12nd Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary
2Momentum Hereditary Endocrine Tumors Research Group, Semmelweis University, Hungary
3National Bionics Program, Semmelweis University
4National Institute of Clinical Neurosciences, Budapest, Hungary
51st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary
6Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary
Introduction: Different methylation patterns have been already in certain pituitary adenoma types. Several studies reported site-specific hypermethylation influencing tumorsupressor genes, oncogenes, cell cycle regulator proteins. To date there is only a few information available regarding the whole genome methylation status and the reverse mechanism, DNA demethylation. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is an easy and accurate method to detect the level of methylcytosine (5mC) and the demethylation intermedier hydroxymethylcytosine (5hmC).
Aims: We investigated whole genome DNA methylation-demethylation status of different types of pituitary adenomas and correlated it with clinicopathological parameters.
Material and Methods: 44 DNA samples, isolated from fresh frozen human pituitary adenoma tissues (29 gonadotroph, 12 somatotroph and 3 corticotroph) were analyzed. Samples were collected and categorized according to the 2017 WHO classification. Cytosine and its derivates (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) were separated with HPLC-MS/MS. Data were analyzed with Statistica software 12.0.
Results: Higher level of 5hmC% and 5hmC%/5mC% ratios were confirmed in hormone-negative (HN) HN-SF1+ and HN-Tpit+ groups compared to FSH/LH+ and GH secreting adenomas. Significant difference between HN-SF1+ gonadotroph and FSH/LH+ gonadotroph adenomas was found but there was no alteration between HN-SF1+ and HN-Tpit+ tumors.
In the samples with higher proliferative index (Ki-67>4%) lower 5hmC (ns) and 5hmC%/5mC% ratio were detected than in slower proliferating tumours (Ki-67 1-2% or 3-4%)(p:0.009).
Conclusion: 5hmC% and 5hmC/5mC ratio indicating demethylation status can be reliably detected by HPLC-MS-MS method. We found correlation between methylation status and hormone-secretion/pituitary adenoma type as well as between methylation status and proliferative behaviour. Adenomas with higher Ki-67 indexes exhibit gradually lower demethylation. 5hmC/5mC ratio may be used as prognostic tissue biomarker but these data need to be further investigated on higher sample set.
Doctoral School of Clinical Medicine
Program: Hormonal Regulations
Supervisor: Henriett Butz