This study confirms and extends previous observations that identification of serum DNA methylation in specific set of genesis a potentially useful approach to detect lung cancer patients. Serum DNA methylation was more frequently observed in patients with lung cancer than those with age-matched controls. Although the sensitivity for the diagnosis of lung cancer was only 35.5% when analyzed by a single gene (DCC), the high specificity (100%) indicates the usefulness of QMSP assay for lung cancer detection. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% but decreased the specificity from 100% to 73%. Addition of gene/genes with reasonable sensitivity and very high specificity with DCC may allow higher diagnostic coverage while still proving 100% specificity. Of note, serum DNA methylation could be identified even in patients in the early stages of lung cancer, whereas conventional serum protein tumor markers were rarely elevated, indicating that this DNA-based method is more sensitive than protein-based method for diagnosis of lung cancer in early stage. In our previous study (37
), DCC methylation was detected in 54% of cases with 100% specificity in evaluation set and fell to 26% in the independent set. In the present cohort DCC methylation was detected in 36% of cases, which compares favorably and suggests that about a third of lung cancer cases could be detected by DCC alone with high specificity.
In former studies, methylation in tumor tissues was detected in 25% to 41% for p16INK4a
, 30% to 40% for RASSF1A
, and 16% to 27% for MGMT
). These results were consistent with our data in 10 tumor tissue samples. The frequency of detecting methylated genes in serum was about half to two thirds compared with that in tumor tissues. However, when we consider that tumor-derived DNA in blood is generally detectable in less than half of cancer patients (32
), the frequency of methylation in serum DNA in our study may be quite reasonable. Laird reviewed the studies examining the methylation status of serum/plasma DNA in patients with various neoplasms and indicated that clinical sensitivity of DNA methylation was ~50% (40
). Esteller et al. did methylation analysis in serum DNA from patients with non–small cell lung cancer for multiple genes and showed 33% to 80% clinical sensitivity by combination analysis of several genes (41
Among various techniques used for methylation analysis, we adopted a simple method of QMSP analysis. The specificity of the primers and probes we used in this study has been verified using genomic sequencing and/or MSP. Recently, several studies showed improved detection rates of methylation status using a nested PCR approach or a quantitative real-time PCR technique (42
). The sensitivity of the Taqman method was reported to be 10-fold higher than conventional qualitative MSP and we feel that more sensitive technologies are very likely to reduce specificity (44
Although promoter methylation was observed predominantly in lung cancer patients, several controls were methylation positive for one of the 5 genes (except DCC) in the independent test set. In methylation pattern analysis in primary tumors and paired serum samples, one lung cancer patient with serum DNA methylation for CALCA and CDH1 did not show the same alteration in the corresponding tumor tissue. We considered the following as possible explanation of these apparent false positive results. Firstly, the methylated serum DNA might be derived from undetected precancerous lesions in these cases. According to previous reports, aberrant promoter methylation is clearly detectable in precancerous lesions, such as dysplasia and nonmalignant lung tissues of patients with lung cancer (45
). In a prospective study Belinsky et al. reported that 3 genes in a panel of 6 were hypermethylated in the sputum of high risk individuals, resulting in a >6 fold risk of developing lung cancer within 18 months (47
). Long term follow up of control subjects with methylated DNA of several genes in serum samples will elucidate the risk of developing lung cancer in these subjects. Secondly, aberrant methylation might be caused by environmental factors, such as smoking, yet may not necessarily lead to clonal expansion (25
). Finally, endogenous factors like the occurrence of other occult malignancies or age-related methylation could be the cause in the control group.
Hypermethylation of the APC and CDH1 promoters in serum DNA from lung cancer patients often occurred together. Given that hypermethylation of these genes is common in lung cancer, these associations could have occurred by chance alone and should be interpreted with caution. However, a possible interaction between these genes in lung cancer deserves further evaluation. CDH1 methylation occurred more often in patients with tumor sizes < 3 cm (20/24) 83% compared to patients with tumors ≥ 3 cm (24/46) 52%. p=0.01. It is well studied that CDH1 inactivation is related to invasion, metastasis and increased tumor size. So our finding of presence of any CDH1 methylation decreased the probability of tumor size ≥ 3 cm (OR=0.22 (95% CI: 0.06, 0.74), p=0.01) need to be further evaluated.
To consider the use of serum DNA methylation as a marker in lung cancer active screening, several issues must be considered. Certainly optimal specificity and sensitivity must be achieved before the approach can be used alone or in combination with imaging. It is tempting to combine methylation with promising methods such as low-dose spiral computed tomography (49
). Because one of the serious limitations of low-dose spiral computed tomography is its poor specificity, a combination with serum DNA methylation may overcome this limitation.
Serum DNA methylation is found in early-stage disease (73% and 77% in stage I and stage II respectively) and thus could be tested before invasive procedures after testing in a larger cohort and in a prospective setting. Although further evaluation is essential, the results in this study indicate the substantial usefulness of methylation marker for detection of lung cancer. Further studies are warranted to confirm the accuracy of the approach and search for the best combination of genes for methylation analysis. Moreover, it is important to investigate prospectively whether methylation-positive non-clinical cancer cases will ultimately develop malignancies in the near future.