We assessed the promoter methylation of the 6 genes in a large cohort of well-characterized NSCLC subjects using Q-MSP technique in the present study, including CALCA, CDH1, DAPK1, IRX2, TIMP3
, and PAX6. CALCA
is known to encode a peptide hormone that plays a role in maintenance of calcium levels in blood serum and T-and B-cell regulation in certain malignancies, which is frequently methylated in multiple types of cancer [16
is a classical cadherin from the cadherin superfamily, which encodes a calcium dependent cell-cell adhesion protein. Epigenetic inactivation of CDH1
is thought to contribute to progression in cancer by increasing proliferation, invasion, and/or metastasis [18
encodes a structurally unique calcium/calmodulin-dependent serine/threonine kinase which acts as a positive regulator of apoptosis. It is frequently methylated in human cancers as a tumor suppressor gene [19
]. IRX2 is a member of the Iroquois homeobox transcription factor family, which is involved in developmental pattern formation in multiple organs such as the brain and heart [20
]. It is highly specific for tumor-associated methylation, and little or no methylation is found in nonmalignant lung tissue [21
]. TIMP3 is an angiogenesis inhibitor, and its epigenetic inactivation is associated with neovascularization and invasion in human malignancy [22
]. PAX6, a transcription factor, has currently been suggested to function as a tumor suppressor in glioblastoma and to act as an early differentitation marker for neuroendocrine cells [23
], which is frequently methylated in human cancers [21
]. Similarly, our findings also showed that 3 of 6 genes had significantly higher methylation level in tumor tissues than nonmalignant lung tissues, including CALCA, CDH1
, and PAX6
genes. Importantly, with 100% diagnostic specificity, excellent sensitivity, ranging from 44.9 to 84.1%, was found for each of the 6 genes. The high specificity and frequency of these methylation markers make them excellent candidates for future applications developed for early diagnosis and prognosis evaluation of lung cancer.
Given smoking plays the central role in lung cancer development, it is somewhat surprising that we did not find significant association between promoter methylation of most genes and smoking history, in agreement with most studies [25
]. However, several previous studies have reported aberrant DNA methylation of tumor-related genes was associated with tobacco smoking [29
]. It is possible that lung cancer as a result of tobacco smoking is a complex disease with many unique genetic and epigenetic features. Better understanding of the molecular mechanisms underlying this disease would undoubtedly improve the outcomes of patients with smoking-associated lung cancer.
Our findings of substantial differences in promoter methylation depending on histologic typing of NSCLC have been reported to some degree in the literatures. Unlike what observed in the present study, a number of others have noted that adenomatous polyposis coli (APC
), cyclin D2 (CCND2
), potassium voltage-gated channel, subfamily H (eag-related), member 5 (KCNH5
), and runt-related transcription factor 2 (RUNX2
) genes were significantly more frequently in ADC compared to SCC [33
]. Similar to the present study, although no statistical significance was observed, promoter methylation of DAPK1
gene was detected with higher frequency in SCC compared to ADC [36
]. Conversely, there was not significant difference in promoter methylation of CDH1
gene between SCC and ADC in this literature [36
]. Anyway, we believe that our findings that promoter methylation of several genes is more frequent in SCC compared to ADC, particularly IRX2
gene, are new to the literature. Moreover, methylation level of DAPK1
genes was significantly associated with lymph node metastasis and invasion/adhesion, suggesting that methylation degree of these genes might contribute to oncologic outcomes of NSCLC patients.
A very recent study showed that a number of important differences in frequency of promoter methylation in females compared to males, suggesting that promoter methylation is associated with gender [36
]. However, these substantial differences have not been consistently noted in the previous literatures [35
]. Similarly, promoter methylation was also not significantly associated with gender in the present study. It is possible that the differences in the promoter methylation associated with gender may be related to the geographical or cultural differences in carcinogen exposures, including cigarette smoking, dietary factors, occupational and environmental chemical exposure [40
]. The discrepant results might also have been due to genetic differences of the study populations. A previous study indicated that patients harboring functional polymorphic variants of glutathione S-transferase pi 1 (GSTP1
) had a higher risk of promoter hypermethylation of O-6-methylguanine-DNA methyltransferase (MGMT
) gene [43
], suggesting that functional variants in the genes involved in the folate metabolism, DNA methylation, carcinogen metabolosim and the repair of methylation may play an important role in the susceptibility to promoter methylation. However, it is possible that these differences are attributable to chance as a result of the relatively small number of the study subjects examined. Therefore, further study with more subjects will be needed.
Interestingly, multivariable analyses revealed that the promoter methylation of a number of genes had signficant higher frequency in patients with pleural indentation compared with without pleural indentation, suggesting that promoter methylation may be a potentially increased risk for pleural indentation of NSCLC patients. Pleural indentation is a well-known imaging sign on chest computed tomography (CT) that suggests a possible pleural invasion by peripheral NSCLC, particularly ADC [44
]. A previous study showed that pleural involvement was significantly correlated with a poor prognosis in NSCLC, suggesting that pleural involvement may be one of most important factors to affect on the prognosis of NSCLC [46
]. Although the degree of pleural invasion is clinically important, the accurate preoperative evaluation is sometimes difficult. CT and magnetic resonance imaging (MRI) are usually used in the evaluation of tumor extent, and diagnosis of chest wall invasion, pleural dissemination and pleural effusion can be easily made preooperatively by this way [47
]. However, pleural dissemination and/or pleural effusion, which were not documented preoperatively, were sometimes revealed during operation. In addition, the accruate visceral pleural invasion can not be made with preoperative CT or MRI. In the present study, our findings suggested that promoter methylation of certain genes can increase the risk of pleural indentation in NSCLC. DNA methylation is the earliest and most frequent molecular events in human tumorigenesis. Detection of aberrant methylation using some high-senstive approaches can be thus used to predict and evaluate the pleural invovlement in NSCLC.