|Year : 2016 | Volume
| Issue : 1 | Page : 340-349
Promoter methylations of RASSF1A and p16 is associated with clinicopathological features in lung cancers
Ji-Chang Han1, Feng Xu1, Na Chen1, Guan-Bin Qi1, Yu-Ju Wei2, Hong-Bing Li1, Yi-Jie Zhang1, Jing-He Li1, Xiu-Li Wang1, Wen Xu1, Xiao-Feng Li1, Li-Fang Jin1, Jiao-Yuan Jia1, Zhong-Sen Ma2
1 Department of Respiration, Huaihe Hospital of Henan University, Kaifeng, People’s Republic of China
2 Department of Gastroenterology, Huaihe Hospital of Henan University, Kaifeng, People’s Republic of China
|Date of Web Publication||13-Apr-2016|
Department of Respiration, Huaihe Hospital of Henan University, Kaifeng
People’s Republic of China
Source of Support: None, Conflict of Interest: None
Objection: The aim of this study is to investigate the association between promoter methylation of RASSF1A and p16 and the clinicopathological features in lung cancers.
Materials and Methods: PubMed, EBSCO, Ovid, Wiley, Web of Science, Wanfang, and VIP databases were searched using combinations of keywords related to RASSF1A, p16, methylation, and lung cancers. After screening for relevant studies, following a strict inclusion and exclusion criteria; the selected studies were incorporated into the present meta.analysis conducted using Comprehensive Meta Analysis 2.0. (CMA 2.0).
Results: We initially retrieved 402 studies, out which 13 studies met the inclusion and exclusion criteria for this meta.analysis, and contained a total of 1,259. patients with lung cancers. The results of this meta.analysis showed that the differences in promoter methylation ratio between the lung cancer patients in tumor, node, metastasis. (TNM) I.II and III.IV were not statistically significant. Based on histological types, patients with adenocarcinoma. (AC) and squamous cell carcinoma. (SCC) showed no significant differences in the promoter methylation ratios of RASSF1A, while the promoter methylation ratio of p16 was significantly higher in SCC patients compared to AC patients. Based on smoking status, the promoter methylation ratios of both RASSF1A and p16 was significantly higher in lung cancer patients with smoking history compared to nonsmokers.
Conclusion: The present meta.analysis provides convincing evidence that the promoter methylation ratio of RASSF1A and p16 is associated with clinicopathological features in lung cancers, and could be used as effective biomarkers in early diagnosis in lung cancers.
Keywords: Biomarker, clinicopathological features, diagnosis, lung cancers, meta-analysis, methylation, p16, RASSF1A
|How to cite this article:|
Han JC, Xu F, Chen N, Qi GB, Wei YJ, Li HB, Zhang YJ, Li JH, Wang XL, Xu W, Li XF, Jin LF, Jia JY, Ma ZS. Promoter methylations of RASSF1A and p16 is associated with clinicopathological features in lung cancers. J Can Res Ther 2016;12:340-9
|How to cite this URL:|
Han JC, Xu F, Chen N, Qi GB, Wei YJ, Li HB, Zhang YJ, Li JH, Wang XL, Xu W, Li XF, Jin LF, Jia JY, Ma ZS. Promoter methylations of RASSF1A and p16 is associated with clinicopathological features in lung cancers. J Can Res Ther [serial online] 2016 [cited 2020 Jan 28];12:340-9. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/340/154926
| > Introduction|| |
Lung cancer accounts for 27% of all cancer-related mortalities in men and women, and majority of patients present at diagnosis with advanced stage or metastatic disease of the lungs., Non-small cell lung cancer (NSCLC) constitutes 80–85% of all lung cancers, with small cell lung cancer (SCLC), adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma as the other main histological types. Epidemiological studies indicate that air pollution, other environmental factors, and cigarette smoking are strongly associated with lung cancers, and 80–90% of patients with NSCLCs are proved to have smoking status., The tumor, node, metastasis (TNM) staging system for lung cancers is used widely as a guide for planning treatment, determining prognosis, evaluating treatment results, and facilitating information exchange between multiple centers., Surgical resection is the most effective treatment in lung cancers when diagnosed early, and majority NSCLC patients have no surgical option since approximately 70% of patients are diagnosed with advanced stage disease. Therefore, chemotherapy treatment is more widespread than surgery, and platinum drugs remain the most efficacious therapy in these patients., Despite enormous progress in early detection and in development of new drugs and innovative treatment strategies in recent years, lung cancer is still a deadly disease with 5-year survival rates at about 17%. It is therefore of critical urgency to evaluate suitable biomarkers for NSCLC prognosis and to establish preventive measures. In the context of lung cancers, tobacco carcinogens and other environmental and genetic factors appear to influence DNA methylation, suggesting that DNA methylation plays an important role in the pathology of lung cancers; and therefore, early detection of DNA methylation changes is a new frontier in lung cancer diagnosis and clinical management.,
DNA methylation profiling identified tumor-specific hyper- and hypomethylation patterns in the promoter and distal regulatory elements of genes involved in epithelial cell differentiation and transformation. Promoter hypermethylation results in gene silencing, and can be a mechanism of tumor-suppressor gene inactivation in cancers. The p16 and RASSF1A are tumor-suppressor genes frequently inactivated by de novo promoter hypermethylation in NSCLC. The tumor-suppressor gene, p16(chromosome 9p21.3), is a cell cycle regulator involved in the inhibition of G1 phase progression in normal cells. The de novo methylation of the p16 promoter was reported to occur at a high frequency in lung cancers associated with cigarette smoke.RASSF1A (chromosome 3p21.3) is a member of the RASSF family and shares 30–50% amino acid homology with other members of the family, and it is inactivated by epigenetic silencing at high frequency in a broad range of tumors.RASSF1A is inactivated early in lung carcinogenesis and might be related to early events in NSCLC progression. Some studies suggest that the hypermethylation status of p16 and RASSF1A genes is associated with a significantly increased risk of lung cancer and the risk of lung cancer increased as the methylation level increased.,,, On the other hand, contradicting data indicate that gene methylation of p16 and RASSF1A were only weakly related to smoking status in lung cancers and have no relationship with lung cancer progression. Therefore, the aim of this study was to investigate the correlation between the promoter methylation status of RASSF1A and p16 and lung cancer progression.
| > Materials and Methods|| |
Published studies related to the association of promoter methylation of RASSF1A and p16 and lung cancers were exhaustively searched using following computerized databases: PubMed, EBSCO, Ovid, Springer Link, Wiley Online Library, The Cancer Genome Atlas More Details (TCGA), Embase, Web of Science, Chinese Biomedical Database, the Chinese Journal Full-Text Database, China National Knowledge Infrastructure (CNKI), Wanfang, and the VIP Database (since inception to October, 2014). Additional pertinent literatures were obtained using manual search of cross-references from highly relevant studies. A combination of keywords and free words was applied in of database searches resulting in a highly efficient and sensitive searching strategy:(”lung neoplasms” or “lung cancers” or “non-small cell lung cancer” or “carcinoma, non-small-cell lung”) and (”cyclin-dependent kinase inhibitor p16” or “multiple tumor suppressor 1”or “MTS”or”cyclin dependent kinase inhibitor p16”or”CDKN2 Protein” or “cyclin-dependent kinase Inhibitor-2A”or”p16INK4A Protein”or”MTS1 protein”or”p16”) and (”RASSF1 protein, human” or “ras association (RalGDS/AF-6) domain family 1 protein, human” or “RASSF1B protein, human” or “RASSF1C protein, human” or “RASSF1A protein, human”) and (”methylation” or “DNA methylation” or “hypermethylation” OR “demethylation” or “methylation”).
Inclusion and exclusion criteria
The following inclusion criteria were considered for selecting published articles for inclusion in the present study: (i) Study types should be cohort studyrelated to promoter methylation of RASSF1A and p16 and lung cancers; (ii) subjects in the enrolled studies should have confirmed diagnosis of lung cancers; (iii) detection method of aberrant methylation should be methylation-specific polymerase chain reaction (MS-PCR); (iv) the outcomes index should be the rate of RASSF1A and p16 gene promoter methylation in patients with lung cancer among clinical parameters such as TNM grading, histological classification; and (v) language was restricted to Chinese and English for selected studies. The exclusion criteria were: (i) Summary and abstracts; (ii) insufficient information; and (iii) duplicate publications.
Data extraction and quality assessment
The data were extracted from each included study by two independent investigators, and the following information was collected: Surname and initials of the first author, year of submission, country, ethnicity, language, age, gender, pathological type, TNM stage, and smoking status. Disagreement on the inclusion of any study was settled by consulting a third investigator. The quality of included trials was assessed by the Critical Appraisal Skills Programme (CASP) for cohort studies (http://www.casp-uk.net). The CASP criteria are standardized as follows: The study address a clearly focused issue (CASP01); the cohort recruited in an acceptable way (CASP02); the exposure accurately measured to minimize bias (CASP03); the outcome accurately measured to minimize bias (CASP04); the author identified all important confounding factors (CASP05); the follow-up subjects complete enough and long enough (CASP06); the result of this study is complete (CASP07); the result is precise (CASP08); the result is reliable (CASP09); the results can be applicable to the local population (CASP10); the result fits with other available evidence (CASP11); and the results have implication for practice (CASP12).
All statistical tests for this meta-analysis were performed with Comprehensive Meta Analysis 2.0 (CMA 2.0). The odds ratio (OR) with 95% confidence interval (95%CI) were estimated by the fixed effects model or random effects model to evaluate the association of promoter methylation of RASSF1A and p16 and clinicopathological features of lung cancers. Z-test was employed to detect the significance of the pooled effect size. Cochran's Q-statistic was used (Ph< 0.05 was considered significant) and I2 tests were employed to quantify heterogeneity among studies. When Ph< 0.05 or I2> 50% indicated heterogeneity, the random effects model was preformed; otherwise, the fixed effects model was applied. In order to evaluate the influence of a single study on the overall estimate, a sensitivity analysis was employed. In addition, potential publication bias was examined by funnel plots, classic fail-safe N, and Egger's linear regression test to ensure the reliability of results (Ph< 0.05 was considered significant).
| > Results|| |
Selection of eligible studies
Firstly, 402 potential articles were identified from the electronic database searches. After excluding duplicates (n = 24), non-human studies (n = 10), letters, reviews, meta-analyses (n = 6), and studies unrelated to research topics (n = 52);310 full-text articles remained. Based on the inclusion and exclusion criteria, we further eliminated 30 studies that were not case-control or cohort, 42 studies for being irrelevant to RASSF1A, 35 studies not relevant to p16, and 178 studies without sufficient data related to clinicopathological features, such as TNM stage and smoking status. Finally, 13 case-control studies ,,,,,,,,,,,, published between 2003 and 2013 were selected for the current meta-analysis and contained a total of 1,259 lung cancer patients. The baseline characteristics and CASP score for the 13 eligible studies are summarized in [Table 1].
|Table 1: Baseline characteristics for the eligible studies associated with promoter methylation of RASSF1A and p16 may be associated with clinicopathological features of lung cancers|
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Meta-analysis of the association with promoter methylation of RASSF1A and p16 and TNM grade of lung cancers patients
Nine studies investigated the association of promoter methylation status of RASSF1A and p16 with TNM grade of lung cancers. Heterogeneity was observed in the meta-analysis (RASSF1A: I2= 79.8%, Ph< 0.001; p16: I2= 76.7%, Ph< 0.001), thus the random effect model was adopted. Our findings clearly demonstrated that promoter methylation ratios, with respect to the two genes, were not significantly different between the lung cancers patients in TNMI–II and III–IV (RASSF1A: OR = 0.646, 95%CI = 0.261–1.602, P = 0.346; p16: OR = 0.495, 95%CI = 0.224–1.094, P = 0.082) [Figure 1]a and [Figure 2]a.
|Figure 1: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for the comparison of TNM I–II and III–IV patients in RASSF1A promoter methylation ratio. TNM = Tumor, node, metastasis, CI = confidence interval|
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|Figure 2: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for the comparison of TNM I-II and III-IV patients in p16 promoter methylation ratio|
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Meta-analysis of the association with promoter methylation of RASSF1A and p16 and histology type of lung cancers patients
Eleven studies investigated the association of promoter methylation of RASSF1A with the histology type of lung cancers. No heterogeneity was observed in the meta-analysis (I2= 36.7%, Ph = 0.106), thus the fixed effect model was adopted. Ten studies investigated the association between promoter methylation of p16 and histology type of lung cancers. Heterogeneity was observed in the meta-analysis (I2= 72.1%, Ph< 0.001), thus random effect model was adopted. The results suggested that the promoter methylation ratio of RASSF1A was not significantly different between SCC and AC patients (OR = 0.854, 95%CI = 0.642–1.135, P = 0.276), butpromoter methylation ratio of p16 was higher in SCC patients compared to AC patients (OR = 2.259, 95%CI = 1.17–4.361, P = 0.015) [Figure 3]a and [Figure 4]a.
|Figure 3: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for the comparison of SCC and AC patients in RASSF1A promoter methylation ratio|
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|Figure 4: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for the comparison of SCC and AC patients in p16 promoter methylation ratio. SCC = squamous cell carcinoma, AC = adenocarcinoma|
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Meta-analysis of the association with promoter methylation of RASSF1A and p16 and smoking status of lung cancers patients
Ten studies investigated the association between promoter methylation of RASSF1A and smoking status in lung cancer patients. Heterogeneity was observed in the meta-analysis (I2= 59.2%, Ph = 0.009), thus random effect model was adopted. Nine studies investigated the association between promoter methylation of p16 and smoking status in lung cancers patients. Heterogeneity was observed in the meta-analysis (I2= 64.1%, Ph = 0.004), thus random effect model was adopted. Our findings demonstrated that the promoter methylation ratio of RASSF1A and p16 is significantly higher in patients with smoking history compared to nonsmoker lung cancer patients (RASSF1A: OR = 0.484, 95%CI = 0.282–0.832, P = 0.009; p16: OR = 0.458, 95%CI = 0.231–0.908, P = 0.025) [Figure 5]a and [Figure 6]a.
|Figure 5: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for comparison of smoking status in RASSF1A promoter methylation ratio|
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|Figure 6: Forest plots (a), sensitivity analysis (b), and funnel plot (c) for comparison of smoking status in p16 promoter methylation ratio|
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Sensitive analysis and publication bias
Sensitivity analyses were performed, and the results in our study demonstrated that no single study had the weight to impact on the overall estimate of OR of promoter methylation ratio of RASSF1A and p16 in TNM grades, histology type, and smoking status [Figure 1]b, [Figure 2]b, [Figure 3]b, [Figure 4]b, [Figure 5]b, and [Figure 6]b. Further, we did not observe any obvious asymmetry from the shapes of the funnel plots, and the Egger's regression test (P > 0.05) and classic fail-safe N suggested the absence of publication bias; thus no significant publication bias was detected in the meta-analysis in our systematic reviews [Figure 1]c, [Figure 2]c, [Figure 3]c, [Figure 4]c, [Figure 5]c, and [Figure 6]c.
| > Discussion|| |
In this study, we assessed methylation of two important lung cancers-associated genes in a carefully selected cohort of patients diagnosed lung cancers, and assessed the correlation between gene methylation with clinicopathological features. We studied the promoter methylation status of p16 and RASSF1A, and correlated the results with clinicopathological parameters. The p16 and RASSF1A are tumor-suppressor genes frequently inactivated by de novo promoter hypermethylation in NSCLC. Our results demonstrated that hypermethylation of RASSF1A and p16 was significantly higher in smokers than in nonsmoker patients with lung cancers, suggesting that tobacco smoke potentially influenced the methylation status of RASSF1A and p16. The p16 gene is commonly inactivated in lung cancers and preinvasive lesions of bronchial epithelium, and the most common mechanism of inactivation is transcriptional silencing by promoter methylation. The p16 gene with promoter CpG island hypermethylation, which is associated with transcriptional silencing, is an early event in multiple human cancers, including lung cancers. Abnormal expression of cell cycle regulators of G1 to S transition phase represents a fundamental event in the onset or progression of lung cancers and, in this respect, silencing of p16 expression should be considered as biomarker for identification of patients likely to develop a recurrent disease, and who might possibly benefit from a more aggressive treatment approach based on p16 status. The RASSF1A methylation density was remarkably responsive to smoking status, with former smokers having higher methylation density versus never-smokers and current smokers. Hypermethylation of the CpG islands within the RASSF1A promoter region, rather than mutations in RASSF1A, was found to be the major cause of loss-of-expression., Overall, epigenetic inactivation of tumor-suppressor genes play a crucial role in the pathogenesis of tumors, including lung cancers and the epigenetic silencing of tumor-suppressor genes, along with genetic alterations such as mutations and deletions, drive tumor progression.
We found significant differences in p16 methylation between SCCs than ACs. This finding could indicate that differential methylation of specific genes may lead to the development of different histologic types and the methylation of specific genes may lead to the development of different histologies in NSCLCs. Based on previous studies, mutations in p16 are more common in SCC than AC. The epigenetic mechanism of p16 hypermethylation of CpG islands in the promoter region is a classic example of inactivation of tumor-suppressor genes, which is a hallmark of human tumors in general, and lung cancer, in particular. Aberrant promoter methylation of promoter CpG islands is an important mechanism for gene silencing and is also a promising tool for the development of accurate molecular biomarkers that are useful to predict the disease course., In our study, we detected p16 methylation of several cancer-related genes that were downregulated in microarray assays. Consistent with our results, aberrant methylation of CpG islands in the promoter region has been associated with the transcriptional inactivation of several tumor-suppressor genes in human cancers. On the other hand, we did not find significant differences in RASSF1A methylation between SCCs than ACs. We also assessed the relationship between hypermethylation of RASSF1A and p16 with TNM stages, but the results did not find statistically significant differences between promoter methylation ratio between the lung cancers patients in TNM I–II and III–IV.
Limitations of this meta-analysis should be acknowledged. Firstly, the sample sizes in several of the included studies were relatively small, which may reduce the strength of our conclusions. Secondly, all eligible studies were written and published in English and Chinese languages indexed by the selected databases. It is possible that published studies in other languages or unpublished studies could have been missed, which might bias the results. Thirdly, although we have searched enough authoritative databases, we may miss some databases out during the process of searching, which might affect the accuracy of our results.
In conclusion, the present meta-analysis provides convincing evidence that the promoter methylation ratios of RASSF1A and p16 may be used to predict the clinicopathological features, and are effective biomarkers for early diagnosis in lung cancers. The detection of methylation status of tumor-suppressor genes has valuable clinical application in early diagnosis of lung cancers. Therefore, future studies with larger sample sizes and including other potential markers will be necessary to confirm our findings and establish a reliable early diagnostic criterion for lung cancers.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]