DNA Methylation Pattern in Somatotroph Pituitary Neuroendocrine Tumors

Abstract

<b><i>Introduction:</i></b> Growth hormone secretion by sporadic somatotroph neuroendocrine pituitary tumors (PitNETs) is a major cause of acromegaly. These tumors are relatively heterogenous in terms of histopathological and molecular features. Our previous transcriptomic profiling of somatotroph tumors revealed three distinct molecular subtypes. This study aimed to investigate the difference in DNA methylation patterns in subtypes of somatotroph PitNETs and its role in distinctive gene expression. <b><i>Methods:</i></b> Genome-wide DNA methylation was investigated in 48 somatotroph PitNETs with EPIC microarrays. Gene expression was assessed with RNAseq. Bisulfite pyrosequencing and qRT-PCR were used for verifying the results of DNA methylation and gene expression. <b><i>Results:</i></b> Clustering tumor samples based on methylation data reflected the transcriptome-related classification. Subtype 1 tumors are densely granulated without <i>GNAS</i> mutation, characterized by high expression of <i>NR5A1</i> (SF-1) and <i>GIPR</i>. The expression of both genes is correlated with specific methylation of the gene body and promoter. This subtype has a lower methylation level of 5′ gene regions and CpG islands than the remaining tumors. Subtype 2 PitNETs are densely granulated and frequently <i>GNAS</i>-mutated, while those in subtype 3 are mainly sparsely granulated. Methylation/expression analysis indicates that ∼50% genes located in differentially methylated regions are those differentially expressed between tumor subtypes. Correlation analysis revealed DNA methylation-controlled genes, including <i>CDKN1B</i>, <i>CCND2</i>, <i>EBF3</i>, <i>CDH4</i>, <i>CDH12</i>, <i>MGMT</i>, <i>STAT5A</i>, <i>PLXND1</i>, <i>PTPRE</i>, and <i>MMP16</i>, and genes encoding ion channels and semaphorins. <b><i>Conclusion:</i></b> DNA methylation profiling confirmed the existence of three molecular subtypes of somatotroph PitNETs. High expression of <i>NR5A1</i> and <i>GIPR</i> in subtype 1 tumors is correlated with specific methylation of both genes.

Pituitary neuroendocrine tumors with PIT1/SF1 co-expression show distinct clinicopathological and molecular features

“…Integrated methylation data consensus cluster analysis confirmed that somatotroph tumors separate into three distinct epigenomic subclusters, as recently described (Fig. 3 a) [ 11 ]. SF1 expression depicted by immunoreactivity (this study) and RNA expression levels (external studies) demonstrated that the three epigenomic subclusters relate to the tumors denoted as DGST-PIT1/SF1, DGST-PIT1 and SGST-PIT1 in this study.…”

“…The methylation data of our 31 in-house samples were integrated into a large reference series of 77 somatotroph PitNETs compiled from three independent publicly available datasets [ 11 , 17 , 23 ]. Integrated methylation data consensus cluster analysis confirmed that somatotroph tumors separate into three distinct epigenomic subclusters, as recently described (Fig.…”

“…Raw methylation array data (idat files) from this study and publicly available deposits [Capper et al (GSE109381) [ 6 ]; Neou et al (E-MTAB-7762) [ 17 ], Kober et al (GSE226764) [ 11 ], and Silva-Júnior et al (GSE207937) [ 23 ]] were processed using the minfi package [ 1 ] in R [ 19 ]. Probes on sex chromosomes, probes with a detection p value of or above 0.01, probes with SNPs at the CpG site, and cross-reactive probes were excluded.…”

“…For evaluation of clinicopathological parameters in DGST-PIT1/SF1, DGST-PIT1, and SGST-PIT1 across studies, we compiled an extended case series consisting of our in-house samples, and publicly available data of somatotroph PitNETs derived from Capper et al [ 6 ] (GSE109381), Neou et al [ 17 ] (E-MTAB-7762, E-MTAB-7768), Silva-Júnior et al [ 23 ] (GSE207937, GSE209903), Kober et al [ 11 ] (GSE226764) and Rymuza et al [ 20 ] (E-MTAB-11889). Extended sample data of the latter study were kindly provided by Mateusz Bujko (Dept.…”

“…Previous transcriptome-based studies confirmed occasional SF1 expression in somatotroph PitNETs, predominantly in the densely granulated subtype [ 17 , 20 ]. Moreover, SF1 expression was recently linked to distinct transcriptomic signatures and methylation profiles among somatotroph PitNETs [ 11 , 20 ]. In summary, the current literature suggests that PIT1/SF1 co-expression associates with the somatotroph lineage.…”