DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps

Colorectal cancer (CRC) screening rates are currently suboptimal. Blood-based screening could improve rates of earlier detection for CRC and adenomatous colorectal polyps. In this study, we evaluated the feasibility of plasma-based detection of early CRC and adenomatous polyps using array-mediated analysis methylation profiling of 56 genes implicated in carcinogenesis. Methylation of 56 genes in patients with Stages I and II CRC (N=30) and those with adenomatous polyps (N=30) were compared with individuals who underwent colonoscopy and were found to have neither adenomatous changes nor CRC. Composite biomarkers were developed for adenomatous polyps and CRC, and their sensitivity and specificity was estimated using five-fold cross validation. Six promoters (CYCD2, HIC1, PAX 5, RASSF1A, RB1 and SRBC) were selected for the biomarker, which differentiated CRC patients and controls with 84% sensitivity and 68% specificity. Three promoters (HIC1, MDG1 and RASSF1A) were selected for the biomarker, which differentiated patients with adenomatous polyps and controls with sensitivity of 55% and specificity of 65%. Methylation profiling of plasma DNA can detect early CRC with significant accuracy and shows promise as a methodology to develop biomarkers for CRC screening.     

K-ras mutations and RASSF1A promoter methylation in colorectal cancer

Human cancer is characterized by genetic and epigenetic alterations. In this study we provide evidence for the interruption of Ras signaling in sporadic colorectal cancer (CRC) by either genetic activation of the K-ras oncogene or epigenetic silencing of the putative tumor suppressor gene RASSF1A. Paraffin embedded tumor tissue samples from 222 sporadic CRC patients were analysed for K-ras codon 12 and codon 13 activating mutations and RASSF1A promoter hypermethylation. Overall, K-ras mutations were observed in 87 of 222 (39%) and RASSF1A methylation was observed in 45 of 222 (20%) of CRCs. Mutation of K-ras alone was detected in 76 of 222 (34%) CRCs. RASSF1A promoter methylation with wild-type K-ras was observed in 34 of 222 (15%) CRCs. In 101 of 222 (46%) CRCs neither K-ras mutations nor RASSF1A methylation was observed and 11 of 222 (5%) CRCs showed both K-ras mutations and RASSF1A methylation. These data show that the majority of the studied CRCs with K-ras mutations lack RASSF1A promoter methylation, an event which occurs predominantly in K-ras wild-type CRCs (P=0.023, Chi-square test).     

Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells

Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.     

Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues

The methylation pattern in the promoter region of p16, DAPK, MGMT and GSTP1 genes was investigated in oral cancer tissues and tumor associated adjacent tissues, using methylation specific PCR assay. The samples constituted 60 primary oral tumors and corresponding adjacent clinically and histopathologically normal mucosa, and buccal epithelial scrapings from 20 normal healthy individuals without any tobacco habits. The incidence of hypermethylation in oral tumor and adjacent mucosa for p16 gene was 66.7 and 50%, for DAPK was 68.3 and 60%, and MGMT gene was 51.7 and 26.7%, respectively. The overall hypermethylation in the three genes in the primary tumor was 86.7%, and corresponding adjacent normal mucosa tissues 76.7%. Hypermethylation was not observed in the promoter region of GSTP1 gene in either the primary tumors or the corresponding adjacent normal mucosa. Absence of aberrant methylation in the four genes was noted in buccal scrapings from normal healthy individuals with no tobacco habits. Thus, a high frequency of promoter region hypermethylation was observed in p16, DAPK and MGMT genes in oral cancer tissues as well as in corresponding adjacent normal mucosa. Our results indicate that epigenetic alteration of these genes is a frequent event in oral cancer, and is an early event observed in normal oral mucosa of the patients, indicating the critical importance of the epigenetic alteration in chewing tobacco associated oral carcinogenesis.     

CHFR promoter hypermethylation in colon cancer correlates with the microsatellite instability phenotype

A subset of sporadic colon cancers has been shown to have microsatellite instability caused by an epigenetic inactivation of the MLH1 gene by hypermethylation of the the CpG island in its promoter region. We report here that in colorectal cancer, inactivation of the MLH1 gene is frequently accompanied by hypermethylation of the CpG island in the promoter of the mitotic gene checkpoint with forkhead and ring finger domains (CHFR). This was first observed in the colon cancer cell lines HCT-116, DLD-1, RKO and HT29. Among the 61 primary colon cancer samples studied, hypermethylation of the MLH1 and the CHFR promoter was found in 31% of the tumors. In 68% of all primary cancers (13/19) with MLH1 promoter hypermethylation, hypermethylation of the CHFR promoter was observed as well (P-value < 0.0001, Fisher's two-sided exact). Hypermethylation of the HLTF, MGMT, RASSF1, APC, p14 and p16 promoter regions were also frequent events, being observed in 48% (28/58), 40% (26/64), 21% (14/64), 50% (31/62), 43% (26/60) and 56% (35/63), respectively. However, methylation of these genes was not associated with methylation of either MLH1 or CHFR. The observed methylation profile was unrelated to Duke's stage. The coordinated loss of both mismatch repair caused by methylation of MLH1 and loss of checkpoint control associated with methylation of CHFR suggests the potential to overcome cell cycle checkpoints, which may lead to an accumulation of mutations.     

Hypermethylation of the RASSF1A tumor suppressor gene in Japanese clear cell renal cell carcinoma

Hypermethylation associated inactivation of RASSF1A tumor suppressor gene at chromosome 3p21.3 has been observed in several human malignancies. Relatively high (91%) or low (23%) frequencies were reported in the methylation status of promoter region of the RASSF1A gene in clear cell renal carcinoma (RCC) depending on the country the report was from. To clarify exact contribution of the hypermethylation of RASSF1A gene in the development of RCC in Japan, we analyzed the methylation status of the RASSF1A promoter region in 50 Japanese clear cell RCC and RCC cell lines. Although relatively high frequency of hypermethylation in RASSF1A promoter (39 of 50 tumors, 78%) was observed, most of matched proximal normal tissue DNA also showed weak methylation. By comparison with methylation level of adapted normal kidney tissue DNA, tumor preferential hypermethylation in RASSF1A promoter was recognized as 40% (20/50 matched sets) of primary clear cell RCCs. Hypermethylation in RASSF1A promoter was observed in 36% (15/42) and 64% (5/8) of stage I-II or III-IV tumors, and also observed in 42% (11/26) and 38% (9/24) of our tumor samples with pathological grade I or II, respectively. In addition, 16 of 19 RCC cell lines (84%) showed complete or partial methylation of RASSF1A promoter region. There was no association between the frequency of RASSF1A methylation and inactivation of VHL tumor suppressor gene in either primary RCCs or RCC cell lines. Our results showed tumor specific RASSF1A promoter hypermethylation in up to 40% of low grade or low stage clear cell RCCs. It is essential to compare the methylation status of RASSF1A promoter in tumor with normal tissue to understand tumor specific hypermethylation. Since considerable cases of normal kidney are hypermethylated, contribution of the RASSF1A for the development and progression of kidney cancer may be more complex than expected.     

Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation

In prostate carcinoma (PCa) increased DNA methylation ('hypermethylation') occurs at specific genes such as GSTP1. Nevertheless, overall methylation can be decreased ('hypomethylation') because methylation of repetitive sequences like LINE-1 retrotransposons is diminished. We analysed DNA from 113 PCa and 36 noncancerous prostate tissues for LINE-1 hypomethylation by a sensitive Southern technique and for hypermethylation at eight loci by methylation-specific PCR. Hypermethylation frequencies for GSTP1, RARB2, RASSF1A, and APC in carcinoma tissues were each >70%, strongly correlating with each other (P<10(-6)). Hypermethylation at each locus was significantly different between tumour and normal tissues (10(-11)82% of PCas. PCa may fall into three classes, that is, with few DNA methylation changes, with frequent hypermethylation, or with additional LINE-1 hypomethylation.     

卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区甲基化的研究

目的 探讨RASSF1A基因启动子区异常甲基化与卵巢上皮性恶性肿瘤发生、发展的关系。方法 应用甲基化特异性PCR方法,检测80例卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区异常甲基化。结果 80例卵巢上皮性恶性肿瘤组织中,RASSF1A基因启动子区甲基化的发生率为52．5％,而相应痛旁正常组织中,RASSF1A基因启动子区均未发生甲基化（P〈0．05）。浆液性癌、黏液性癌和内膜样癌中,RASSF1A基因启动子区甲基化的发生率分别为54．2％、52．4％和45．5％,差异尤统计学意义。临床Ⅰ期、Ⅱ期卵巢上皮性恶性肿瘤RASSF1A基因启动子区甲基化的发生率分别为21．4％和16．7％,明显低于临床Ⅲ期（66．7％）和Ⅳ期（77．8％）。高分化组和中分化组RASSFlA基因启动子区甲基化的发牛率分别为34．5％和35．0％,均低于低分化组（80．6％）。结论 卵巢上皮性恶性肿瘤组织中存在RASSF1A基因启动子区的异常甲基化,甲基化与卵巢上皮性恶性肿瘤的临床分期和组织学分级有关。     

Identification of epigenetic aberrant promoter methylation of RASSF1A in serum DNA and its clinicopathological significance in lung cancer

RASSF1A is a novel putative tumor suppressor gene located in 3p21.3 region. The most common inactivation mechanism of RASSF1A is promoter hypermethylation, which is observed in multiple solid tumors including lung cancer. In the present study, we identified the methylation status of RASSF1A in lung cancer sera using methylation-specific PCR and analyzed its clinicopathological significance. Hypermethylation of RASSF1A was detected in 27 of 80 (33.8%) cancer patients but no benign pulmonary disease patients or healthy donors (P<0.001). RASSF1A hypermethylation was preferentially observed in small cell lung cancer (P=0.042), while no statistical difference was found among methylation frequencies of different subtypes of non-small cell lung cancer. RASSF1A methylation status was associated with differentiation (P=0.009) and stage (P=0.013), but not with gender, age or treatment. These findings suggest that serum RASSF1A hypermethylation is a promising molecular biomarker for lung cancer diagnosis.     

Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours

Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16(INK4A), APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARbeta and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P=0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASSF1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P=0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs.     

Aberrant promoter methylation in pleural fluid DNA for diagnosis of malignant pleural effusion

Accumulating evidence implicates epigenetic changes such as hypermethylation in carcinogenesis. We investigated whether DNA methylation of 5 tumor suppressor genes in pleural fluid samples could aid in diagnosis of malignant effusion. In samples from 47 patients with malignant pleural effusions and 34 with nonmalignant effusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of the promoters of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a), ras association domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK), and retinoic acid receptor beta (RARbeta). Promoter hypermethylation was associated with malignant effusion for MGMT (Odds ratio (OR) = infinity), p16(INK4a) (OR = infinity), RASSF1A (OR = 13.8; CI, 1.71-112), and RARbeta (OR = 3.17; CI, 1.10-9.11), but not for DAPK. Instead, DAPK methylation was associated with the length of smoking (p < 0.05). Patients with hypermethylation of MGMT, p16(INK4a), RASSF1A or RARbeta were 5.68 times more likely to have malignant effusions than patients without methylation (p = 0.008). Methylations per patient were more numerous for lung cancer than nonmalignant pulmonary disease (0.915 vs. 0.206, p < 0.001). Sensitivity, specificity, and positive predictive value of methylation in one or more genes for diagnosis of malignant effusion were 59.6%, 79.4%, and 80.0% respectively. In conclusion, aberrant promoter methylation of tumor suppressor genes in pleural fluid DNA could be a valuable diagnostic marker for malignant pleural effusion.