Quantitative assessment of gene promoter methylation in non‑small cell lung cancer using methylation‑sensitive high‑resolution melting

Abstract

DNA methylation is closely associated with aberrant epigenetic changes. Previous studies have identified various genes associated with non-small cell lung cancer (NSCLC), but the precise combination responsible for its etiology is still debated. The aim of the present study was to select a new set of NSCLC-related genes using methylation-sensitive high-resolution melting. The promoter methylation status of six selected genes, consisting of protocadherin γ subfamily B, 6 (PCDHGB6), homeobox A9 (HOXA9), O6-methylguanine-DNA methyltransferase (MGMT), microRNA (miR)-126, suppressor of cytokine signaling 3 (SOCS3) and Ras association domain family member 5, also termed NORE1A, was evaluated in 54 NSCLC patients. From these samples, genome-wide DNA was extracted and bisulfite conversion was performed along with fluorogenic quantitative polymerase chain reaction to detect methylation values of the six selected promoters. The present results revealed frequent methylation on PCDHGB6, HOXA9 and miR-126, which contrasted with infrequent methylation on MGMT. The results indicated no methylation on either SOCS3 or NORE1A. The sensitivity and specificity of the methylation assessment were 85.2 and 81.5%, respectively, and the analysis results were validated by pyrosequencing. Furthermore, minute comparison of the association between DNA methylation and clinical features was performed. Overall, these results may provide potential information for the development of better clinical diagnostics and more targeted and effective therapies for NSCLC.

DNA methylation analysis with methylation‐sensitive high‐resolution melting (MS‐HRM) reveals gene panel for glioma characteristics

“…However, when pyrosequencing and MS‐HRM results were dichotomized into methylated and unmethylated groups (taking 10% methylation as a cutoff value) the results were fully concordant. Similar results were reported by several groups 52‐54 …”

Subtyping Lung Cancer Using DNA Methylation in Liquid Biopsies

“…This discrepancy may be due to methodological differences, since conventional methylation-specific PCR (MSP), a qualitative analysis, was used in those studies, whereas we employed qMSP, a quantitative method with higher sensitivity and specificity [42]. High HOXA9 methylation levels were previously reported in NSCLC [19,20,21,27], but to the best of our knowledge, this is the first study demonstrating higher HOXA9 methylation levels in SCLC tissue and ccfDNA samples. Moreover, higher HOXA9 methylation levels were found in SCC compared to AdC in tissue samples, confirming previous studies [43,44].…”

“…Furthermore, the detection of circulating cell-free DNA (ccfDNA) methylation in plasma/serum samples may better represent tumor heterogeneity than tissue biopsy, being also less invasive [16]. Thus, we sought to evaluate the feasibility of using methylation of four gene promoters, previously characterized as hypermethylated in cancer [15,16,17,18,19], to discriminate among the major LCa subtypes in ccfDNA extracted from plasma, by means of quantitative methylation-specific PCR (qMSP). Selection of candidate genes APC , HOXA9 , RARβ2, and RASSF1A was based on published literature [17,20,21,22], including our previous experience [15], since methylation levels disclosed differences among LCa subtypes, suggesting a role as biomarkers.…”

Methods for Early Detection of Lung Cancer (Review)

“…Были отобраны 54 пары опухолей и окружающих тканей для определения статуса метилирования промоторов возможных генов, ассоциированных с НМРЛ (PCDHGB6, HOXA9, MGMT, miR-126, SOCS3, NORE1A). Комбинация PCDHGB6, HOXA9, MGMT и miR-126 показала чувствительность 85,2 % и специфичность 81,5 %, что указывает на возможность ранней диагностики путем мониторинга метилирования промотора с использованием эффективной комбинации родственных генов [20].…”