Prognostic value of apoptosis-associated speck-like protein containing a CARD gene promoter methylation in resectable non-small-cell lung cancer

BACKGROUND: The apoptosis-associated speck-like protein containing a CARD (ASC) is a newly identified tumor suppressor gene that is frequently inactivated by de novo promoter methylation in non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We studied 79 patients with NSCLC who had undergone surgery with curative intent. The ASC promoter methylation status was determined by methylation-specific polymerase chain reaction. Statistical analyses (all 2-sided) were performed to determine the prognostic effect of hypermethylation on various clinical parameters. RESULTS: ASC promoter hypermethylation was detectable by methylation-specific polymerase chain reaction in 9 of 70 (12.9%) normal lungs and in 33 of 70 (47.1%) matching tumor samples of patients with NSCLC. The mean ASC methylation level was significantly higher in tumor than in matching normal tissue (P < 0.001). ASC methylation in normal tissue was always accompanied with ASC methylation in matching tumor tissue. Patients without ASC promoter hypermethylation showed a significantly better survival than patients with ASC promoter hypermethylation (P = 0.007). Multivariate analysis revealed ASC promoter methylation as an independent unfavorable prognostic factor (P = 0.009). CONCLUSION: Our results indicate that ASC promoter hypermethylation is a common event in patients with primary NSCLC, and this epigenetic alteration is associated with inferior survival, suggesting that ASC promoter hypermethylation might be an important biomarker for a biologic, aggressive disease in NSCLC.     

痰标本p16和MGMT基因甲基化对肺癌的诊断价值

目的：分析肺癌患者痰标本中p16和MGMT基因启动子区甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标志物的价值。方法：运用甲基化特异性PCR技术,检测77例原发性肺癌患者痰标本和部分对应肿瘤组织(53例),以及30例正常对照者痰标本中p16和MGMT基因启动子区域的甲基化改变。结果：49例(63．6%)肺癌患者痰标本中检测到了pi6基因异常甲基化,34例(44．2%)检测到了MGMT基因异常甲基化,77例患者痰标本中2个基因中至少有1个基因出现甲基化为64例(83．1%),对肿瘤的检出灵敏度较高。长期吸烟史是影响肺鳞状细胞癌痰标本p16(P=0．001)基因启动子区甲基化的因素。随TNM分期增高,肺鳞状细胞癌患者痰标本中p16基因甲基化比例增高(P=0．021)；随TNM分期增高,肺腺癌患者痰标本MGMT基因甲基化比例增高(P=0．023)。对照组正常人痰液标本未发现p16和MGMT基因启动子区甲基化。结论：痰标本中p16和MGMT基因甲基化是临床肺癌辅助诊断的有效生物标志物之一。     

Prognostic Value of Apoptosis-Associated Speck-like Protein Containing a CARD Gene Promoter Methylation in Resectable Non-Small-Cell Lung Cancer

BackgroundThe apoptosis-associated speck-like protein containing a CARD (ASC) is a newly identified tumor suppressor gene that is frequently inactivated by de novo promoter methylation in non-small-cell lung cancer (NSCLC).Patients and MethodsWe studied 79 patients with NSCLC who had undergone surgery with curative intent. The ASC promoter methylation status was determined by methylation-specific polymerase chain reaction. Statistical analyses (all 2-sided) were performed to determine the prognostic effect of hypermethylation on various clinical parameters.ResultsASC promoter hypermethylation was detectable by methylation-specific polymerase chain reaction in 9 of 70 (12.9%) normal lungs and in 33 of 70 (47.1%) matching tumor samples of patients with NSCLC. The mean ASC methylation level was significantly higher in tumor than in matching normal tissue (P < 0.001). ASC methylation in normal tissue was always accompanied with ASC methylation in matching tumor tissue. Patients without ASC promoter hypermethylation showed a significantly better survival than patients with ASC promoter hypermethylation (P = 0.007). Multivariate analysis revealed ASC promoter methylation as an independent unfavorable prognostic factor (P = 0.009).ConclusionOur results indicate that ASC promoter hypermethylation is a common event in patients with primary NSCLC, and this epigenetic alteration is associated with inferior survival, suggesting that ASC promoter hypermethylation might be an important biomarker for a biologic, aggressive disease in NSCLC.     

Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer

BACKGROUND: Death-associated protein (DAP) kinase is a serine/threonine kinase that is important in ligand-induced programmed cell death and plays an important role in lung cancer metastasis in animal models. Hypermethylation of the promoter represses the expression of the DAP kinase gene. Our purpose was to determine whether the hypermethylation status of the DAP kinase promoter influences the prognosis of non-small-cell lung cancer (NSCLC). METHODS: We retrospectively studied 135 patients with pathologic stage I NSCLC who had undergone curative surgery. Methylation-specific polymerase chain reaction was used to determine the methylation status of the DAP kinase promoter in resected specimens from patients with primary NSCLC. Statistical analyses, all two-sided, were performed to determine the prognostic effect of methylation status on various clinical parameters. RESULTS: Hypermethylation of the DAP kinase promoter was found in 59 (44%) of the 135 tumors. Patients whose tumors exhibited such hypermethylation had a statistically significantly poorer probability of overall survival at 5 years after surgery than those without such hypermethylation (.46 versus.68; P: =.007). Moreover, the groups with and without hypermethylation of the DAP kinase promoter showed a striking difference in the probability of disease-specific survival; i.e., among people who died of lung cancer-related causes specifically, the probability of 5-year survival was.56 for those with such hypermethylation and.92 for those without it (P:<.001). Multivariate analysis indicated that hypermethylation of the DAP kinase promoter is the only independent predictor for disease-specific survival among clinical and histologic parameters tested. CONCLUSIONS: Hypermethylation of the DAP kinase promoter is a common abnormality in early-stage NSCLC. This abnormality is strongly associated with survival, suggesting that DAP kinase plays an important role in determining the biologic aggressiveness of early-stage NSCLC.     

O(6)-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancer.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes mutagenic adducts from the O6 position of guanine, thereby protecting the genome against G to A transition mutations. MGMT is inactivated by promoter hypermethylation in many human cancers and has been associated with G to A mutations in K-ras in colorectal cancer. We hypothesized that MGMT promoter hypermethylation would be associated with an increase in G to A transitions in the p53 gene in non-small cell lung cancer (NSCLC). p53 mutations were detected by both dideoxy sequencing and p53 GeneChip analysis in 92 patients with primary NSCLC. Methylation of the promoter region of the MGMT gene was determined using methylation-specific PCR and was present in 27 of 92 (29%) tumors. Hypermethylation of the MGMT promoter was more common in adenocarcinoma than in other histological types of NSCLC and was also more common in poorly differentiated tumors. MGMT promoter hypermethylation was present significantly more often in tumors with a G to A mutation in p53 (9 of 14; 64%) than in tumors with other types of p53 mutations (11 of 41; 27%; P = 0.02) or in tumors with wild-type p53 (7 of 37; 18%; P = 0.006). MGMT promoter hypermethylation was also strongly associated with G to A transitions at CpG sites. Inactivation of the MGMT gene by promoter hypermethylation alters the pattern of p53 mutation in NSCLC.     

Correlation between insulin-like growth factor-binding protein-3 promoter methylation and prognosis of patients with stage I non-small cell lung cancer.

PURPOSE: The activities of insulin-like growth factors (IGFs) in regulating cell proliferation,differentiation, and apoptosis are modulated by a family of high-affinity specific IGF-binding proteins (IGFBPs), especially IGFBP-3, the most abundant IGFBP in circulation. Hypermethylation of the promoter represses the expression of the IGFBP-3 gene. The purpose of this study was to determine whether the methylation status of IGFBP-3 promoter influences the prognosis of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Eighty-three patients with pathological stage I NSCLC who had undergone curative surgery were investigated for promoter hypermethylation of IGFBP-3 by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of methylation status of the IGFBP-3 promoter on various clinical parameters. IGFBP-3 was the only molecular parameter tested on these tissues in this study. RESULTS: Hypermethylation of the IGFBP-3 promoter was found in 51 (61.5%) of the 83 tumors. The clinicopathological factors, such as age, histological type, histological grade, gender, and smoking status, of corresponding patients, did not exhibit statistically significant association with the methylation status of IGFBP-3 promoter. However, patients with a hypermethylated IGFBP-3 promoter had a significantly lower 5-year disease-specific, disease-free, and overall survival rate than did those without a methylated IGFBP-3 promoter (53.1% versus 86.1%, P = 0.006; 36.5% versus 76.2%, P = 0.007; and 38.9% versus 64.0%, P = 0.022, respectively). Moreover, multivariate analysis indicated that hypermethylation of the IGFBP-3 promoter was the only independent predictor for disease-free and disease-specific survival among the clinical and histological parameters tested. CONCLUSIONS: Hypermethylation of the IGFBP-3 promoter, as measured by methylation-specific PCR, is a frequent phenomenon and strongly associated with poor prognosis among patients with stage I NSCLC.     

Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer.

PURPOSE AND EXPERIMENTAL DESIGN: The etiologic association and prognostic significance of mismatch repair gene/protein alterations have never been examined in nonsmoking lung cancer. Therefore, we investigated protein expression and promoter hypermethylation of hMLH1 and hMSH2 genes in the tumor specimens from 105 nonsmoking female non-small cell lung cancer (NSCLC) patients. Immunohistochemistry and restriction enzyme-based multiplex PCR were used to examine the protein expression and promoter hypermethylation, respectively. The occurrence of gene/protein alteration for each gene was compared with the patients' clinicopathologic variables as well as the overall survival and cancer-specific survival rates. RESULTS: Protein expression alteration and promoter hypermethylation were observed in 66% to 67% and 30% to 34% of tumor specimens for hMLH1 and hMSH2 genes, respectively. Loss of hMLH1 and hMSH2 protein expression was significantly associated with their promoter hypermethylation (P < 0.0001 and P = 0.049). The overall survival and cancer-specific survival rates were significantly lower in patients with promoter hypermethylation of hMSH2 gene than in those without hypermethylation (P = 0.038 and P = 0.004). The poor prognosis was still especially significant in adenocarcinoma (P = 0.035 and P = 0.061) and early-stage NSCLC patients (P = 0.067 and P = 0.041). CONCLUSION: Our data suggest that hMLH1 is the major altered mismatch repair gene involved in nonsmoking NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMSH2 gene may be a potential prognostic factor in nonsmoking female lung cancer.     

Hypermethylation of O6-methylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases.

PURPOSE: Because O(6)-methylguanine-DNA methyltransferase (MGMT) plays an essential role in repairing DNA damage caused by environmental alkylating chemicals, we were interested in determining whether we could see any obvious changes in the properties of colorectal cancers (CRCs) in which the MGMT gene had been silenced by hypermethylation and hence in which very few MGMT protein molecules were being produced. EXPERIMENTAL DESIGN: We used a methylation-specific PCR assay to determine the methylation status of the MGMT promoter in the DNA molecules extracted from CRC and nontumor tissue samples from 116 patients who had undergone CRC surgery and for whom clinical outcome information was available on file. RESULTS: We found evidence of MGMT promoter hypermethylation in 26 of 90 CRC cases, and we noted that the later the stage at which a tumor was diagnosed, the less likely its MGMT promoter was to be methylated (P = 0.03, adjusting for chemotherapy), especially for stage D patients (P = 0.01). We also found that CRC patients with unmethylated MGMT promoters were much more likely to experience recurrence within 36 months than patients with hypermethylated MGMT promoters (crude odds ratio, 14.0; 95% confidence interval, 2.42-81.01). After adjustment for stage, CRC patients with unmethylated MGMT promoters who had been exposed to chemotherapy were found to have a 5.3-fold greater risk of recurrence than those who had no exposure to chemotherapy (95% confidence interval, 1.15-30.92). CONCLUSIONS: Hypermethylation of the MGMT promoter may be predictive of a low risk of recurrence in CRC patients receiving adjuvant chemotherapy.     

Methylation status of insulin-like growth factor-binding protein-3 promoter in prostate cancer tissues

Background: It has been postulated that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is a mediator of growth suppression signals. Recently, a correlation between the promoter hypermethylation of IGFBP‐3 and cancer risk was reported in hepatocellular cancer and in non‐small‐cell lung cancer (NSCLC). We investigated whether the methylation status of IGFBP‐3 promoter in prostate tissues influences the progression and prognosis of prostate cancer. Methods: We conducted a case‐control study consisting of 38 prostate cancer patients and 57 control subjects, and assessed the relationship of promoter hypermethylation at IGFBP‐3 with the clinical and pathological tumor characteristics of these patients. The methylation status was analyzed by two methylation‐specific PCR techniques. Results: Hypermethylation of the IGFBP‐3 promoter was found in 13 (34.2%) of the 38 cases, and 11 (19.3%) of the 57 controls with one method using a hepatocellular primer as described by Hanafusa et al. With other methods using a NSCLC‐primer, as described by Chang et al., hypermethylation was found in four (10.5%) of the cases and two (3.5%) of the controls. No significant differences were observed in the methylation frequencies between the controls and the cases. When the cases were stratified based on their clinical stage (localized or metastatic) and pathological grade (Gleason score of <7 or ≥7), the promoter hypermethylation status of IGFBP‐3 also showed no significant differences. Conclusions: Hypermethylation of the IGFBP‐3 promoter was observed in prostate tissues, but we found no significant association of IGFBP‐3 promoter hypermethylation with the risk of developing prostate cancer.     

Aberrant Promoter Methylation Profile in Pleural Fluid DNA and Clinicopathological Factors in Patients with Non-small Cell Lung Cancer

The aim of this study was to investigate the prognostic value of hypermethylation of tumor suppressor genesin patients with non-small cell lung cancer (NSCLC). In samples from 34 lung patients with malignant pleuraleffusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of thepromoters of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, rasassociation domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK),and retinoic acid receptor ß (RARß).There is no association between methylation status of five tumor suppressorgenes including MGMT, p16INK4a, RASSF1A, DAPK and RARß in pleural fluid DNA and clinicopathologicalparameters including clinical outcome. Aberrant promoter methylation of tumor suppressor genes in pleuralfluid DNA could not be a valuable prognostic marker of NSCLC patients with malignant pleural effusion.     

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.     

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.     

Methylation of the O6-methylguanine-DNA methyltransferase promoter suppresses expression in mouse skin tumors and varies with the tumor induction protocol

Hypermethylation of CpG sites within the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene occurs frequently in human cancer, preventing both MGMT expression and repair of alkylation damage. To assess the role of MGMT in the development of mouse skin tumors induced by initiation-promotion protocols, methylation of the MGMT promoter was examined in tumor DNA using methylation-specific PCR. To determine whether MGMT promoter methylation was affected by the tumor induction protocol, tumors were initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein. Although the MGMT promoter was not methylated in normal skin, promoter methylation was found in 56 of 136 papillomas (41.2%) and in 19 of 37 squamous cell carcinomas (51.4%). When methylation of the MGMT promoter was compared in the 4 treatment groups, hypermethylation was found more frequently in tumors initiated by DMBA and promoted by mezerein, a protocol associated with a high frequency of malignant conversion. Methylation was found in some tumors as early as 5 weeks after initiation, but the methylation frequency increased with time. MGMT promoter methylation reduced MGMT expression as determined by immunohistochemistry. Although MGMT promoter methylation was not generally correlated with ras mutations, the frequency of MGMT methylation was higher in MNNG-initiated, mezerein-promoted papillomas with mutations in Ha-ras compared to papillomas with Ki-ras. Methylation of the MGMT promoter, associated with reduced MGMT expression, is found in nearly half of mouse skin tumors, but varies with both the tumor initiator and tumor promoter, and may be a key step in the progression from papillomas to carcinomas.     

DNA修复基因MGMT启动子区过甲基化与食管鳞状细胞癌

目的：O^6－甲基鸟嘌呤DNA甲基转移酶（MGMT）可以转移DNA加合物O^6－甲基鸟嘌呤中的甲基,从而修复DNA损伤,许多肿瘤中发现MGMT基因启动子过甲基化导致该基因失活,我们研究了MGMT基因启动子甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应及测序方法分析食管癌。癌旁组织和正常食管上皮中MGMT启动子甲基化状态。结果：在检测的199例食管癌组织中,46例（38．7％）有MGMT基因启动子过甲基化,相应癌旁组织22例中也有5例（22．7％）出现MGMT基因甲基化,而21例正常食管上皮均无此种改变。结论：MGMT基因启动子过甲基化是食管癌中常见的分子事件,可能发生在癌过程的早期阶段。     

Quantitative detection of multiple gene promoter hypermethylation in tumor tissue, serum, and cerebrospinal fluid predicts prognosis of malignant gliomas

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the malignant transformation in gliomas. We hypothesized that quantitative analysis of methylated genes will provide prognostic values in malignant glioma patients. We used an immunocapturing approach followed by real-time polymerase chain reaction analysis to detect altered patterns of promoter methylation in O-6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, tissue inhibitor of metalloproteinase-3 (TIMP-3), and thrombospondin 1 (THBS1). The tumor tissue and paired serum as well as cerebrospinal fluid (CSF) from 66 patients with malignant gliomas were studied. Serum and CSF from 20 age-matched noncancer individuals were used as control. Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1 was detected at high frequencies in tumor tissue, serum, and CSF. None of the control serum or CSF showed aberrant methylation. Hypermethylation in serum and CSF DNA was all accompanied with methylation in the corresponding tumor tissues with 100% specificity. Highly elevated MGMT, p16INK4a, and THBS1 methylation levels in gliomas serum were the sole independent factors predicting inferior overall survival in this cohort. For progression-free survival, hypermethylation of MGMT and THBS1 in CSF were the independent prognostic factors. Multiple gene promoter hypermethylation analysis appears to be promising as a prognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSF DNA. Evaluation of these changes may help in selecting glioma patients for optimal adjuvant treatments and modifying chemotherapy.     

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.