Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Abstract

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a "Multiplex Methylation Specific PCR" (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.

Homeobox A9 directly targeted by miR‐196b regulates aggressiveness through nuclear Factor‐kappa B activity in non‐small cell lung cancer cells

“…Therefore, our data provide evidence suggesting that HOXA9 is controlled by miR‐196b in NSCLC cells. Collectively, these data strongly suggest that HOXA9 expression in lung cancer tissues and cancer cell lines is controlled by miRNAs as well as by the methylation of the promoter, which is another form of epigenetic modification .…”

Clinicopathological Significance and Diagnostic Value of <i>DLEC1</i> Hypermethylation in Lung Cancer: A Meta-analysis

“…Nowadays, a number of potential biomarkers have been reported, but very few have reached clinical standards to be an efficient index due to small study sizes and lack of assay optimization 22,23 . The aberrant of DLEC1 methylation had been reported in several cancers, such as prostate cancer 24 , gastric cancer 25 , and lung cancer 18 .…”

DNA methylation analysis of selected genes for the detection of early-stage lung cancer using circulating cell-free DNA

“…The modified DNA was amplified using real-time quantitative methylation-specific PCR (QMSP) as described by Nawaz et al 12 The target genes included CDH13, WT1, CDKN2A, HOXA9, PITX2, CALCA, RASSF1A, and DLEC1. The relative methylation level of the target genes was determined with β-actin as a reference gene.…”

“…One study found that the combined detection of multigene methylation can increase the sensitivity to 93%, but the specificity is decreased. 12 Even so, the concentration and integrity of free DNA in patients with cancer are higher than in healthy subjects, and detecting the methylation of lung cancer-associated genes by extracting free DNA in plasma is the trend in the early diagnosis of lung cancer in recent years.…”