Hypermethylation of adjacent CpG sites is negatively correlated with the expression of lineage oncogene in pulmonary neuroendocrine tumors

Abstract

Achaete-scute homolog 1 is a lineage oncogene of high-grade pulmonary neuroendocrine tumors. Due to the relatively few studies investigating the epigenetic regulation of achaete-scute homolog 1 expression, we wanted to address whether DNA methylation of the achaete-scute homolog 1 CpG island is associated with clinicopathological features in pulmonary neuroendocrine tumors and to investigate its effect on the expression of this gene. Here, We performed multiplex immunohistochemistry (PerkinElmer, Waltham, MA, USA) to check for achaete-scute homolog 1 and Notch homolog 1 expression in 139 pulmonary neuroendocrine tumor samples. Quantitative measurements of achaete-scute homolog 1 CpG island methylation were conducted using the MassARRAY EpiTYPER (Sequenom, San Diego, CA, USA). The correlation between immunohistochemistry data, methylation data, and clinicopathological information was analyzed. Achaete-scute homolog 1 methylation levels were increased in pulmonary neuroendocrine tumors compared to those in normal controls (0.107 vs 0.061, p < 0.001), and among the achaete-scute homolog 1 CpG island, only CpG_6 and CpG_7.8 showed higher methylation levels in pulmonary neuroendocrine tumors (0.208 and 0.135, respectively) compared to those in normal lung tissues (0.072 and 0.087, respectively; p < 0.001). Moreover, the methylation level of CpG_6.7.8 was higher in patients with stage I pulmonary neuroendocrine tumors than in patients with stage II/III pulmonary neuroendocrine tumors (0.19 ± 0.16 vs 0.14 ± 0.07, p = 0.012). The hypermethylation of CpG_6.7.8 showed an inverse correlation with achaete-scute homolog 1 protein expression (r = −0.408, p = 0.007, Spearman test). Finally, we found that CpG_6.7.8 of the achaete-scute homolog 1 CpG island is frequently hypermethylated in early-stage pulmonary neuroendocrine tumors, and this aberrant hypermethylation is negatively correlated with achaete-scute homolog 1 expression in this tumor spectrum.