Application of a methylation gene panel by quantitative PCR for lung cancers

Abstract

Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.

Aberrant Methylation of Genes in Sputum Samples as Diagnostic Biomarkers for Non-small Cell Lung Cancer: a Meta-analysis

“…Twelve of these studies (Destro et al, 2004;Konno et al, 2004;Belinsky et al, 2005;Olaussen et al, 2005;Georgiou et al, 2007;Shivapurkar et al, 2007;Liu et al, 2008;Vander et al, 2008;Hu et al, 2009;Kand et al, 2011Hwang et al, 2011Hubers et al, 2013) had QUADAS score ≥ 8, six (Wang et al, 2004;Zhang et al, 2004;Cirincione et al, 2006;Hus et al, 2007;Cao et al, 2008;Sun et al, 2013) had QUADAS score =7 and four (Wang et al, 2006;Guo et al, 2008;Peng et al, 2010;Zhang et al, 2012) had a QUADAS score ≤ 6.…”

“…The systematic literature search yielded a total of 22 studies including a total of 1208 patients and 1200 controls for final analysis (Destro et al, 2004;Zhang et al, 2004;Konno et al, 2004;Wang et al, 2004;Belinsky et al, 2005;Olaussen et 2007; Shivapurkar et al, 2007;Liu et al, 2008;Van der Drift et al, 2008;Guo et al, 2008;Cao et al, 2008;Hu et al, 2009;Peng et al, 2010;Kang et al, 2011;Hwang et al, 2011;Zhang et al, 2012;Hubers et al, 2013;Sun et al, 2013) (Figure 1). The baseline characteristics of the included studies were presented in Table 1.…”

Differential Methylation of a Short CpG-Rich Sequence within Exon 1 of TCF21 Gene: A Promising Cancer Biomarker Assay

“…Genomic DNA was extracted from cell lines, primary tumors and nonmalignant cells, sputum samples as described previously (9). Sodium bisulfite treatment was done as described previously (13).…”

“…qPCR analysis was done using the Chromo4 MJ Research Real-time PCR System. Sodium bisulfite -treated genomic DNA was amplified by fluorescence-based real-time methylationspecific PCR using TaqMan technology as described previously (9,15,16). In brief, primers (forward 5 ¶-CGAGGAGAGTTTTAATTGCGAGA-3 ¶ and reverse 5 ¶-CCTAACTAACCCCGCTCAAAAAA-3 ¶) and probe (5 ¶-FAM-TAGAAGGGTCGCGGCGGTTTGGBHQ-1-3 ¶) were designed to specifically amplify bisulfite-converted DNA within the region of the test genes that was differentially methylated between expression-positive and expression-negative cell lines (Fig.…”

“…Recently, we reported quantitative methylation-specific PCR analysis of sputum DNA based on a panel of methylated genes (9). The panel separated patients with lung cancer from those without lung cancer, showing the potential of the quantitative methylation-specific PCR analysis of sputum as an effective biomarker assay.…”