DNA methylation in small lung adenocarcinoma with bronchioloalveolar carcinoma components

Abstract

We examined the methylation status in 100 specimens of lung adenocarcinomas measuring 2 cm or less and with bronchioloalveolar carcinoma (BAC) components (Noguchi types A–C) and then compared the methylation status between noninvasive tumors (Noguchi type A or B) and invasive tumors (Noguchi type C). Methylation-specific PCR was used to determine the methylation statuses of p16^INK4a, RASSF1A, CDH13, RARβ, and Cyclin D2. The methylation index that was regarded as representing the degree of methylation was calculated. We also determined the mutational statuses of EGFR exons 19 and 21 using a PCR-based method. A multivariate analysis showed that the aberrant methylation of p16^INK4a, RASSF1A, and CDH13 was significantly more frequent in invasive tumors than in noninvasive tumors [p16^INK4a, 36.5% versus (vs.) 8.3%, P = 0.0023; RASSF1A, 46.2% vs. 14.6%, P = 0.0012; CDH13, 42.3% vs. 10.4%, P = 0.0006]. The methylation index was significantly higher in invasive tumors than in noninvasive tumors (P = 0.004). The methylation of p16^INK4a was significantly more frequent in EGFR wild-type tumors than in EGFR mutant tumors (P = 0.021). Our results indicate the involvement of epigenetic alterations in the progression of adenocarcinoma with BAC components.

Introduction

Lung cancer is the leading cause of cancer deaths worldwide. Adenocarcinoma is one of the most common histologic types of lung carcinomas, and its incidence is still increasing in Japan. Recently, advances in diagnostic imaging techniques have enabled small peripheral adenocarcinomas to be recognized and treated. In 1995, Noguchi et al. developed an independent classification consisting of six tumor subtypes for small peripheral adenocarcinomas measuring 2 cm or less [1]. According to Noguchi's criteria, Noguchi types A–C tumors consist entirely or predominantly of a bronchioloalveolar carcinoma (BAC) component (“replacement tumors”). Noguchi types A and B tumors consist of BAC tumors without (type A) or with (type B) areas of alveolar collapse and subsequent fibrosis, whereas Noguchi type C tumors are BAC tumors with foci or fibroblastic proliferation. The prognosis of types A and B, in which metastasis is rare, is superior to that of type C. The World Health Organization (WHO) defined the major subtypes of adenocarcinoma as BAC, acinar, papillary, or solid with mucin. In general, the majority of tumors consist of mixtures of two or more subtypes. For practical purposes, Noguchi types A and B tumors are regarded as the equivalent of the WHO's BAC subtype (i.e., noninvasive) and Noguchi type C tumors are regarded as the equivalent of the WHO's mixed (i.e., invasive) tumors with prominent BAC components. The WHO classification also recognizes a peripheral lesion, atypical adenomatous hyperplasia (AAH), as a precursor to BAC. These classifications suggest a multistage progression model for most peripheral adenocarcinomas that gives rise to noninvasive BAC tumors that, in turn, later become invasive (mixed histology adenocarcinomas with BAC components), and have a poor patient prognosis, compared with pure BACs. The aberrant methylation of various tumor suppressor genes has been established as an important mechanism in human carcinogenesis [2]. Frequent methylation of cyclin dependent kinase inhibitor 4A gene (p16^INK4a), Ras association domain family 1 gene (RASSF1A), H-cadherin gene (CDH13), retinoic acid receptor-beta gene (RARβ), and cyclin D2 gene (Cyclin D2) have been reported to occur in lung adenocarcinomas, and the aberrant methylation of these genes is associated with lung carcinogenesis [3], [4], [5], [6], [7].

Regarding the involvement of aberrant methylation in carcinogenesis, the aberrant methylation of genes appears to increase significantly in the adenoma–carcinoma sequence of colorectal carcinogenesis, and the frequencies at which specific genes are methylated vary at different steps of carcinogenesis in a gene-specific fashion [8].

In this study, we examined the methylation statuses of five genes (p16^INK4a, RASSF1A, CDH13, RARβ, and Cyclin D2) that are frequently methylated in lung adenocarcinoma to investigate epigenetic alterations in the multistep carcinogenesis of small lung adenocarcinomas with BAC components.