assay using the primer set p16-M2, but not p16-M, correlated with that of DHPLC. These results imply that the primer set p16-M2 might be more suitable than p16-M to detect p16 methylation in gastric tissues. In conclusion, the present data indicates that p16 methylation correlates with progression of GCs significantly.

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

Background H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. Methodology/Principal Findings In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. Conclusion/Significance These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter.

Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

BackgroundMany miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression.MethodsMethylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed.ResultsMethylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02).ConclusionsIn conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.

Genetic Polymorphisms of the E-Cadherin Promoter and Risk of Sporadic Gastric Carcinoma in Chinese Populations

Frequent mutations and loss of expression of E-cadherin have been reported in a number of cancers. E-cadherin germ line mutations lead to a high risk of familial diffused gastric carcinoma. In the present study, to evaluate the effect of genetic polymorphisms in the E-cadherin promoter on the risk of sporadic gastric carcinoma (SGC), a comprehensive study was conducted in two populations with high and low risk of SGC in China, respectively. Five hundred seventy-two SGC cases and 625 controls from low-risk area and 589 individuals enrolled in a long-term follow-up survey in high-risk area were studied. Polymorphisms of E-cadherin around transcription start site (−437 to +314) were analyzed by sequencing. Five variations of −347del>A, −160C>A, −73A>C, +178T>C, and +234 13N ins>del were linked tightly. The −347del/del and its strongly linked +178T/T, +234 13N ins/ins genotypes increased male SGC risk in the high-risk area significantly [odds ratio (OR), 2.22; 95% confidence intervals (95% CI), 1.10-4.46] and correlated with the severity of gastric lesions. A synergetic effect was also observed between −347del/del genotype and Helicobacter pylori infection (OR, 4.93; 95% CI, 1.65-14.71). Compared with −347del-containing haplotypes, the −347A-containing haplotype [A(−347)-C(−160)-A(−73)-C(+178)-13N del(+234)] decreased the risk of SGC among male subjects (OR, 0.61; 95% CI, 0.37-1.01). Such correlation could not be observed among subjects from the low-risk area. The present data suggest that the −347del allele of E-cadherin strongly links with the +178T and +234 13N ins alleles. The −347del/del genotype may increase the susceptibility of SGC among males in the high-risk area of China. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2402–8)

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

BackgroundAlu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation.MethodsBisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0.ResultsFrom the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%).ConclusionsMost Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.

BRCA1 promoter methylation associated with poor survival in Chinese patients with sporadic breast cancer.

Transcriptional inactivation of breast cancer gene 1 (BRCA1) by DNA methylation is a frequent event in sporadic breast cancers. To investigate whether BRCA1 methylation is associated with survival in Chinese patients with sporadic breast cancer, BRCA1 methylation was determined using methylation specific PCR in 536 sporadic breast cancers. Survival curves for patients with methylated and unmethylated BRCA1 were compared using the log‐rank tests. Twenty‐six percent (139/536) of patients exhibited BRCA1 methylation in their tumors. The degree of BRCA1 methylation was correlated with clinical stages of breast cancer, but was not significant. Patients with BRCA1 methylated tumors had a significantly worse 5‐year disease‐free survival (DFS) and 5‐year disease‐specific survival (DSS) than did patients with unmethylated tumors (DFS: 73.2%vs 82.6%, P = 0.045; DSS 80.5%vs 87%, P = 0.038, two‐sided). In conclusions, BRCA1 methylation is a frequent event in breast cancer and is associated with poor clinical outcome in Chinese women with breast cancer. (Cancer Sci 2009; 100: 1663–1667)