DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type

Abstract

Background

We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC.

Methods

We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.

Results

Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (percentage methylation reference (PMR) ≥ 4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level.

Conclusions

Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.

Introduction

Primary lung cancer is a major cause of death worldwide [1] and remains the leading cause of cancer death in the United States, with an estimated 215,020 new patients diagnosed and 161,840 deaths expected in 2008 [2]. Non-small cell lung cancer (NSCLC) accounts for 80% of these new cases and includes the following histologic types: adenocarcinoma, squamous cell carcinoma, large cell carcinoma and mixed histologies. Approximately 25–33% of NSCLC patients present with stage I or II disease, which permits surgical resection with curative intent. However, despite a complete and presumably curative resection, approximately 40–50% of patients with resected NSCLC die of recurrent disease [3].

In recent years, much attention has focused on whether women and men differ with respect to the epidemiologic and clinicopathologic features of NSCLC. Recent trends in the United States show that while incidence in men has declined during the past decades, lung cancer has continued to increase in women [4]. Further, much of the current evidence suggests that women have an increased susceptibility to lung cancer, are younger at age of diagnosis, are more likely to have adenocarcinoma compared to squamous cell carcinomas, and have better response to therapy and improved survival [5], [6], [7], [8], [9]. Identifying the molecular profiles of these patients could provide insight into differential pathways to malignancy in females compared to males and could lead to strategies for better treatment of individuals with NSCLC.

It has recently become clear that epigenetic alterations play an important role in cancer development and result in changes in gene function that occur without changes in nucleotide sequence [10]. One of the most well studied epigenetic changes is DNA methylation, which adds a methyl group to cytosines preceding guanidines (also called CpG dinucleotides). Methylation of CpG islands in the promoter region of the gene leads to gene silencing and inactivation, and it has been proposed that DNA methylation of promoter regions of tumor suppressor genes plays an important role in tumor development [11], [12]. A number of genes appear to be aberrantly methylated in tumor as opposed to non-tumor tissues from NSCLC patients [13], [14], [15], [16], [17], [18], [19]. Multiple studies have attempted to assess the clinical significance of one gene, or a small set of genes, in differentiating the clinicopathological features of these tumors [16], [18], [20], [21], [22], [23], [24], [25]. However, most of these studies utilized qualitative methylation-specific PCR (MSP) in order to detect DNA methylation, a method that can sometimes lead to false positive results and does not distinguish between low and high level methylation [26]. Furthermore, results in these studies have been inconsistent due to varying methylation detection protocols, PCR primers, and study populations, and none have comprehensively studied more than 10 genes.

In the present study, we chose to quantify methylation of genes which were potentially important in lung cancer, many of which have been assessed by others but some which have not been previously evaluated (Appendix 1). The selected genes affect apoptosis (DAPK1, RUNX3, TMS1, PTEN, and SOCS3), cell adhesion, invasion and/or metastasis (OPCML, CDH13, BVES, APC, CDH1, IGSF4, KCNH5, and PCSK6), cell cycle control (CCND2, RASSF1, APC, FHIT, CDKN2A, CDKN2B, P14, and PTEN), cell proliferation and/or differentiation (RARB, IGFBP3, KCNH5, KCNH8, SOCS3 and PTGS2), and DNA-repair and/or detoxification of DNA adducts (FHIT, MLH1, MGMT, FANCF, and GSTP1). In order to assess more broadly the clinical significance of gene hypermethylation in NSCLC, we determined the DNA methylation status of these 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.